Multifaceted HIV integrase functionalities and therapeuticstrategies for their inhibitionPublished, Papers in Press, August 29, 2019, DOI 10.1074/jbc.REV119.006901

X Alan N. Engelman1

From the Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215 and theDepartment of Medicine, Harvard Medical School, Boston, Massachusetts 02115

Edited by Karin Musier-Forsyth

Antiretroviral inhibitors that are used to manage HIV infec-tion/AIDS predominantly target three enzymes required forvirus replication: reverse transcriptase, protease, and integrase.Although integrase inhibitors were the last among this group tobe approved for treating people living with HIV, they have sincerisen to the forefront of treatment options. Integrase strandtransfer inhibitors (INSTIs) are now recommended componentsof frontline and drug-switch antiretroviral therapy formula-tions. Integrase catalyzes two successive magnesium-dependentpolynucleotidyl transferase reactions, 3� processing and strandtransfer, and INSTIs tightly bind the divalent metal ions andviral DNA end after 3� processing, displacing from the integraseactive site the DNA 3�-hydroxyl group that is required for strandtransfer activity. Although second-generation INSTIs presenthigher barriers to the development of viral drug resistance thanfirst-generation compounds, the mechanisms underlying thesesuperior barrier profiles are incompletely understood. A sepa-rate class of HIV-1 integrase inhibitors, the allosteric integraseinhibitors (ALLINIs), engage integrase distal from the enzymeactive site, namely at the binding site for the cellular cofactorlens epithelium-derived growth factor (LEDGF)/p75 that helpsto guide integration into host genes. ALLINIs inhibit HIV-1 rep-lication by inducing integrase hypermultimerization, whichprecludes integrase binding to genomic RNA and perturbs themorphogenesis of new viral particles. Although not yetapproved for human use, ALLINIs provide important probesthat can be used to investigate the link between HIV-1 integraseand viral particle morphogenesis. Herein, I review the mecha-nisms of retroviral integration as well as the promises and chal-lenges of using integrase inhibitors for HIV/AIDS management.

Combination antiretroviral therapy (cART)2 treats patientswith a mixture of drugs to inhibit different steps of the HIV-1

replication cycle (1) (Fig. 1). Unique among animal viruses is therequirement for retroviruses to integrate their genetic informa-tion into the genome of the host cell that they infect. Integrationis mediated by the viral protein integrase (IN), which is incor-porated into fledgling viral particles alongside the other viralenzymes reverse transcriptase (RT) and protease (PR). PR ini-tiates virus particle maturation by cleaving viral Gag and Gag-Pol polyprotein precursors into separate viral structural pro-teins and enzymes, which is required to form the viral core(reviewed in Ref. 2). The core consists of the viral ribonucleo-protein (RNP) complex, which contains two copies of the RNAgenome bound by viral nucleocapsid, IN, and RT proteins,encased within a fullerene shell composed of the viral capsidprotein (reviewed in Ref. 3). RT converts retroviral RNA into asingle molecule of linear DNA containing a copy of the virallong terminal repeat (LTR) at each end (Figs. 1 and 2) (reviewedin Ref. 4). The linear DNA, comprised of U3 and U5 terminalsequences in respective upstream and downstream LTRs, is thesubstrate for IN-mediated viral DNA insertion into chromo-somal DNA (5–7).

Four classes of antiretroviral drugs, nucleoside RT inhibitors(NRTIs), nonnucleoside RT inhibitors (NNRTIs), PR inhibitors(PIs), and IN strand transfer inhibitors (INSTIs) have in recentyears comprised frontline cART formulations (8). Highlightingthe success of the INSTI drug class, current guidelines recom-mend the use of a second-generation INSTI (dolutegravir(DTG) or bictegravir (BIC)) co-formulated with two NRTIs totreat most people living with HIV (PLHIV) who have not pre-viously failed an INSTI-containing regimen (9, 10). INSTIsinhibit IN strand transfer activity and thus specifically block theintegration step within the HIV-1 life cycle (11) (Fig. 1). A sep-arate class of inhibitors, the allosteric IN inhibitors (ALLINIs),

This work was supported by National Institutes of Health Grants R37 AI039394and R01 AI070042. The author has received fees from ViiV Healthcare Co.within the past 12 months. The content is solely the responsibility of theauthor and does not necessarily represent the official views of the NationalInstitutes of Health.

1 To whom correspondence should be addressed: Dept. of Cancer Immunol-ogy and Virology, Dana-Farber Cancer Institute, Boston, MA 02215.Tel.: 617-632-4381; Fax: 617-632-4338; E-mail: alan_engelman@dfci.harvard.edu.

2 The abbreviations used are: cART, combination antiretroviral therapy;ALLINI, allosteric integrase inhibitor; BIC, bictegravir; CAB, cabotegravir;CCD, catalytic core domain; CIC, conserved intasome core; cryo-EM, cryo-

genic EM; CTD, C-terminal domain; CSC, cleaved synaptic complex; DTG,dolutegravir; EVG, elvitegravir; FDA, Food and Drug Administration; IBD,integrase-binding domain; IN, integrase; INSTI, integrase strand transferinhibitor; KPN, karyopherin; LA, long-acting; LEDGF, lens epithelium-de-rived growth factor; LRA, latency reversal agent; LTR, long terminal repeat;MMTV, mouse mammary tumor virus; MVV, Maedi-visna virus; NNRTI; non-nucleoside reverse transcriptase inhibitor NRTI, nucleoside reverse tran-scriptase inhibitor; NTD, N-terminal domain; PDB, Protein Data Bank; PFV,prototype foamy virus; PI, protease inhibitor; PIC, preintegration complex;PLHIV, people living with HIV; PPT, polypurine tract; PR, protease; PrEP,pre-exposure prophylaxis; RAL, raltegravir; RANBP, Ran-binding protein;RNP, ribonucleoprotein; RT, reverse transcriptase; SHIV, simian-humanimmunodeficiency virus; SSC, stable synaptic complex; t-BSF, tert-butylsul-fonamide; TCC, target capture complex; TNPO, transportin; SH3, Src homo-logy 3.

croREVIEWS

J. Biol. Chem. (2019) 294(41) 15137–15157 15137© 2019 Engelman. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

by contrast inhibit particle maturation (12, 13) (see below). Tofully understand the nature of these different types of inhibi-tors, it is important to appreciate the different steps of HIV-1replication (Fig. 1) as well as the mechanistic and structuralbases of retroviral DNA integration.

Mechanism of retroviral integrationIN is a polynucleotidyl transferase composed of three con-

served protein domains: the N-terminal domain (NTD) withconserved His and Cys residues (HHCC motif) that coordinateZn2� binding for 3-helix bundle formation; the catalytic coredomain (CCD), which adopts an RNase H fold and harbors theenzyme active site composed of invariant carboxylate residues(DDE motif); and the C-terminal domain (CTD), which adoptsan SH3 fold (reviewed in Ref. 14). The role of the DDE residuesin catalysis is to coordinate the positions of two divalent cat-ions, which under physiological conditions are almost certainlymagnesium, to deprotonate attacking oxygen nucleophiles anddestabilize scissile phosphodiester bonds for one-step transes-terification chemistry (15, 16). Similar functionalities existacross a large superfamily of polynucleotidyl transferases that

includes related enzymes such as transposase proteins andRNase H (reviewed in Ref. 17).

Two different IN activities, 3� processing and strand transfer,are required for integration (Fig. 2). During 3� processing, INprepares the linear reverse transcript for integration by hydro-lyzing the DNA ends 3� of conserved CA dinucleotides, whichmost often liberates a dinucleotide from each end (18 –20).However, symmetrical DNA processing is not required for inte-gration; the upstream terminus of spumaviral DNA is 5�-TG,obfuscating the need for U3 end processing by IN (21), whereasa trinucleotide is processed from the U5 end of some primatelentiviruses (22, 23), including HIV-2 (24). During strand trans-fer, IN uses the CAOH-3� hydroxyl groups to cut chromosomalDNA in a staggered fashion, which, due to the nature of SN2chemistry, simultaneously joins the viral DNA ends to the5�-phosphate groups of the dsDNA cut (6, 7, 15). The resultinggapped DNA intermediate with unjoined viral DNA 5� ends isrepaired by host cell machineries to yield the integrated provi-rus flanked by the sequence duplication of the host DNA cut,which for HIV-1 is most often 5 bp (25, 26) (Fig. 2).

Figure 1. HIV replication cycle. After entry into a susceptible target cell, RT converts genomic RNA into linear DNA within the confines of the reversetranscription complex (RTC) (272). Processing of the viral DNA ends by IN yields the PIC (30), which can integrate the endogenous DNA made by reversetranscription into recombinant target DNA in vitro (5). Following nuclear import and integration, the provirus (flanked by composite cyan/yellow/magentaLTRs) serves as a transcriptional template to produce viral mRNAs for translation of viral proteins as well as nascent viral genomes that co-assemble with viralproteins to form immature virions that bud out from the infected cell (2). Shown is a generalized scheme that depicts the major steps of HIV-1 replication,although it is important to note that deviations from this plan exist throughout Retroviridae. Most notably, spumavirus reverse transcription occurs during thesecond half of the infectious cycle (after integration), and spumaviral particles accordingly predominantly contain dsDNA (104, 273). The primary steps in theHIV-1 replication cycle that are inhibited by the two major classes of IN inhibitors discussed herein are indicated.

JBC REVIEWS: HIV integrase mechanisms and inhibition

15138 J. Biol. Chem. (2019) 294(41) 15137–15157

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Intasome structure and function

Integration in cells is mediated by the preintegration com-plex (PIC), which is a large nucleoprotein complex derived fromthe core of the infecting virion (27, 28). Within the confines ofthe PIC, IN functions as part of the intasome nucleoproteincomplex, which is comprised of a multimer of IN and the viralDNA ends (29 –35) (Figs. 1 and 3). A series of X-ray crystallo-graphic and single-particle cryogenic electron microscopy(cryo-EM) structures determined over the past decade has clar-ified that the number of IN molecules required to build theintasome differs depending on the type of retrovirus (reviewedin Ref. 36). Seven retroviral genera are grouped into two sub-families of Retroviridae: Spumavirinae, solely harboring thespumaviruses, and Orthoretrovirinae, which encompass thelentiviruses, such as HIV-1, as well as �-, �-, �-, �-, and �-ret-roviruses. X-ray crystal structures of the spumavirus prototypefoamy virus (PFV) intasome provided initial high-resolutionviews of the functional IN-viral DNA architecture as well ascritical insight into the mechanism of INSTI action (see below)(35, 37, 38).

The PFV intasome is composed of an IN tetramer with thefollowing division of labor. Two extended, intertwined IN mol-ecules (Fig. 3A, blue and green) harbor operational active sitesand thus catalyze 3� processing and strand transfer activities,whereas the other two IN molecules (Fig. 3A, cyan) with non-operational active sites serve as bookends to truss the DNA-bound IN protomers together (35). The interwoven nature ofthe two catalytically active IN molecules, with their NTDsmutually swapped between CCDs, was observed previously incrystal structures of two-domain lentiviral IN NTD-CCD con-structs in the absence of DNA (39, 40). Prior to these structures,the NTD from one IN protomer had been shown to function intrans with the active site of a separate IN molecule within theactive HIV IN multimer (41, 42). The interwoven NTD-CCDarrangement at the heart of the machine leverages the partici-pation of both viral DNA ends in intasome assembly and DNArecombination.

Four types of intasomes describe the ground states and prod-uct complexes associated with IN 3� processing and strandtransfer activities. IN processes the viral DNA ends in the con-text of the initial stable synaptic complex (SSC), yielding thecleaved synaptic complex (CSC) after viral DNA hydrolysis.The target capture complex (TCC) describes the CSC boundto target or host DNA, whereas strand transfer yields the strandtransfer complex (16, 35–37, 43). The overall conformation ofthe PFV intasome structure changes little as the complexmorphs from the SSC to the strand transfer complex and pro-motes IN 3� processing and strand transfer activities (16, 35, 37,43, 44). Although integration occurs largely throughout animalcell genomes (45, 46), host DNA sequences that contort to fitthe target DNA-binding interface within the CSC are preferredtargets (37, 44, 47–49) (for a detailed review, see Ref. 50). Thus,

Figure 2. DNA cutting and joining steps of retroviral integration. Thelinear viral reverse transcript (lavender lines; plus-strands shaded more darklythan same-colored minus-strands throughout the cartoon) contains a copy ofthe LTR at each end composed of cyan U3, yellow R repeat, and magenta U5sequences. The upstream LTR is abutted by the primer-binding site (PBS; pur-ple box), whereas the downstream element is abutted by the polypurine tract(PPT; lavender box). During 3� processing, IN hydrolyzes the DNA adjacent toinvariant CA dinucleotides, which for HIV-1 liberates the pGTOH dinucleotide

from each end. After nuclear localization, the intasome interacts with hosttarget DNA (gray lines with targeted green sequence) to promote DNA strandtransfer. The DNA gaps that persist after strand transfer are repaired by hostcell machinery to yield a target site duplication (thin green lines) flanking theintegrated provirus.

JBC REVIEWS: HIV integrase mechanisms and inhibition

J. Biol. Chem. (2019) 294(41) 15137–15157 15139

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

strand transfer proceeds without gross rearrangements in inta-some architecture.

Studies of additional retroviral intasomes unveiled a com-mon structural feature at the hearts of the machines that wascoined the conserved intasome core (CIC) (51) (Fig. 3, A–C).However, as mentioned, different viruses utilize different num-bers of IN protomers to form the CIC. The tetrameric IN archi-tecture of the PFV intasome defines the basic features of theCIC, including two active protomers with CCDs and NTDsswapped across a synaptic interface where two CTDs engagetarget DNA for integration (37) and two additional IN mole-cules that bookend the active subunits (35). Whereas four PFVIN molecules suffice to build the CIC, �- and �-retroviral inta-somes require eight IN molecules (52, 53), and the lentivirusesHIV-1 and Maedi-visna virus (MVV) require 12 and 16 INprotomers, respectively (51, 54). The requirement for the dif-ferent numbers of IN molecules stems from evolutionary con-straints on the amino acid composition of the linker that con-nects the CCD and CTD (52). The linker in PFV IN, composedof �50 residues, is sufficiently long to allow the CTDs of theactive IN protomers to assume the positions required for hostDNA binding (Fig. 3D). The analogous linker in �- and �-ret-roviruses, at only �8 residues, precludes the necessary CTDpositioning (Fig. 3E). These viruses accordingly use two flank-ing IN dimers to position the critical CTDs, resulting in overall

IN octamers. The lentiviral IN CCD-CTD linker, althoughcomposed of �15–20 residues, adopts �-helical conformation(51, 55) that likewise imposes a distance constraint to precludeproper IN CTD positioning from the catalytically active INmolecules (Fig. 3F). In the MVV intasome, IN tetramers donatethe required CTDs, resulting in an overall IN hexadecamer (51).The HIV-1 intasome structure employed a fusion composed ofheterologous Sso7d protein appended onto the IN N terminus,which significantly improved IN solubility and enzyme activity(54, 56). Flanking Sso7d-IN dimers were seen to donate therequired CTDs to complete the CIC structure, resulting in anoverall IN dodecamer. On the one hand, it seems likely thatsome flexibility is tolerated in terms of the multimeric characterof the flanking IN oligomer that donates the CTD to the CICstructure, minimally requiring an IN dimer. On the other hand,it is possible that the use of the heterologous protein domainprecluded high occupancy of flanking IN tetramers in theSso7d-IN intasome structure. Additional structures derivedfrom WT HIV-1 or related primate lentiviral IN proteins mayfurther inform the IN-to-viral DNA stoichiometry necessarilyfor HIV-1 IN function.

A key unanswered question in retroviral integration researchis the mechanism of intasome assembly. DNA-based tetramer-ization of the predominant IN species in solution has been pro-posed (51) based on observations that PFV IN in the absence of

Figure 3. Retroviral intasome structures. A–C, representative intasomes from the spumavirus PFV (A; protein database (PDB) accession code 3OY9), �-ret-rovirus MMTV (B; PDB code 3JCA), and lentivirus MVV (C; PDB code 5M0Q) are color-coded to highlight the CIC. Green and blue, catalytically active IN protomers;cyan, supporting IN CCDs; black, DNA strands. Whereas four PFV IN molecules suffice to form the CIC, both MMTV and MVV require six IN protomers. For MMTV,critical CTDs (magenta) are donated by flanking IN dimers, leading to an overall IN octamer. In MVV, flanking IN tetramers provide the critical CTDs, resulting inan overall IN hexadecamer. Gray coloring in B and C deemphasizes IN elements that do not compose the CIC. D–F, resected CCD and CTD domains from abovegreen IN protomers, oriented to highlight the different CCD-CTD linker regions (dark gray). Associated magenta CTDs from separate IN protomers in E and Fassume similar positions as the green CTD in D. Red sticks, DDE catalytic triad residues.

JBC REVIEWS: HIV integrase mechanisms and inhibition

15140 J. Biol. Chem. (2019) 294(41) 15137–15157

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

DNA is monomeric (35), �- and �-retroviral INs are predomi-nantly dimeric (52, 53), and lentiviral INs are predominantlytetrameric although with evidence for additional lower- andhigher-order forms (40, 42, 51, 57– 62). However, the relation-ship between protein behavior in solution and the multimericstate of IN in virions or during reverse transcription is largelyunknown. Because HIV-1 IN binds genomic RNA in virions(63), it seems possible that RNA-bound IN may transfer to theDNA ends as they form during reverse transcription to initiateSSC formation. The IN tail region, which is composed of theamino acids C-terminal from the CTD SH3 fold, varies inlength from about 5 residues in the lentivirus equine infectiousanemia virus to 55 residues in MMTV. The tail region in �-ret-roviral IN, which is 19 residues, can regulate DNA-dependentIN octamer formation (64, 65). Although implicating a role forthis region of IN in intasome assembly, tail regions are unre-solved in all IN and intasome structures solved to date, limitingthe interpretation of how the tail might regulate nucleoproteincomplex formation.

INSTIs

Research in the mid-1980s first established a role for the 3�region of the pol gene, which encodes for IN, in retroviral rep-lication (66 –69), and the extension of this requirement toHIV-1 highlighted IN as a high-value antiviral target (70). How-ever, a scant number of promising preclinical lead compoundswere known by the time RT and PR inhibitors were adminis-tered to patients in cART formulations (71–73), calling intoquestion whether IN inhibitors would ever make it to the clinic.Indeed, around this time, I can recall one of the more promi-nent researchers in our field espousing the view at a nationalmeeting that clinical IN inhibitors were unattainable. The rea-soning here was based on the observation that an equal numberof IN, RT, and PR molecules are packaged into each virion par-ticle, which one can estimate as 120 based on the 20:1 synthesisratio of Gag to Gag-Pol (74) and circa 2,400 Gag molecules pervirion (75). Per replication cycle, RT and PR catalyze roughly19,400 and 12,900 chemical reactions, respectively. However,the same population of IN molecules performs only four chem-ical reactions. How then could one effectively inhibit IN in theface of this seemingly large excess of available enzyme? Fortu-itously, my colleague turned out to be incorrect. What wasunknown at the time of our discussion was the utility of mole-cules designed to inhibit IN strand transfer activity. WhereasHIV-1 IN processes the viral DNA ends to yield the CSC con-comitant with or soon after reverse transcription (30, 76) (Fig.1), integration into chromosomal DNA does not occur untilhours to days (76 –79) or, in some extreme cases, weeks later(80). The comparatively long-lived CSC intasome replicationintermediate is a pharmacological HIV-1 Achilles’ heel that isleveraged fully by the INSTI class of antiretroviral compounds.

Because HIV-1 IN purified from recombinant sources dis-played 3� processing and strand transfer activities in vitro (15,20, 81, 82), systems to search for inhibitory molecules of HIV-1IN activity were readily scalable (83–85). However, due to sub-optimal assay designs, few early leads turned out to specificallyinhibit HIV-1 integration under physiological conditions (86,87). Consider the following example. A compound such as

ethidium bromide that would likely score as a hit if test com-pounds were comixed together with IN and viral DNA is highlyunlikely to specifically inhibit integration in infected cells.Numerous early compounds accordingly lacked specificity toinhibit integration during HIV-1 infection (reviewed in Ref. 88).A key turning point in IN inhibitor development came fromreformulating the design of the in vitro assay to prebind IN to asynthetically preprocessed viral DNA end substrate (87) andscreen for inhibitors of strand transfer activity, which led to thediscovery of first-in-class INSTIs (11). Although these diketoacid compounds were never licensed to treat PLHIV, they nev-ertheless served as important molecules with which to probeINSTI mechanisms of action. INSTIs harbor two commonali-ties across otherwise diverse pharmacophores (Figs. 4A and 5).At the hearts of the compounds are three adjacent heteroatoms(most usually oxygen; red in Fig. 5, B and C), whereas a terminalhalogenated benzene ring connects to the rest of the moleculevia a flexible linker (Fig. 4A; blue in Fig. 5, B and C). The com-pounds avidly bound IN-viral DNA complexes yet failed toappreciably bind HIV-1 IN in the absence of viral DNA (89),and subsequent work revealed the importance of the terminaldeoxyadenylate residue at the 3� end of processed viral DNA inthe regulation of INSTI binding and dissociation (90, 91). Theconserved INSTI heteroatoms engage the divalent metal ionsthat are bound by the DDE active-site residues (35, 92).

Raltegravir (RAL) in 2007 was the first INSTI licensed by theUnited States Food and Drug Administration (FDA) (93) andelvitegravir (EVG) in 2012 became the second licensed INSTI(94). (Fig. 4A). Although prior work demonstrated the impor-tance of divalent metal ion and viral DNA sequence for INSTIbinding (90 –92), the field lacked a detailed view of how INSTIsinhibited IN strand transfer activity. Fortuitously, both NRTIsand INSTIs, which target respective RT and IN active sitescomposed of invariant amino acid residues, inhibit a wide rangeof retroviruses (95–103) including spumaviruses (104, 105).Thus, the PFV intasome could serve as a model system to inves-tigate INSTI mechanism of action. Co-crystal structures withRAL or EVG revealed that the halobenzyl groups assumed theposition of the purine rings of the 3�-deoxyadenylate residue,supplanting the terminal nucleoside from the IN active site (Fig.5, A and B). INSTI binding accordingly inactivates the intasomecomplex by displacing from the enzyme active site the DNA3�-OH group that is required to cut chromosomal DNA forstrand transfer activity (35).

Second-generation INSTI compounds physically expandupon first generation scaffolds while maintaining both metal-chelating and DNA-supplanting drug functions. Such modifi-cations include increasing the length of the linker between themetal-chelating and halobenzyl moieties (106 –108), increasingthe number of central ring moieties to three (106, 107, 109),and, akin to EVG, derivatization of a second ring that lies distalfrom the halobenzyl group (110 –113) (Fig. 4A). Second-gener-ation INSTIs more fully occupy the IN active-site region thatspans from the DNA-binding pocket on the one side to theconnector sequence that links IN secondary structural ele-ments �4 and �2 on the other (112–115) (Fig. 5C, �4-�2connector).

JBC REVIEWS: HIV integrase mechanisms and inhibition

J. Biol. Chem. (2019) 294(41) 15137–15157 15141

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Overlaying the structures of INSTI-bound PFV intasomes tothose of the SSC and TCC yielded important insight into themechanism of drug action (16, 112, 113). The RAL metal-chelating oxygen atom distal from the halobenzyl group coin-cided with the nucleophilic water molecule for IN 3� processingactivity (red sphere in Fig. 5D), whereas the RAL-chelating oxy-gen proximal to the halobenzyl coincided with the scissile phos-phodiester bond in viral DNA (Fig. 5D). For strand transfer, thehalobenzyl-proximal oxygen coincided with the nucleophilic3�-oxygen of processed viral DNA, whereas the distal RAL oxy-gen overlapped with the scissile phosphodiester bond in targetDNA (16) (Fig. 5E). These observations first identified INSTIsas IN substrate mimics, which was subsequently expandedthrough the broader concept of substrate envelope. Previouslyespoused for HIV-1 PR and the mechanism of PI action, thesubstrate envelope is defined as the space occupied by the sub-strate (peptide in the case of PR; DNA for IN) in an enzymeactive site. Because the enzyme must interact with the substratefor catalysis, drugs that interfere with enzyme–substrate inter-actions should be inhibitory and might impart relatively highresistance barriers (116 –118). Indeed, second-generationINSTI elements distal from the halobenzyl groups coincidewith the position of host DNA in PFV intasome structures (16,112–114) (Fig. 5E), likely accounting for the competitionbetween target DNA and INSTIs for binding to HIV-1 IN-viralDNA complexes (89). Compound modifications that furtherinterfere with HIV-1 IN–substrate interactions could improveINSTI potency and increase the barrier to acquire drug-resist-ant mutations (119).

Second-generation INSTIs are currently undergoing exten-sive safety evaluation due to their planned global rollout for

HIV/AIDS treatment. Although DTG was initially deemed safefor pregnant women (120), follow-up work highlighted agreater frequency of neural tube defect in infants born toBotswanan mothers who were taking DTG-containing cARTsince the time of conception (4 of 426; 0.94%) versus frequenciesobserved in the general population (86 of 87,755; 0.1%) or ininfants from mothers taking other cART regimens (14 of11,300; 0.12%) (121). Such observations prompted several reg-ulatory agencies including the FDA and the World HealthOrganization in 2018 to issue alerts regarding possibleincreased risk of neural tube defect in infants born to motherstaking DTG-containing cART at the time of conception (122).Subsequent retrospective analyses have failed to detect a linkbetween DTG usage and neural tube birth defect, althoughsuch studies were generally limited by sample size (123, 124).Retrospective analysis has also failed to detect an increase in thefrequency of neural tube defect in infants born to mothers onRAL-based drug regimens (125). Recent follow-up work thatincreased the number of patients from 426 to 1,683 in theBotswanan cohort revised the neural tube defect frequencyfrom 0.94% to 0.3%, which was still 0.2% greater than the fre-quencies observed in control populations (126). It will beinformative to ascertain whether BIC, which is the otherlicensed second-generation INSTI and is structurally related toDTG (Fig. 4A), also influences neural tube defect frequencyversus control populations.

Mechanisms of HIV resistance to INSTIs

Substitutions of HIV-1 IN residues Gln-148, Asn-155, orTyr-143 were recognized early on as separate genetic pathwaysto clinical RAL resistance (127), and mutant viral strains har-

Figure 4. INSTI structures and ALLINI chemotypes. A, diagrams of the four FDA-licensed INSTIs as well as investigational second-generation compound 6p(113, 119). B, representative ALLINI chemotypes. Asterisks mark common positions of t-butoxyacid moieties.

JBC REVIEWS: HIV integrase mechanisms and inhibition

15142 J. Biol. Chem. (2019) 294(41) 15137–15157

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

boring changes such as Q148H/G140S or N155H conveyed sig-nificant resistance to EVG as well (reviewed in Ref. 128).Although structure-based studies with PFV intasomes in-formed the mechanisms of drug resistance (35, 38), partialamino acid identity between PFV and HIV-1 INs limits theextent of information that can be gleaned from the model sys-tem. PFV and HIV-1 IN are overall 18.4% identical, whereastheir respective CCDs share 22.4% identity (35). Fortuitously,two of the three clinically relevant amino acids, Tyr-143 andAsn-155, are conserved as Tyr-212 and Asn-224 in PFV IN (Fig.5A). The binding mode of RAL to the PFV intasome in partic-ular informed the Tyr-143 resistance pathway, as the methyl-

oxadiazole constituent of this drug stacked against the p-cresolside chain of IN residue Tyr-212 (Fig. 5B). Changes that wouldreduce the aromatic nature of HIV-1 IN residue Tyr-143 wouldaccordingly result in loss of an important RAL binding contact.The structure accounted for the relative specificity of RAL resis-tance to Tyr-143 changes in IN (129, 130), as only RAL amongthe clinical INSTIs harbors the methyl-oxadiazole constituent(Fig. 4A). Although Asn-224 similarly resides near the INSTI-binding pocket, it does not directly contact bound drugs. Themutant His side chain in the intasome structure derived fromPFV IN N224H interacted with the 3�-deoxyadenylate–bridging phosphate, which was disrupted by second-generation

Figure 5. Mechanisms of INSTI action. A, close-up view of one PFV IN active site in the CSC intasome structure (PDB code 3OY9) with IN secondary elementslabeled. Additional labels highlight the conserved CA dinucleotide of the transferred DNA strand (magenta sticks) and associated G nucleotide of the non-transferred strand (orange), the 3�-OH nucleophile of the terminal deoxyadenylate used by IN to cut chromosomal DNA, as well as IN residues (in sticks) thatcompose the catalytic triad (Asp-128, Asp-185, and Glu-221) and that when changed confer INSTI resistance (Tyr-212 and Asn-224). Blue and red stick colorsdenote nitrogen and oxygen atoms, respectively. Gray spheres, divalent metal ions; �, �, and � denote �-helix, �-strand, and 310-helix, respectively. B, RAL (cyansticks)-bound PFV intasome structure (PDB code 3OYA) oriented as in A to highlight the mode of INSTI binding. RAL binding results in a greater than 6-Ådisplacement of the 3�-OH of the terminal deoxyadenylate from the IN active site. The position of the RAL methyl-oxadiazole group is highlighted in the cartoonon the left as well as the chemical diagram on the right, which was reconfigured from Fig. 4A to accentuate the position within the crystal structure. Other colorsand labeling are the same as in A or as described under “INSTIs.” C, same as in B, except with DTG bound (PDB code 3S3M). The position of the IN �4-�2connector is noted; other labeling is the same as in A and B. D, RAL (magenta) from the drug-bound PFV CSC structure (PDB code 3OYA) is overlaid with the PFVSSC structure (green; PDB code 4E7I) to highlight mimicry between drug oxygen atoms and oxygen atoms critical for IN 3� processing activity (red sphere,nucleophilic water (W) molecule; red bridge in viral DNA (vDNA), scissile phosphodiester bond). E, similar to D; RAL is superimposed onto the PFV TCC structure(IN and target DNA in cyan and vDNA in blue; PDB code 4E7K) to highlight similarly positioned drug oxygen atoms with the vDNA 3�-oxygen and scissilephosphodiester bond in target DNA (tDNA) critical for strand transfer activity. Other labeling is the same as in A and B.

JBC REVIEWS: HIV integrase mechanisms and inhibition

J. Biol. Chem. (2019) 294(41) 15137–15157 15143

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

INSTI MK-2048 binding (38). Although disruption of the His–DNA interaction could contribute to the mechanism of clinicalINSTI resistance to HIV-1 N155H (38), intasome structureswith INs that share greater amino acid identity to HIV-1 areexpected to more completely inform INSTI resistance mecha-nisms outside of the Tyr-143 pathway. Of note, although MVVis a lentivirus, its IN also shares limited amino acid sequenceidentity with HIV-1 IN (27.4% overall; 34.3% between CCDs).Given fast-paced advancements in single-particle cryo-EM(131, 132), one can optimistically expect comparatively high-resolution structures of INSTIs bound to the HIV-1 intasomein the not too distant future. Such structures should criticallyinform mechanisms of INSTI drug resistance as well as how topotentially improve INSTI potencies moving forward.

Clinical (133–135) as well as in vitro (136) studies have high-lighted the superior resistance profiles of second-generationINSTIs such as DTG compared with predecessor first-genera-tion compounds. Whereas selection of HIV-1 resistance in cellculture invariably leads to changes in IN that can confer �100-fold resistance to RAL and EVG, such resistance is much harderto come by for DTG, and the selected changes in IN, such asR263K, engender just a few-fold resistance to the compound(137). Whereas cART formulations typically comprise threedistinct compounds, such observations inspired clinical evalu-ation of DTG as a monotherapy or as dual therapy in conjunc-tion with an NRTI, NNRTI, or PI (138 –143). Based on rates ofvirological failure, the use of DTG as a monotherapy for PLHIVis contra-indicated, whereas the evaluation of dual therapyoptions is ongoing (143) (reviewed in Ref. 144). Current guide-lines recommend the use of DTG, BIC, or RAL with two NRTIsfor most PLHIV (9, 10).

The rates at which INSTIs dissociate from IN-viral DNAcomplexes in vitro have informed the mechanisms of drugaction and drug resistance. Consistent with its comparativelyhigh resistance barrier, the dissociative half-life of DTG, 71 h,was significantly longer than the corresponding RAL and EVGvalues of about 9 and 3 h, respectively (145). Although analysesof mutant IN-viral complexes failed to identify a direct correla-tion between drug dissociation half-life and antiviral potencyand resistance, HIV-1 was generally sensitive to INSTIs whencompound dissociative half-life was greater than 4 h and resist-ant to inhibition when half-lives were less than 1 h (145). Thus,INSTI dissociative half-life is a useful predictor of drug potencyand drug resistance. Whereas some IN amino acid substitu-tions, such as Y143R/K/C, increase dissociation by altering adirect IN-INSTI contact (35), changes such as Q148H/G140Sseemingly act indirectly by altering the conformation of the INactive-site region (38).

Alterations of viral DNA sequence, especially the terminaldeoxyadenylate residue, also alter INSTI dissociative half-life(91), although to date, LTR sequence changes have not beenimplicated in INSTI resistance. Reverse transcription initiateswith minus-strand DNA synthesis via a co-packaged hosttRNALys3 primer that engages the primer-binding site near the5� end of the viral RNA genome (see Ref. 4 for review). Synthesisof the plus-strand of retroviral DNA is primed via an oligonu-cleotide derived from the 3� polypurine (PPT) tract. In the RNAgenome, the 3� PPT abuts the U3 sequence that will form the

upstream viral DNA terminus after reverse transcription.Selection of DTG resistance in cell culture has revealed changesin the HIV-1 3� PPT, which was unexpected because thissequence abuts the downstream LTR distal from the viral DNAtermini (Fig. 2) (146). One possible explanation is that the alter-ations lead to misprocessing of the PPT during reverse tran-scription and accordingly extend the U3 DNA terminus, whichwould be a suboptimal sequence for IN binding (147). However,sequencing of 2-LTR circles, which form at low frequency in thecell nucleus via DNA ligation and thus provide a snapshot ofviral DNA end sequences, failed to identify the hypothesizedPPT extension (148). PPT mutations have been recorded in onepatient who received DTG monotherapy (149), indicating thatsuch changes may very well be clinically relevant. Additionalwork is required to more fully document the frequency of PPTchanges in patients that fail DTG therapy as well as how suchchanges engender drug resistance.

Other changes outside of the IN coding region, including theHIV-1 env gene, can confer resistance to DTG (150). HIV-1 caninfect cells through fusing directly with the cellular plasmamembrane or through the virological synapse that formsbetween an infected cell and an uninfected cell (151) (reviewedin Ref. 152). The env mutations effectively increased the multi-plicity of HIV-1 infection by significantly increasing the effi-ciency of cell-to-cell infection (150). This mechanism of drugresistance, which is indirect because it is highly unlikely toinfluence the dissociative half-life of DTG from the HIV-1 inta-some, is reminiscent of prior reports that HIV-1 infectionthrough the virological synapse reduced the efficacy of certaincART compounds (153, 154). Additional work is required todetermine whether changes in HIV-1 env can confer resistanceto DTG in the clinical setting (150).

The investigational second-generation INSTI cabotegravir(CAB) formulated as a crystalline nanoparticle conferred long-acting (LA) protection against challenge by chimeric simian-HIV (SHIV) in the macaque model of HIV/AIDS (155–157).LA-CAB administered as a monotherapy or in combinationwith LA-rilpivirine, which is a long-acting NNRTI, is beingevaluated as a pre-exposure prophylaxis (PrEP) to preventHIV-1 infection (reviewed in Ref. 158). One of the biggest fac-tors contributing to the emergence of anti-HIV drug resistanceis dosing protocol compliance, which for oral cART formula-tions is one to several pills daily. LA regimens largely obfuscatethe need for end-user dose monitoring, which could increasecompliance, although at the same time such regimens requireregular injections to maintain plasma trough concentrationsabove values required to inhibit HIV-1 replication. Macaquesthat were positive for SHIV RNA but seronegative at the time ofinfection could develop resistance to LA-CAB, and the associ-ated IN changes conferred potent cross-resistance to alllicensed INSTIs (159). Such observations highlight the need tocarefully monitor patients to avoid initiating PrEP duringunrecognized acute HIV-1 infection. CAB in cell culture is mar-ginally less effective at inhibiting infection by certain INSTI-resistant viruses than is either DTG or BIC (160), and both DTG(161) and BIC (162) have been formulated as LA compounds.Future research will evaluate the efficacy of LA INSTIs to treatat risk patients with PrEP as well as PLHIV.

JBC REVIEWS: HIV integrase mechanisms and inhibition

15144 J. Biol. Chem. (2019) 294(41) 15137–15157

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

ALLINIs

Despite relatively high barriers, second-generation INSTIsdo select for resistance (149, 159, 163). As exemplified by theclinical successes of the NRTIs and NNRTIs, it would accord-ingly be highly beneficial to have additional drug classes thatinhibit IN activity through novel mechanisms of action.

Whereas a number of different types of IN-targeting mole-cules have been described in the literature, the class of com-pounds collectively known as ALLINIs has advanced thefurthest. Predecessor compounds of potent ALLINIs were dis-covered via two different means, including a high-throughputscreen for inhibitors of IN 3� processing activity (164 –166) andstructure-guided modeling of the amino acid contacts thatmediate the interaction of HIV-1 IN with the host integrationtargeting cofactor lens epithelium-derived growth factor(LEDGF)/p75 (167). In addition to ALLINI (168), such com-pounds have been referred to as LEDGIN for LEDGF-interac-tion site (167), NCINI for noncatalytic site IN inhibitor (165,169), IN-LAI for IN-LEDGF allosteric inhibitor (170), andMINI for multimeric IN inhibitor (171).

Although HIV-1 in large part integrates throughout thehuman genome (46), it does so in nonrandom fashion, on aver-age favoring active genes that reside within relatively gene-dense regions of chromosomes (172). This targeting preferenceis largely dictated through specific interactions of two PIC-as-sociated proteins, IN and capsid, with respective host factorsLEDGF/p75 and cleavage and polyadenylation specificity factor6 (173, 174) (for a recent review, see Ref. 50). LEDGF/p75 is achromosome-associated (59, 175, 176) transcriptional co-acti-vator (177) that harbors two globular domains, an N-terminalPWWP (for Pro-Trp-Trp-Pro) chromatin reader with affinityfor histone 3 Lys-36 trimethylation (178 –180), and a down-stream region that was termed the IN-binding domain (IBD)because it mediated the binding of LEDGF/p75 to HIV-1 IN invitro (181). The LEDGF/p75 IBD is a PHAT domain (for pseu-do-HEAT repeat analogous topology) composed of two helix-hairpin-helix HEAT repeats (182) (Fig. 6A, left). The IN CCDdimerizes via an extensive interface with DDE catalytic triadspositioned at distal apices (183). LEDGF/p75 hotspot interac-tion residues Ile-365, Asp-366, and Phe-406 within the IBDhairpins engage both IN monomers at the CCD dimer interface(182, 184). Whereas Asp-366 hydrogen-bonds with the back-bone amides of residues Glu-170 and His-171 within one INmonomer, Ile-365 occupies a hydrophobic pocket composed ofIN residues from both IN monomers (Fig. 6A, right). Electro-positive residues within IBD �1 make additional contacts withelectronegative residues within the HIV IN NTD (185). HIV-1IN subunits undergo dynamic exchange in solution (61), andLEDGF/p75 binding accordingly stabilized HIV-1 IN dimersand tetramers (40, 61, 186, 187) and significantly stimulated INcatalytic activities in vitro (40, 59, 61, 185, 188). The LEDGF/p75-IN interaction is specific to the lentivirus genus of Retro-viridae (189 –191).

Cellular depletion of LEDGF/p75 predominantly limitsHIV-1 infection by reducing the level of integrated viral DNA(192–197). Expression of fusion proteins in susceptible targetcells comprised of GFP and the LEDGF/p75 IBD also inhibited

integration (192, 198), an effect that was exacerbated signifi-cantly by RNAi-mediated knockdown of LEDGF/p75 expres-sion (199). Such observations highlighted the antiviral potentialof small molecules designed to inhibit the interaction betweenHIV-1 IN and LEDGF/p75. Although inhibition of IN-LEDGF/p75 binding was initially espoused as the antiviral mechanismof action (167), there has since been little evidence to suggestthat inhibition of the protein–protein interaction significantlycontributes to ALLINI potency.

ALLINI compounds are built around heterocyclic cores,such as pyridine (171, 200), thiophene (201), quinoline (165,167, 168, 170, 197, 202–204), isoquinoline (205), thienopyri-dine (167, 206) (Fig. 4B), or naphthyridine (207); additional che-motypes have been described in the patent literature (reviewedin Ref. 208). Potent compounds contain a 2-carbon arm harbor-ing t-butoxy and carboxylic acid that is commonly connected tothe ring two positions from the heteroatom (Fig. 4B; also seeFig. 6, B and C). As espoused in the initial LEDGIN paper (167),the binding modes of these compounds to HIV-1 IN in largepart mimic LEDGF/p75 binding. Co-crystal structures with theHIV-1 IN CCD dimer revealed that ALLINI carboxylic acidsmimic the carboxylate side chain of LEDGF/p75 residue Asp-366 by making similar contacts with the backbone amides of INresidues Glu-170 and His-171 (Fig. 6, cyan IN monomer). Thet-butoxy moiety additionally interacts with IN residue Thr-174from this same monomer. ALLINI central rings mimic LEDGF/p75 residue Ile-365 by occupying a hydrophobic pocket com-posed of residues from both IN monomers. The benzimidazolemoiety in pyridine ALLINI KF116 additionally interacts withIN residue Thr-125 of the green IN monomer (Fig. 6C). Similarbinding modes can explain why quinoline ALLINIs effectivelyinhibited the LEDGF/p75-IN interaction in vitro (167, 170, 202,204, 206). By contrast, pyridine (171) and isoquinoline (205)ALLINIs are comparatively weak inhibitors of the virus– hostinteraction.

ALLINIs can inhibit HIV-1 IN 3� processing and strandtransfer activities in vitro in a LEDGF/p75-independent man-ner (168, 170, 202, 205, 206). Accordingly, quinoline ALLINIsinhibited HIV-1 infection at the integration step when the com-pounds were added to susceptible target cells at the time ofvirus infection (12, 167, 170, 202). However, inhibition of inte-gration is arguably a side effect of ALLINI antiviral potency (13,169, 171), as the compounds display much greater potenciesduring the late phase of HIV-1 replication (12, 169 –171, 204,209, 210). Retroviral RNP complexes appear electron-dense innegatively stained thin sections due to the comparative inabilityof electrons to pass through these structures. The underlyingbasis for ALLINI antiviral activity is IN hypermultimerization(12, 169 –171, 204, 209 –214), which inhibits IN binding toRNA in virions (63) and elicits the formation of eccentric HIV-1particles with viral RNP complexes situated outside of compar-atively electron-translucent and often deformed capsid shells(12, 13, 169 –171, 201, 209, 210, 212, 215). The morphologydefect is reminiscent of what is seen via a variety of mutations inthe IN region of HIV-1 pol that yield so-called class II INmutant viruses (216) (reviewed in Refs. 217 and 218). Sucheccentric viral particles are noninfectious due to their inabilityto promote reverse transcription in target cells (12, 13, 169 –

JBC REVIEWS: HIV integrase mechanisms and inhibition

J. Biol. Chem. (2019) 294(41) 15137–15157 15145

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

171, 209, 217, 218). Both IN and viral RNA are rapidly degradedafter cell entry, likely due to their exposure to the cell cytoplasmoutside the confines of the protective capsid shell (214, 219).

ALLINI potency during the late phase of HIV-1 replication isindependent of cellular LEDGF/p75 content (12, 169, 220).Remarkably, LEDGF/p75 depletion significantly increased thepotency at which the quinoline ALLINI BI-D inhibited HIV-1integration (12, 197). Instead of being the antiviral target,engagement of IN by LEDGF/p75 during the early phase ofHIV-1 infection protects the virus from the inhibitory action ofthe compounds (221). Accordingly, quinoline and thienopyri-dine ALLINIs can significantly diminish the extent of HIV-1integration into genes in LEDGF/p75-expressing cells (171,204, 221, 222).

Cells that comprise the latent HIV-1 reservoir show variablegrowth characteristics, with integrations near growth-promot-

ing genes linked in some cases to cellular proliferation (223–225) (reviewed in Ref. 226). Because LEDGF/p75 depletionresults in global shifts of HIV-1 integration sites toward gene 5�end regions (173, 195, 227), treating patients with quinolineALLINIs could cause unwanted side effects from promoter-proximal integration if growth promotion was sufficiently up-regulated to seed tumorigenesis. However, preliminary work inthis area has revealed that HIV-1 proviruses formed in theabsence of LEDGF/p75 are transcriptionally repressed. The useof HIV-1 reporter vectors that express two different fluoro-phores— one from the LTR and another from a constitutivelyactive internal promoter—indicated that LEDGF/p75 deple-tion or treatment with thienopyridine ALLINI CX014442 spe-cifically decreased LTR activity and accordingly increased theproportion of latent proviruses that form during the early phaseof HIV-1 infection in cell culture (222). Moreover, such provi-

Figure 6. ALLINI mimicry of LEDGF/p75 binding to the HIV-1 IN CCD dimer. A, solution structure of the LEDGF/p75 IBD (left; PDB code 1Z9E) and IBD-CCDco-crystal structure (right; PDB code 2B4J) highlight the locations of hotspot-interacting residues Ile-365 and Asp-366 in the hairpin that connects �-helices 1and 2 and Phe-406 in the �4-�5 hairpin (left). Whereas Asp-366 interacts with the backbone amide groups of IN residues Glu-170 and His-171 of the cyan INmonomer (dashed lines), Ile-365 occupies a hydrophobic pocket composed of IN residues from each monomer (i.e. Trp-132 of the green IN monomer andMet-178 of the cyan monomer; right). Other colorings denote atoms of interacting amino acid residues: nitrogen (blue), sulfur (yellow), and oxygen (red). B,quinoline ALLINI BI 224436 (left, chemical diagram) bound to the IN CCD dimer (right, PDB code 6NUJ). The interactions between the compound carboxylic acidand backbone amides within the IN cyan monomer are analogous to those shown in A for LEDGF/p75 residue Asp-366. Thr-174 of the IN cyan monomeradditionally interacts with the t-butoxy moiety of the drug. Other labeling is the same as in A. C, binding of pyridine ALLINI KF116 (left, chemical structure) tothe IN CCD dimer (right, PDB code 4O55). This view, rotated down �90° from A and B, is shown to accentuate the drug-binding pocket. In addition to thecontacts described in B, Thr-125 of the green IN monomer interacts with the benzimidazole moiety of KF116. Other labeling is as defined in A and B.

JBC REVIEWS: HIV integrase mechanisms and inhibition

15146 J. Biol. Chem. (2019) 294(41) 15137–15157

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

ruses were refractory to transcriptional activation by latencyreversal agents (LRAs) (222). Such observations have promptedthe notion that quinoline or thienopyridine ALLINIs could beused as part of PrEP regimens to limit the size of the latent viralreservoir that forms during the acute phase of HIV-1 infection(228). Curiously, LEDGF/p75 can repress the transcription ofestablished HIV-1 proviruses (229). The mechanistic connec-tion between the transcriptional competency of newly formedHIV-1 proviruses and IN-LEDGF/p75 binding is currentlyunclear. It should be informative to fine map the positions ofthese proviruses as well as their responses to LRA treatment.

Both an X-ray crystal structure (212) and molecular model-ing (213, 214) have yielded clues as to the nature of ALLINI-induced IN hypermultimerization. Quinoline ALLINIs boundat the LEDGF/p75-binding pocket of an IN dimer engaged theCTD of a separate IN dimer, thereby templating the polymeri-zation of IN dimers through successive interdimeric CTD-ALLINI-CCD bridge contacts (212, 213). Whereas quinolineALLINIs similarly hypermultimerized IN dimers and tetram-ers, the pyridine ALLINI KF116 specifically multimerized INtetramers (214). Modeling revealed that hypermultimerizationin this case occurred through successive CTD-ALLINI-CCDbridge contacts whose formation specifically required INtetramers (214). KF116 potency tracked hand-in-hand withHIV-1 IN tetramerization, indicating that the tetramer is thepredominant form of IN in HIV-1 virions (214).

Cell-free virions are recalcitrant to ALLINI treatment,revealing that inhibition requires HIV-1 exposure to ALLINIsin the confines of virus-producing cells (12, 169, 170). Thien-opyridine ALLINI CX05045 enhanced the multimerization ofpurified HIV-1 Pol protein in vitro, indicating that the antiviraltarget during HIV-1 infection could be Gag-Pol (209). Geneticexperiments, however, revealed that this need not be the case.Although usually incorporated into HIV-1 particles via Gag-Pol, IN can be supplied in trans as a fusion protein with theaccessory protein Vpr (230). HIV-1 harboring IN expressedsolely from Vpr-IN remained fully sensitive to ALLINI inhibi-tion, revealing that Gag-Pol need not be the initial site ofALLINI engagement (12, 13). ALLINI-mediated multimeriza-tion of Pol protein in vitro minimally suggests that the CCDdimer interface within IN is present in Pol. Additional work isrequired to ascertain whether ALLINIs may first gain access toIN via engaging Gag-Pol under normal infection conditions.Conceivably, initial engagement via Gag-Pol or IN coulddepend on the type of compound, as the pyridine ALLINIKF116 specifically targeted tetrameric IN (214), and it isunknown whether IN could tetramerize in the context ofGag-Pol.

Similar to the NNRTIs, which engage an allosteric bindingpocket on RT (reviewed in Ref. 231), ALLINIs are specific forHIV-1 and do not inhibit closely related primate lentivirusessuch as HIV-2 or simian immunodeficiency virus from rhesusmacaques (167, 201, 209). Eccentric HIV-1 particles producedby exposure to the thiophene ALLINI MUT-A displayedimmunoreactivity characteristics similar to mock-treated viri-ons, indicating a potential novel avenue for chemically inacti-vated immunogens as vaccine candidates (201). Given the spec-ificity of ALLINIs for HIV-1 IN, intensive safety evaluations

necessitated by such approaches will likely require chimericSHIV strains that carry HIV-1 IN (232).

The role of IN in HIV-1 particle morphogenesis is an ongoingarea of investigation. IN, as a free protein or as part of Gag-Pol,could nucleate the formation of the capsid shell around theRNP (13). Disruption of IN–RNA binding (63) and/or IN–INdynamics through mutations or ALLINIs would then yield non-infectious particles with eccentric electron density. Such mod-els invoke IN as a molecule tether or communicator betweenthe RNP and the capsid; although IN can directly interact withdifferent components of the RNP, including RT (233–237) andgenomic RNA (63), interactions with the virus capsid proteinhave not been reported. ALLINIs are important compoundswith which to further probe the role of IN in HIV-1 particlemorphogenesis.

HIV-1 resistance to ALLINI compounds

Resistance against quinoline and thienopyridine ALLINIs isreadily selected in cell culture, with most changes mapping toIN residues in the vicinity of the LEDGF/p75-binding pocket(165, 167, 201–203, 212). Whereas some of these, such as Thr-174, are invariant among circulating HIV-1 strains, others, suchas Ala-124 and Ala-125, are highly polymorphic in nature(238 –241) (reviewed in Ref. 242). Because Asn is often found atposition 124 and A124D conferred significant resistance to thequinoline ALLINI BI-D (165), recent studies have tested anti-viral activities of compounds against HIV-1 strains harboringrepresentative 124/125 polymorphisms, such as Thr/Thr, Thr/Ala, Ala/Thr, Ala/Ala, Asn/Thr, and Asn/Ala (200, 207, 215).Whereas Asn-124 and Ala-125 each conferred �50-fold resis-tance to pyridine ALLINI compound 20 (200) and thiopheneALLINI MUT-A (215), respectively, naphthyridine ALLINIcompound 23 remained active against such strains (207). Coun-terscreening against representative polymorphic strains such asthese, as well as viruses containing changes such as A128T andT174I that are commonly selected during virus passage (165,167, 171, 201–203, 212), is important to the ongoing develop-ment of the ALLINI drug class.

Similar to INSTI resistance (35, 38), some changes in IN thatconfer ALLINI resistance, such as T174I, alter a direct bindingcontact (Fig. 6, B and C), whereas others work via less directmechanisms. The A128T change in IN conferred significantresistance to the hypermultimerization activities of quinolineALLINIs without affecting the binding of LEDGF/p75 to themutant IN protein (211). IN CCD co-crystal structures revealedthat the bulky Thr substituent shifted the position of ALLINIbinding, lowering its propensity to hypermultimerize themutant IN (211). On the flip side, thiophene ALLINI MUT-Aefficiently inhibited LEDGF/p75 binding to Ala-125–containing IN yet in large part lost the ability to hypermultim-erize this polymorphic variant. In this case, downstream inter-actions of the CCD-engaged ALLINI with the CTD of anotherIN oligomer seemed to underlie the loss of hypermultimeriza-tion activity (215). Thus, resistance to ALLINIs can be instilledby loss of direct IN binding contact, a shift in compound posi-tion within the CCD-binding pocket, or inability of bound com-pound to mediate downstream interactions.

JBC REVIEWS: HIV integrase mechanisms and inhibition

J. Biol. Chem. (2019) 294(41) 15137–15157 15147

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Development of resistance to the pyridine ALLINI KF116required successive changes in IN that initiated with T124N,followed by T174I, and culminated with T124N/V165I/T174I(171). Whereas recombinant T124N virus was, as expected,similarly infectious as the WT, the infectivity of T124N/T174Iwas reduced almost 1,000-fold, and T124N/T174I virions failedto mature due to processing defects in both Gag and Gag-Polprecursor proteins (243). These data highlight how a singleamino acid substitution in IN can exert catastrophic conse-quences on the late stages of HIV-1 replication. Remarkably,the added V165I change restored polyprotein processing andboosted infectivity about 120-fold, to 17% of the level of the WT(243). Because resistance required the virus to pass through anearly noninfectious mutational bottleneck, KF116 harbors ahigher genetic barrier to resistance compared with predecessorquinoline ALLINIs (171, 243). Assessment of genetic resistancebarrier is another important consideration in the developmentof clinical ALLINI compounds.

Antiretroviral compounds display class-specific slopes intheir dose–response curves. For example, whereas INSTIs andNRTIs display slopes close to 1, NNRTIs and PIs display steeperslopes of �1.7 and from 1.8 to 4.5, respectively (244). Slope isrelated to the Hill coefficient, which is a measure of the intra-molecular cooperativity of ligand binding to a multivalentreceptor. Although �120 molecules of IN enter a susceptibletarget cell, only the two that engage the ends of HIV-1 DNAwithin the confines of a single intasome are sensitive to INSTIaction. Likewise, bystander RT molecules that are not activelyengaged in DNA synthesis are unseen by NRTI compounds.The limited number of enzyme–substrate targets per replica-tion cycle accounts for the comparatively shallow slopes ofINSTI and NRTI dose–response curves (244). By contrast,NNRTIs and PIs target apoenzymes. Reasons for steeper dose–response curve slopes in these cases include the requirement toinhibit the full complement of viral enzymes, which in thissense likens the enzyme population to a multivalent receptor(244), or the inhibition of multiple steps within the viral repli-cation cycle (245). ALLINIs like PIs display comparatively steepdose–response curve slopes (m � 4) (168, 203). Because eccen-tric HIV-1 particles made in the presence of ALLINIs are defec-tive for reverse transcription, the compounds potently inhibitminimally two steps of the viral replication cycle, which couldaccount for the steep slopes. Determining ALLINI-to-IN stoi-chiometries required to inhibit HIV-1 replication would beexpected to further inform the cooperative nature of drugaction.

Other IN functionalities

IN has been proposed to play additional roles in the HIV-1lifecycle, including virus particle uncoating after cell entry (246)and PIC nuclear import (247, 248). IN accordingly has beenshown to interact with numerous karyopherin (KPN) nucleartransport receptors, including KPNA2/importin �1 (247, 249–251), KNPB1/importin �1 (249, 251), KPNB2/transportin-1(TNPO1) (249), KPNA4/importin �3 (252, 253), importin 7(249, 254), Ran-binding protein (RANBP) 9/importin 9 (255),RANBP4/importin 4 (256), and TNPO3 (257). Genetic map-ping experiments at the same time have highlighted the capsid

protein as the key mediator of HIV-1 PIC nuclear import (258)(reviewed in Ref. 3), and numerous follow-up studies havequestioned purported roles for IN nuclear localization signals(259 –262) and/or IN– host factor interactions (263–267) inHIV-1 nuclear import. Compounds that inhibited IN bindingto TNPO3 (268) or KPNA2/KPNB1 (269) in vitro displayedcomparatively weak antiviral activities of less than 50% inhibi-tion at 100 �M and 50% inhibition at �50 –100 �M, respectively.To establish IN-KPN interactions as bona fide antiviral targets,it will be important to show that resistance to compounds with10 –100-fold greater potencies maps to the IN region of HIV-1pol.

Novel tert-butylsulfonamide (t-BSF) compound 1 inhibitedthe late phase of HIV-1 infection 6-fold more potently than theearly phase, indicating bona fide ALLINI activity (270).Whereas the T174I substitution in the LEDGF/p75 IBD bind-ing pocket conferred about 500-fold resistance to a controlquinoline ALLINI compound, the mutant virus was 5-foldmore sensitive to inhibition by t-BSF ALLINI compound 1,indicating engagement of the IN CCD dimer at a location otherthan the LEDGF/p75-binding pocket (270). The binding of apredecessor ALLINI compound, which inhibited IN subunitexchange in solution, mapped to the CCD dimer interface adja-cent to the LEDGF/p75-binding pocket (271). These studiesestablished that regions of the CCD dimer interface outside ofthe LEDGF/p75-binding pocket are potential targets for novelALLINI development. Structural determination of the INregion within Pol or the tetrameric form of IN within virions(214) may reveal additional IN-IN interfaces for the develop-ment of novel ALLINI compounds.

Conclusions

IN inhibitors have come a long way since the early days whensome of the most notable individuals in the field felt the goal ofclinical IN inhibition was unattainable. The impressive poten-cies and resistance barriers of second-generation INSTIs haveprompted worldwide rollouts, although safety profiles for preg-nant women at the time of conception require careful monitor-ing and comprehensive follow-up. The assessment of second-generation INSTI-containing dual therapy regimens is ongoingfor both oral administration for PLHIV and LA formulationsfor PrEP. The odds-on bet is that second-generation INSTIswill be a mainstay part of cART formulations for the foreseeablefuture.

The elucidation of retroviral intasome structures by X-raycrystallography and single-particle cryo-EM over the past dec-ade has provided unprecedented insight into the mechanism ofretroviral integration. The initial PFV intasome structuresadditionally provided important insight into the mechanisms ofINSTI action, and ongoing cryo-EM work with HIV-1 andrelated primate lentiviral intasomes is expected to furtheradvance our understanding of drug action and drug resistancemechanisms. Such high-resolution structures should criticallyinform the future development of these drugs to deal with resis-tance mutations prevalent from prior failure to first-generationINSTI-containing regimens as well as de novo resistance thatwill inevitably arise from global second-generation INSTIrollouts.

JBC REVIEWS: HIV integrase mechanisms and inhibition

15148 J. Biol. Chem. (2019) 294(41) 15137–15157

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

ALLINIs provide a clear example of how drugs against a viralenzyme can primarily inhibit virus replication at a step that isdistinct from where catalytic function transpires. ALLINIs inlarge part recapitulate the class II HIV-1 IN mutant phenotype,revealing a remarkable example of pharmacological mimicry ofbiological phenotype. ALLINI and INSTI potencies are addi-tive/synergistic (203, 206), and ALLINIs retain their potency inthe face of clinically relevant INSTI resistance mutations (167,170, 203). Thus, ALLINIs have the potential to fill the neededrole of a second clinical class of anti-IN compounds with novelmechanism of action. Ongoing work to improve bioavailablechemotypes with broad antiviral activity against polymorphicHIV-1 variants (207) should further advance the evaluation ofthis promising drug class.

References1. Arts, E. J., and Hazuda, D. J. (2012) HIV-1 antiretroviral drug therapy.

Cold Spring Harb. Perspect. Med. 2, a007161 CrossRef Medline2. Sundquist, W. I., and Kräusslich, H.-G. (2012) HIV-1 assembly, budding,

and maturation. Cold Spring Harb. Perspect. Med. 2, a006924 Medline3. Yamashita, M., and Engelman, A. N. (2017) Capsid-dependent host fac-

tors in HIV-1 infection. Trends Microbiol. 25, 741–755 CrossRefMedline

4. Engelman, A. (2010) Reverse transcription and integration. In Retrovi-ruses: Molecular Biology, Genomics and Pathogenesis (Kurth, R., andBannert, N., eds) pp. 129 –159, Caister Academic Press, Norfolk, UK

5. Brown, P. O., Bowerman, B., Varmus, H. E., and Bishop, J. M. (1987)Correct integration of retroviral DNA in vitro. Cell 49, 347–356 CrossRefMedline

6. Fujiwara, T., and Mizuuchi, K. (1988) Retroviral DNA integration: struc-ture of an integration intermediate. Cell 54, 497–504 CrossRef Medline

7. Brown, P. O., Bowerman, B., Varmus, H. E., and Bishop, J. M. (1989)Retroviral integration: structure of the initial covalent product and itsprecursor, and a role for the viral IN protein. Proc. Natl. Acad. Sci. U.S.A.86, 2525–2529 CrossRef Medline

8. Cihlar, T., and Fordyce, M. (2016) Current status and prospects of HIVtreatment. Curr. Opin. Virol. 18, 50 –56 CrossRef Medline

9. Saag, M. S., Benson, C. A., Gandhi, R. T., Hoy, J. F., Landovitz, R. J.,Mugavero, M. J., Sax, P. E., Smith, D. M., Thompson, M. A., Buchbinder,S. P., Del Rio, C., Eron, J. J., Jr., Fätkenheuer, G., Günthard, H. F., Molina,J.-M., Jacobsen, D. M., and Volberding, P. A. (2018) Antiretroviral drugsfor treatment and prevention of HIV infection in adults: 2018 recom-mendations of the International Antiviral Society–U.S.A. panel. JAMA320, 379 –396 CrossRef Medline

10. Panel on Antiretroviral Guidelines for Adults and Adolescents (2019)Guidelines for the Use of Antiretroviral Agents in Adults and Adolescentswith HIV, Department of Health and Human Services, Washington,D. C.

11. Hazuda, D. J., Felock, P., Witmer, M., Wolfe, A., Stillmock, K., Grobler,J. A., Espeseth, A., Gabryelski, L., Schleif, W., Blau, C., and Miller, M. D.(2000) Inhibitors of strand transfer that prevent integration and inhibitHIV-1 replication in cells. Science 287, 646 – 650 CrossRef Medline

12. Jurado, K. A., Wang, H., Slaughter, A., Feng, L., Kessl, J. J., Koh, Y., Wang,W., Ballandras-Colas, A., Patel, P. A., Fuchs, J. R., Kvaratskhelia, M., andEngelman, A. (2013) Allosteric integrase inhibitor potency is determinedthrough the inhibition of HIV-1 particle maturation. Proc. Natl. Acad.Sci. U.S.A. 110, 8690 – 8695 CrossRef Medline

13. Fontana, J., Jurado, K. A., Cheng, N., Ly, N. L., Fuchs, J. R., Gorelick, R. J.,Engelman, A. N., and Steven, A. C. (2015) Distribution and redistributionof HIV-1 nucleocapsid protein in immature, mature, and integrase-in-hibited virions: a role for integrase in maturation. J. Virol. 89, 9765–9780CrossRef Medline

14. Lesbats, P., Engelman, A. N., and Cherepanov, P. (2016) Retroviral DNAintegration. Chem. Rev. 116, 12730 –12757 CrossRef Medline

15. Engelman, A., Mizuuchi, K., and Craigie, R. (1991) HIV-1 DNA integra-tion: mechanism of viral DNA cleavage and DNA strand transfer. Cell 67,1211–1221 CrossRef Medline

16. Hare, S., Maertens, G. N., and Cherepanov, P. (2012) 3�-Processing andstrand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31,3020 –3028 CrossRef Medline

17. Nowotny, M. (2009) Retroviral integrase superfamily: the structural per-spective. EMBO Rep. 10, 144 –151 CrossRef Medline

18. Roth, M. J., Schwartzberg, P. L., and Goff, S. P. (1989) Structure of theterminiofDNAintermediatesintheintegrationofretroviralDNA:depen-dence on IN function and terminal DNA sequence. Cell 58, 47–54CrossRef Medline

19. Katzman, M., Katz, R. A., Skalka, A. M., and Leis, J. (1989) The avianretroviral integration protein cleaves the terminal sequences of linearviral DNA at the in vivo sites of integration. J. Virol. 63, 5319 –5327Medline

20. Sherman, P. A., and Fyfe, J. A. (1990) Human immunodeficiency virusintegration protein expressed in Escherichia coli possesses selective DNAcleaving activity. Proc. Natl. Acad. Sci. U.S.A. 87, 5119 –5123 CrossRefMedline

21. Juretzek, T., Holm, T., Gartner, K., Kanzler, S., Lindemann, D., Herch-enroder, O., Picard-Maureau, M., Rammling, M., Heinkelein, M., andRethwilm, A. (2004) Foamy virus integration. J. Virol. 78, 2472–2477CrossRef Medline

22. Randolph, C. A., and Champoux, J. J. (1993) The majority of simianimmunodeficiency virus/Mne circle junctions result from ligation of un-integrated viral DNA ends that are aberrant for integration. Virology 194,851– 854 CrossRef Medline

23. Du, Z., Ilyinskii, P. O., Lally, K., Desrosiers, R. C., and Engelman, A. (1997)A mutation in integrase can compensate for mutations in the simianimmunodeficiency virus att site. J. Virol. 71, 8124 – 8132 Medline

24. Whitcomb, J. M., and Hughes, S. H. (1991) The sequence of humanimmunodeficiency virus type 2 circle junction suggests that integrationprotein cleaves the ends of linear DNA asymmetrically. J. Virol. 65,3906 –3910 Medline

25. Vincent, K. A., York-Higgins, D., Quiroga, M., and Brown, P. O. (1990)Host sequences flanking the HIV provirus. Nucleic Acids Res. 18,6045– 6047 CrossRef Medline

26. Vink, C., Groenink, M., Elgersma, Y., Fouchier, R. A., Tersmette, M., andPlasterk, R. H. (1990) Analysis of the junctions between human immu-nodeficiency virus type 1 proviral DNA and human DNA. J. Virol. 64,5626 –5627 Medline

27. Bowerman, B., Brown, P. O., Bishop, J. M., and Varmus, H. E. (1989) Anucleoprotein complex mediates the integration of retroviral DNA.Genes Dev. 3, 469 – 478 CrossRef Medline

28. Farnet, C. M., and Haseltine, W. A. (1990) Integration of human immu-nodeficiency virus type 1 DNA in vitro. Proc. Natl. Acad. Sci. U.S.A. 87,4164 – 4168 CrossRef Medline

29. Murphy, J. E., and Goff, S. P. (1992) A mutation at one end of Moloneymurine leukemia virus DNA blocks cleavage of both ends by the viralintegrase in vivo. J. Virol. 66, 5092–5095 Medline

30. Miller, M. D., Farnet, C. M., and Bushman, F. D. (1997) Human immu-nodeficiency virus type 1 preintegration complexes: studies of organiza-tion and composition. J. Virol. 71, 5382–5390 Medline

31. Wei, S.-Q., Mizuuchi, K., and Craigie, R. (1997) A large nucleoproteinassembly at the ends of the viral DNA mediates retroviral DNA integra-tion. EMBO J. 16, 7511–7520 CrossRef Medline

32. Chen, H., Wei, S.-Q., and Engelman, A. (1999) Multiple integrase func-tions are required to form the native structure of the human immuno-deficiency virus type I intasome. J. Biol. Chem. 274, 17358 –17364CrossRef Medline

33. McCord, M., Chiu, R., Vora, A. C., and Grandgenett, D. P. (1999) Retro-virus DNA termini bound by integrase communicate in trans for full-siteintegration in vitro. Virology 259, 392– 401 CrossRef Medline

34. Li, M., Mizuuchi, M., Burke, T. R., Jr., and Craigie, R. (2006) RetroviralDNA integration: reaction pathway and critical intermediates. EMBO J.25, 1295–1304 CrossRef Medline

JBC REVIEWS: HIV integrase mechanisms and inhibition

J. Biol. Chem. (2019) 294(41) 15137–15157 15149

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

35. Hare, S., Gupta, S. S., Valkov, E., Engelman, A., and Cherepanov, P. (2010)Retroviral intasome assembly and inhibition of DNA strand transfer.Nature 464, 232–236 CrossRef Medline

36. Engelman, A. N., and Cherepanov, P. (2017) Retroviral intasomes arising.Curr. Opin. Struct. Biol. 47, 23–29 CrossRef Medline

37. Maertens, G. N., Hare, S., and Cherepanov, P. (2010) The mechanism ofretroviral integration through X-ray structures of its key intermediates.Nature 468, 326 –329 CrossRef Medline

38. Hare, S., Vos, A. M., Clayton, R. F., Thuring, J. W., Cummings, M. D., andCherepanov, P. (2010) Molecular mechanisms of retroviral integrase in-hibition and the evolution of viral resistance. Proc. Natl. Acad. Sci. U.S.A.107, 20057–20062 CrossRef Medline

39. Wang, J.-Y., Ling, H., Yang, W., and Craigie, R. (2001) Structure of atwo-domain fragment of HIV-1 integrase: implications for domain orga-nization in the intact protein. EMBO J. 20, 7333–7343 CrossRef Medline

40. Hare, S., Di Nunzio, F., Labeja, A., Wang, J., Engelman, A., and Che-repanov, P. (2009) Structural basis for functional tetramerization of len-tiviral integrase. PLoS Pathog. 5, e1000515 CrossRef Medline

41. van Gent, D. C., Vink, C., Groeneger, A. A. M. O., and Plasterk, R. H. A.(1993) Complementation between HIV integrase proteins mutated indifferent domains. EMBO J. 12, 3261–3267 CrossRef Medline

42. Engelman, A., Bushman, F. D., and Craigie, R. (1993) Identification ofdiscrete functional domains of HIV-1 integrase and their organizationwithin an active multimeric complex. EMBO J. 12, 3269 –3275 CrossRefMedline

43. Yin, Z., Lapkouski, M., Yang, W., and Craigie, R. (2012) Assembly ofprototype foamy virus strand transfer complexes on product DNA by-passing catalysis of integration. Protein Sci. 21, 1849 –1857 CrossRefMedline

44. Maskell, D. P., Renault, L., Serrao, E., Lesbats, P., Matadeen, R., Hare, S.,Lindemann, D., Engelman, A. N., Costa, A., and Cherepanov, P. (2015)Structural basis for retroviral integration into nucleosomes. Nature 523,366 –369 CrossRef Medline

45. Engelman, A. (1994) Most of the avian genome appears available forretroviral DNA integration. Bioessays 16, 797–799 CrossRef Medline

46. Carteau, S., Hoffmann, C., and Bushman, F. (1998) Chromosome struc-ture and human immunodeficiency virus type 1 cDNA integration: cen-tromeric alphoid repeats are a disfavored target. J. Virol. 72, 4005– 4014Medline

47. Pryciak, P. M., and Varmus, H. E. (1992) Nucleosomes, DNA-bindingproteins, and DNA sequence modulate retroviral integration target siteselection. Cell 69, 769 –780 CrossRef Medline

48. Serrao, E., Krishnan, L., Shun, M.-C., Li, X., Cherepanov, P., Engelman,A., and Maertens, G. N. (2014) Integrase residues that determine nucle-otide preferences at sites of HIV-1 integration: implications for themechanism of target DNA binding. Nucleic Acids Res. 42, 5164 –5176CrossRef Medline

49. Pasi, M., Mornico, D., Volant, S., Juchet, A., Batisse, J., Bouchier, C.,Parissi, V., Ruff, M., Lavery, R., and Lavigne, M. (2016) DNA minicirclesclarify the specific role of DNA structure on retroviral integration. Nu-cleic Acids Res. 44, 7830 –7847 CrossRef Medline

50. Engelman, A. N., and Singh, P. K. (2018) Cellular and molecular mecha-nisms of HIV-1 integration targeting. Cell. Mol. Life Sci. 75, 2491–2507CrossRef Medline

51. Ballandras-Colas, A., Maskell, D. P., Serrao, E., Locke, J., Swuec, P., Jóns-son, S. R., Kotecha, A., Cook, N. J., Pye, V. E., Taylor, I. A., Andrésdóttir,V., Engelman, A. N., Costa, A., and Cherepanov, P. (2017) A supramo-lecular assembly mediates lentiviral DNA integration. Science 355,93–95 CrossRef Medline

52. Ballandras-Colas, A., Brown, M., Cook, N. J., Dewdney, T. G., Demeler,B., Cherepanov, P., Lyumkis, D., and Engelman, A. N. (2016) Cryo-EMreveals a novel octameric integrase structure for betaretroviral intasomefunction. Nature 530, 358 –361 CrossRef Medline

53. Yin, Z., Shi, K., Banerjee, S., Pandey, K. K., Bera, S., Grandgenett, D. P.,and Aihara, H. (2016) Crystal structure of the Rous sarcoma virus inta-some. Nature 530, 362–366 CrossRef Medline

54. Passos, D. O., Li, M., Yang, R., Rebensburg, S. V., Ghirlando, R., Jeon, Y.,Shkriabai, N., Kvaratskhelia, M., Craigie, R., and Lyumkis, D. (2017)

Cryo-EM structures and atomic model of the HIV-1 strand transfercomplex intasome. Science 355, 89 –92 CrossRef Medline

55. Chen, J. C.-H., Krucinski, J., Miercke, L. J. W., Finer-Moore, J. S., Tang,A. H., Leavitt, A. D., and Stroud, R. M. (2000) Crystal structure of theHIV-1 integrase catalytic core and C-terminal domains: a model for viralDNA binding. Proc. Natl. Acad. Sci. U.S.A. 97, 8233– 8238 CrossRefMedline

56. Li, M., Jurado, K. A., Lin, S., Engelman, A., and Craigie, R. (2014) Engi-neered hyperactive integrase for concerted HIV-1 DNA integration.PLoS One 9, e105078 CrossRef Medline

57. van Gent, D. C., Elgersma, Y., Bolk, M. W. J., Vink, C., and Plasterk,R. H. A. (1991) DNA binding properties of the integrase proteins ofhuman immunodeficiency viruses types 1 and 2. Nucleic Acids Res. 19,3821–3827 CrossRef Medline

58. Lee, S. P., Xiao, J., Knutson, J. R., Lewis, M. S., and Han, M. K. (1997) Zn2�

promotes the self-association of human immunodeficiency virus type-1integrase in vitro. Biochemistry 36, 173–180 CrossRef Medline

59. Cherepanov, P., Maertens, G., Proost, P., Devreese, B., Van Beeumen, J.,Engelborghs, Y., De Clercq, E., and Debyser, Z. (2003) HIV-1 integraseforms stable tetramers and associates with LEDGF/p75 protein in humancells. J. Biol. Chem. 278, 372–381 CrossRef Medline

60. Faure, A., Calmels, C., Desjobert, C., Castroviejo, M., Caumont-Sarcos,A., Tarrago-Litvak, L., Litvak, S., and Parissi, V. (2005) HIV-1 integrasecrosslinked oligomers are active in vitro. Nucleic Acids Res. 33, 977–986CrossRef Medline

61. McKee, C. J., Kessl, J. J., Shkriabai, N., Dar, M. J., Engelman, A., andKvaratskhelia, M. (2008) Dynamic modulation of HIV-1 integrase struc-ture and function by cellular lens epithelium-derived growth factor(LEDGF) protein. J. Biol. Chem. 283, 31802–31812 CrossRef Medline

62. Pandey, K. K., Bera, S., and Grandgenett, D. P. (2011) The HIV-1 inte-grase monomer induces a specific interaction with LTR DNA for con-certed integration. Biochemistry 50, 9788 –9796 CrossRef Medline

63. Kessl, J. J., Kutluay, S. B., Townsend, D., Rebensburg, S., Slaughter, A.,Larue, R. C., Shkriabai, N., Bakouche, N., Fuchs, J. R., Bieniasz, P. D., andKvaratskhelia, M. (2016) HIV-1 integrase binds the viral RNA genomeand is essential during virion morphogenesis. Cell 166, 1257–1268.e12CrossRef Medline

64. Pandey, K. K., Bera, S., Shi, K., Aihara, H., and Grandgenett, D. P. (2017)A C-terminal “tail” region in the Rous sarcoma virus integrase provideshigh plasticity of functional integrase oligomerization during intasomeassembly. J. Biol. Chem. 292, 5018 –5030 CrossRef Medline

65. Bera, S., Pandey, K. K., Aihara, H., and Grandgenett, D. P. (2018) Differ-ential assembly of Rous sarcoma virus tetrameric and octameric inta-somes is regulated by the C-terminal domain and tail region of integrase.J. Biol. Chem. 293, 16440 –16452 CrossRef Medline

66. Donehower, L. A., and Varmus, H. E. (1984) A mutant murine leukemiavirus with a single missense codon in pol is defective in a function affect-ing integration. Proc. Natl. Acad. Sci. U.S.A. 81, 6461– 6465 CrossRefMedline

67. Panganiban, A. T., and Temin, H. M. (1984) The retrovirus pol geneencodes a product required for DNA integration: Identification of a ret-rovirus int locus. Proc. Natl. Acad. Sci. U.S.A. 81, 7885–7889 CrossRefMedline

68. Schwartzberg, P., Colicelli, J., and Goff, S. P. (1984) Construction andanalysis of deletion mutations in the pol gene of moloney murine leuke-mia virus: a new viral function required for productive infection. Cell 37,1043–1052 CrossRef Medline

69. Quinn, T. P., and Grandgenett, D. P. (1988) Genetic evidence that theavian retrovirus DNA endonuclease domain of pol is necessary for viralintegration. J. Virol. 62, 2307–2312 Medline

70. LaFemina, R. L., Schneider, C. L., Robbins, H. L., Callahan, P. L., LeGrow,K., Roth, E., Schleif, W. A., and Emini, E. A. (1992) Requirement of activehuman immunodeficiency virus type 1 integrase enzyme for productiveinfection of human T-lymphoid cells. J. Virol. 66, 7414 –7419 Medline

71. Collier, A. C., Coombs, R. W., Schoenfeld, D. A., Bassett, R. L., Timpone,J., Baruch, A., Jones, M., Facey, K., Whitacre, C., McAuliffe, V. J., Fried-man, H. M., Merigan, T. C., Reichman, R. C., Hooper, C., and Corey, L.(1996) Treatment of human immunodeficiency virus infection with

JBC REVIEWS: HIV integrase mechanisms and inhibition

15150 J. Biol. Chem. (2019) 294(41) 15137–15157

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

saquinavir, zidovudine, and zalcitabine. N. Engl. J. Med. 334, 1011–1017CrossRef Medline

72. D’Aquila, R. T., Hughes, M. D., Johnson, V. A., Fischl, M. A., Sommad-ossi, J.-P., Liou, S.-H., Timpone, J., Myers, M., Basgoz, N., Niu, M., andHirsch, M. S. (1996) Nevirapine, zidovudine, and didanosine comparedwith zidovudine and didanosine in patients with HIV-1 infection: a ran-domized, double-blind, placebo-controlled trial. Ann. Intern. Med. 124,1019 –1030 CrossRef Medline

73. Staszewski, S., Miller, V., Rehmet, S., Stark, T., De Crée, J., De Brabander,M., Peeters, M., Andries, K., Moeremans, M., De Raeymaeker, M.,Pearce, G., Van den Broeck, R., Verbiest, W., and Stoffels, P. (1996) Vi-rological and immunological analysis of a triple combination pilot studywith loviride, lamivudine and zidovudine in HIV-1-infected patients.AIDS 10, F1–F7 CrossRef Medline

74. Jacks, T., Power, M. D., Masiarz, F. R., Luciw, P. A., Barr, P. J., and Var-mus, H. E. (1988) Characterization of ribosomal frameshifting in HIV-1gag-pol expression. Nature 331, 280 –283 CrossRef Medline

75. Carlson, L.-A., Briggs, J. A. G., Glass, B., Riches, J. D., Simon, M. N.,Johnson, M. C., Müller, B., Grünewald, K., and Kräusslich, H.-G. (2008)Three-dimensional analysis of budding sites and released virus suggests arevised model for HIV-1 morphogenesis. Cell Host Microbe 4, 592–599CrossRef Medline

76. Munir, S., Thierry, S., Subra, F., Deprez, E., and Delelis, O. (2013) Quan-titative analysis of the time-course of viral DNA forms during the HIV-1life cycle. Retrovirology 10, 87 CrossRef Medline

77. Butler, S. L., Hansen, M. S., and Bushman, F. D. (2001) A quantitativeassay for HIV DNA integration in vivo. Nat. Med. 7, 631– 634 CrossRefMedline

78. Pierson, T. C., Zhou, Y., Kieffer, T. L., Ruff, C. T., Buck, C., and Siliciano,R. F. (2002) Molecular characterization of preintegration latency in hu-man immunodeficiency virus type 1 infection. J. Virol. 76, 8518 – 8531CrossRef Medline

79. Mohammadi, P., Desfarges, S., Bartha, I., Joos, B., Zangger, N., Muñoz,M., Günthard, H. F., Beerenwinkel, N., Telenti, A., and Ciuffi, A. (2013)24 hours in the life of HIV-1 in a T cell line. PLoS Pathog. 9, e1003161CrossRef Medline

80. Cardozo, E. F., Andrade, A., Mellors, J. W., Kuritzkes, D. R., Perelson, A. S.,and Ribeiro, R. M. (2017) Treatment with integrase inhibitor suggests a newinterpretation of HIV RNA decay curves that reveals a subset of cells withslow integration. PLoS Pathog. 13, e1006478 CrossRef Medline

81. Bushman, F. D., Fujiwara, T., and Craigie, R. (1990) Retroviral DNAintegration directed by HIV integration protein in vitro. Science 249,1555–1558 CrossRef Medline

82. Bushman, F. D., and Craigie, R. (1991) Activities of human immunode-ficiency virus (HIV) integration protein in vitro: specific cleavage andintegration of HIV DNA. Proc. Natl. Acad. Sci. U.S.A. 88, 1339 –1343CrossRef Medline

83. Craigie, R., Mizuuchi, K., Bushman, F. D., and Engelman, A. (1991) Arapid in vitro assay for HIV DNA integration. Nucleic Acids Res. 19,2729 –2734 CrossRef Medline

84. Hazuda, D. J., Hastings, J. C., Wolfe, A. L., and Emini, E. A. (1994) A novelassay for the DNA strand-transfer reaction of HIV-1 integrase. NucleicAcids Res. 22, 1121–1122 CrossRef Medline

85. Vink, C., Banks, M., Bethell, R., and Plasterk, R. H. A. (1994) A high-throughput, non-radioactive microtiter plate assay for activity of the hu-man immunodeficiency virus integrase protein. Nucleic Acids Res. 22,2176 –2177 CrossRef Medline

86. Farnet, C. M., Wang, B., Lipford, J. R., and Bushman, F. D. (1996) Differ-ential inhibition of HIV-1 preintegration complexes and purified inte-grase protein by small molecules. Proc. Natl. Acad. Sci. U.S.A. 93,9742–9747 CrossRef Medline

87. Hazuda, D. J., Felock, P. J., Hastings, J. C., Pramanik, B., and Wolfe, A. L.(1997) Differential divalent cation requirements uncouple the assemblyand catalytic reactions of human immunodeficiency virus type 1 inte-grase. J. Virol. 71, 7005–7011 Medline

88. Pommier, Y., Johnson, A. A., and Marchand, C. (2005) Integrase inhibi-tors to treat HIV/Aids. Nat. Rev. Drug Discov. 4, 236 –248 CrossRefMedline

89. Espeseth, A. S., Felock, P., Wolfe, A., Witmer, M., Grobler, J., Anthony,N., Egbertson, M., Melamed, J. Y., Young, S., Hamill, T., Cole, J. L., andHazuda, D. J. (2000) HIV-1 integrase inhibitors that compete with thetarget DNA substrate define a unique strand transfer conformation forintegrase. Proc. Natl. Acad. Sci. U.S.A. 97, 11244 –11249 CrossRefMedline

90. Dicker, I. B., Samanta, H. K., Li, Z., Hong, Y., Tian, Y., Banville, J., Remi-llard, R. R., Walker, M. A., Langley, D. R., and Krystal, M. (2007) Changesto the HIV long terminal repeat and to HIV integrase differentially im-pact HIV integrase assembly, activity, and the binding of strand transferinhibitors. J. Biol. Chem. 282, 31186 –31196 CrossRef Medline

91. Langley, D. R., Samanta, H. K., Lin, Z., Walker, M. A., Krystal, M. R., andDicker, I. B. (2008) The terminal (catalytic) adenosine of the HIV LTRcontrols the kinetics of binding and dissociation of HIV integrase strandtransfer inhibitors. Biochemistry 47, 13481–13488 CrossRef Medline

92. Grobler, J. A., Stillmock, K., Hu, B., Witmer, M., Felock, P., Espeseth,A. S., Wolfe, A., Egbertson, M., Bourgeois, M., Melamed, J., Wai, J. S.,Young, S., Vacca, J., and Hazuda, D. J. (2002) Diketo acid inhibitor mech-anism and HIV-1 integrase: implications for metal binding in the activesite of phosphotransferase enzymes. Proc. Natl. Acad. Sci. U.S.A. 99,6661– 6666 CrossRef Medline

93. Summa, V., Petrocchi, A., Bonelli, F., Crescenzi, B., Donghi, M., Ferrara,M., Fiore, F., Gardelli, C., Gonzalez Paz, O., Hazuda, D. J., Jones, P.,Kinzel, O., Laufer, R., Monteagudo, E., Muraglia, E., Nizi, E., Orvieto, F.,Pace, P., Pescatore, G., Scarpelli, R., Stillmock, K., Witmer, M. V., andRowley, M. (2008) Discovery of raltegravir, a potent, selective orally bio-available HIV-integrase inhibitor for the treatment of HIV-AIDS infec-tion. J. Med. Chem. 51, 5843–5855 CrossRef Medline

94. Sato, M., Motomura, T., Aramaki, H., Matsuda, T., Yamashita, M., Ito, Y.,Kawakami, H., Matsuzaki, Y., Watanabe, W., Yamataka, K., Ikeda, S.,Kodama, E., Matsuoka, M., and Shinkai, H. (2006) Novel HIV-1 integraseinhibitors derived from quinolone antibiotics. J. Med. Chem. 49,1506 –1508 CrossRef Medline

95. Ruprecht, R. M., O’Brien, L. G., Rossoni, L. D., and Nusinoff-Lehrman, S.(1986) Suppression of mouse viraemia and retroviral disease by 3�-azido-3�-deoxythymidine. Nature 323, 467– 469 CrossRef Medline

96. Tsai, C. C., Follis, K. E., Sabo, A., Beck, T. W., Grant, R. F., Bischofberger,N., Benveniste, R. E., and Black, R. (1995) Prevention of SIV infection inmacaques by (R)-9-(2-phosphonylmethoxypropyl)adenine. Science 270,1197–1199 CrossRef Medline

97. North, T. W., North, G. L., and Pedersen, N. C. (1989) Feline immuno-deficiency virus, a model for reverse transcriptase-targeted chemother-apy for acquired immune deficiency syndrome. Antimicrob. Agents Che-mother. 33, 915–919 CrossRef Medline

98. Roquebert, B., Damond, F., Collin, G., Matheron, S., Peytavin, G., Benard,A., Campa, P., Chene, G., Brun-Vezinet, F., Descamps, D., and FrenchANRS HIV-2 Cohort (ANRS CO 05 VIH-2) (2008) HIV-2 integrase genepolymorphism and phenotypic susceptibility of HIV-2 clinical isolates tothe integrase inhibitors raltegravir and elvitegravir in vitro. J. Antimicrob.Chemother. 62, 914 –920 CrossRef Medline

99. Shimura, K., Kodama, E., Sakagami, Y., Matsuzaki, Y., Watanabe, W.,Yamataka, K., Watanabe, Y., Ohata, Y., Doi, S., Sato, M., Kano, M., Ikeda,S., and Matsuoka, M. (2008) Broad antiretroviral activity and resistanceprofile of the novel human immunodeficiency virus integrase inhibitorelvitegravir (JTK-303/GS-9137). J. Virol. 82, 764 –774 CrossRef Medline

100. Lewis, M. G., Norelli, S., Collins, M., Barreca, M. L., Iraci, N., Chirullo, B.,Yalley-Ogunro, J., Greenhouse, J., Titti, F., Garaci, E., and Savarino, A.(2010) Response of a simian immunodeficiency virus (SIVmac251) toraltegravir: a basis for a new treatment for simian AIDS and an animalmodel for studying lentiviral persistence during antiretroviral therapy.Retrovirology 7, 21 CrossRef Medline

101. Paprotka, T., Venkatachari, N. J., Chaipan, C., Burdick, R., Delviks-Fran-kenberry, K. A., Hu, W.-S., and Pathak, V. K. (2010) Inhibition of xeno-tropic murine leukemia virus-related virus by APOBEC3 proteins andantiviral drugs. J. Virol. 84, 5719 –5729 CrossRef Medline

102. Smith, R. A., Gottlieb, G. S., and Miller, A. D. (2010) Susceptibility of thehuman retrovirus XMRV to antiretroviral inhibitors. Retrovirology 7, 70CrossRef Medline

JBC REVIEWS: HIV integrase mechanisms and inhibition

J. Biol. Chem. (2019) 294(41) 15137–15157 15151

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

103. Koh, Y., Matreyek, K. A., and Engelman, A. (2011) Differential sensitivi-ties of retroviruses to integrase strand transfer inhibitors. J. Virol. 85,3677–3682 CrossRef Medline

104. Moebes, A., Enssle, J., Bieniasz, P. D., Heinkelein, M., Lindemann, D.,Bock, M., McClure, M. O., and Rethwilm, A. (1997) Human foamy virusreverse transcription that occurs late in the viral replication cycle. J. Virol.71, 7305–7311 Medline

105. Valkov, E., Gupta, S. S., Hare, S., Helander, A., Roversi, P., McClure, M.,and Cherepanov, P. (2009) Functional and structural characterization ofthe integrase from the prototype foamy virus. Nucleic Acids Res. 37,243–255 CrossRef Medline

106. Min, S., Song, I., Borland, J., Chen, S., Lou, Y., Fujiwara, T., and Piscitelli,S. C. (2010) Pharmacokinetics and safety of S/GSK1349572, a next-gen-eration HIV integraseinhibitor, in healthy volunteers. Antimicrob. AgentsChemother. 54, 254 –258 CrossRef Medline

107. Tsiang, M., Jones, G. S., Goldsmith, J., Mulato, A., Hansen, D., Kan, E.,Tsai, L., Bam, R. A., Stepan, G., Stray, K. M., Niedziela-Majka, A., Yant,S. R., Yu, H., Kukolj, G., Cihlar, T., et al. (2016) Antiviral activity ofbictegravir (GS-9883), a novel potent HIV-1 integrase strand transferinhibitor with an improved resistance profile. Antimicrob. Agents Che-mother. 60, 7086 –7097 CrossRef Medline

108. Naidu, B. N., Walker, M. A., Sorenson, M. E., Ueda, Y., Matiskella, J. D.,Connolly, T. P., Dicker, I. B., Lin, Z., Bollini, S., Terry, B. J., Higley, H.,Zheng, M., Parker, D. D., Wu, D., Adams, S., Krystal, M. R., and Mean-well, N. A. (2018) The discovery and preclinical evaluation of BMS-707035, a potent HIV-1 integrase strand transfer inhibitor. Bioorg. Med.Chem. Lett. 28, 2124 –2130 CrossRef Medline

109. Wiscount, C. M., Williams, P. D., Tran, L. O., Embrey, M. W., Fisher,T. E., Sherman, V., Homnick, C. F., Donnette Staas, D., Lyle, T. A., Wai,J. S., Vacca, J. P., Wang, Z., Felock, P. J., Stillmock, K. A., et al. (2008)10-Hydroxy-7,8-dihydropyrazino[1�,2�:1,5]pyrrolo[2,3-d]pyridazine-1,9(2H,6H)-diones: potent, orally bioavailable HIV-1 integrase strand-transfer inhibitors with activity against integrase mutants. Bioorg. Med.Chem. Lett. 18, 4581– 4583 CrossRef Medline

110. Egbertson, M. S., Wai, J. S., Cameron, M., and Hoerrner, R. S. (2011)Discovery of MK-0536: a potential second-generation HIV-1 integrasestrand transfer inhibitor with a high genetic barrier to mutation. in An-tiviral Drugs (Kazmierski, W. M., ed) pp. 163–180, John Wiley & Sons,Inc., New York

111. Raheem, I. T., Walji, A. M., Klein, D., Sanders, J. M., Powell, D. A., Abey-wickrema, P., Barbe, G., Bennet, A., Childers, K., Christensen, M., Clas,S. D., Dubost, D., Embrey, M., Grobler, J., Hafey, M. J., et al. (2015)Discovery of 2-pyridinone aminals: a prodrug strategy to advance a sec-ond generation of HIV-1 integrase strand transfer inhibitors. J. Med.Chem. 58, 8154 – 8165 CrossRef Medline

112. Zhao, X. Z., Smith, S. J., Maskell, D. P., Metifiot, M., Pye, V. E., Fesen, K.,Marchand, C., Pommier, Y., Cherepanov, P., Hughes, S. H., and Burke,T. R., Jr. (2016) HIV-1 integrase strand transfer inhibitors with reducedsusceptibility to drug resistant mutant integrases. ACS Chem. Biol. 11,1074 –1081 CrossRef Medline

113. Zhao, X. Z., Smith, S. J., Maskell, D. P., Métifiot, M., Pye, V. E., Fesen, K.,Marchand, C., Pommier, Y., Cherepanov, P., Hughes, S. H., and Burke,T. R. (2017) Structure-guided optimization of HIV integrase strandtransfer inhibitors. J. Med. Chem. 60, 7315–7332 CrossRef Medline

114. Hare, S., Smith, S. J., Métifiot, M., Jaxa-Chamiec, A., Pommier, Y.,Hughes, S. H., and Cherepanov, P. (2011) Structural and functional anal-yses of the second-generation integrase strand transfer inhibitor dolute-gravir (S/GSK1349572). Mol. Pharmacol. 80, 565–572 CrossRef Medline

115. DeAnda, F., Hightower, K. E., Nolte, R. T., Hattori, K., Yoshinaga, T.,Kawasuji, T., and Underwood, M. R. (2013) Dolutegravir interactionswith HIV-1 integrase-DNA: structural rationale for drug resistance anddissociation kinetics. PLoS ONE 8, e77448 CrossRef Medline

116. King, N. M., Prabu-Jeyabalan, M., Nalivaika, E. A., and Schiffer, C. A.(2004) Combating susceptibility to drug resistance: lessons from HIV-1protease. Chem. Biol. 11, 1333–1338 CrossRef Medline

117. Prabu-Jeyabalan, M., King, N. M., Nalivaika, E. A., Heilek-Snyder, G.,Cammack, N., and Schiffer, C. A. (2006) Substrate envelope and drugresistance: crystal structure of RO1 in complex with wild-type human

immunodeficiency virus type 1 protease. Antimicrob. Agents Chemother.50, 1518 –1521 CrossRef Medline

118. Shen, Y., Altman, M. D., Ali, A., Nalam, M. N. L., Cao, H., Rana, T. M.,Schiffer, C. A., and Tidor, B. (2013) Testing the substrate-envelope hy-pothesis with designed pairs of compounds. ACS Chem. Biol. 8,2433–2441 CrossRef Medline

119. Smith, S. J., Zhao, X. Z., Burke, T. R., Jr., and Hughes, S. H. (2018) HIV-1integrase inhibitors that are broadly effective against drug-resistant mu-tants. Antimicrob. Agents Chemother. 62, e01035-01018 Medline

120. Zash, R., Jacobson, D. L., Diseko, M., Mayondi, G., Mmalane, M., Essex,M., Gaolethe, T., Petlo, C., Lockman, S., Holmes, L. B., Makhema, J., andShapiro, R. L. (2018) Comparative safety of dolutegravir-based or efa-virenz-based antiretroviral treatment started during pregnancy in Bot-swana: an observational study. Lancet Glob. Health 6, e804-e810CrossRef Medline

121. Zash, R., Makhema, J., and Shapiro, R. L. (2018) Neural-tube defects withdolutegravir treatment from the time of conception. N. Engl. J. Med. 379,979 –981 CrossRef Medline

122. Nakkazi, E. (2018) Changes to dolutegravir policy in several Africancountries. Lancet 392, 199 CrossRef Medline

123. Chouchana, L., Beeker, N., and Treluyer, J. M. (2019) Is there a safetysignal for dolutegravir and integrase inhibitors during pregnancy? J. Ac-quir. Immune Defic. Syndr. 81, 481– 486 CrossRef Medline

124. Vannappagari, V., and Thorne, C., for APR EPPICC (2019) Pregnancyand neonatal outcomes following prenatal exposure to dolutegravir. J.Acquir. Immune Defic. Syndr. 81, 371–378 CrossRef Medline

125. Shamsuddin, H., Raudenbush, C. L., Sciba, B. L., Zhou, Y. P., Mast, T. C.,Greaves, W. L., Hanna, G. J., Leong, R., and Straus, W. (2019) Evaluationof neural tube defects (NTDs) after exposure to raltegravir during preg-nancy. J. Acquir. Immune Defic. Syndr. 81, 247–250 CrossRef Medline

126. Zash, R., Holmes, L., Diseko, M., Jacobson, D. L., Brummel, S., Mayondi,G., Isaacson, A., Davey, S., Mabuta, J., Mmalane, M., Gaolathe, T., Essex,M., Lockman, S., Makhema, J., and Shapiro, R. L. (2019) Neural-tubedefects and antiretroviral treatment regimens in Botswana. N. Engl.J. Med. 381, 827– 840

127. Cooper, D. A., Steigbigel, R. T., Gatell, J. M., Rockstroh, J. K., Katlama, C.,Yeni, P., Lazzarin, A., Clotet, B., Kumar, P. N., Eron, J. E., Schechter, M.,Markowitz, M., Loutfy, M. R., Lennox, J. L., Zhao, J., et al. (2008) Sub-group and resistance analyses of raltegravir for resistant HIV-1 infection.N. Engl. J. Med. 359, 355–365 CrossRef Medline

128. McColl, D. J., and Chen, X. (2010) Strand transfer inhibitors of HIV-1integrase: bringing IN a new era of antiretroviral therapy. Antiviral Res.85, 101–118 CrossRef Medline

129. Métifiot, M., Vandegraaff, N., Maddali, K., Naumova, A., Zhang, X., Rho-des, D., Marchand, C., and Pommier, Y. (2011) Elvitegravir overcomesresistance to raltegravir induced by integrase mutation Y143. AIDS 25,1175–1178 CrossRef Medline

130. Huang, W., Frantzell, A., Fransen, S., and Petropoulos, C. J. (2013) Mul-tiple genetic pathways involving amino acid position 143 of HIV-1 inte-grase are preferentially associated with specific secondary amino Aacidsubstitutions and confer resistance to raltegravir and cross-resistance toelvitegravir. Antimicrob. Agents Chemother. 57, 4105– 4113 CrossRefMedline

131. Lyumkis, D. (2019) Challenges and opportunities in cryo-EM single-particle analysis. J. Biol. Chem. 294, 5181–5197 CrossRef Medline

132. Mitra, A. K. (2019) Visualization of biological macromolecules at near-atomic resolution: cryo-electron microscopy comes of age. Acta Crystal-logr. F Struct. Biol. Commun. 75, 3–11 CrossRef Medline

133. Cahn, P., Pozniak, A. L., Mingrone, H., Shuldyakov, A., Brites, C., An-drade-Villanueva, J. F., Richmond, G., Buendia, C. B., Fourie, J., Ramgo-pal, M., Hagins, D., Felizarta, F., Madruga, J., Reuter, T., Newman, T., etal. (2013) Dolutegravir versus raltegravir in antiretroviralexperienced,integrase-inhibitor-naive adults with HIV: week 48 results from the ran-domised, double-blind, non-inferiority SAILING study. Lancet 382,700 –708 CrossRef Medline

134. Raffi, F., Jaeger, H., Quiros-Roldan, E., Albrecht, H., Belonosova, E., Ga-tell, J. M., Baril, J. G., Domingo, P., Brennan, C., Almond, S., Min, S., andextended SPRING-2 Study Group (2013) Once-daily dolutegravir versus

JBC REVIEWS: HIV integrase mechanisms and inhibition

15152 J. Biol. Chem. (2019) 294(41) 15137–15157

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

twice-daily raltegravir in antiretroviral-naive adults with HIV-1 infection(SPRING-2 study): 96 week results from a randomised, double-blind,non-inferiority trial. Lancet Infect. Dis. 13, 927–935 CrossRef Medline

135. Raffi, F., Rachlis, A., Stellbrink, H. J., Hardy, W. D., Torti, C., Orkin, C.,Bloch, M., Podzamczer, D., Pokrovsky, V., Pulido, F., Almond, S., Marg-olis, D., Brennan, C., Min, S., and SPRING-2 Study Group (2013) Once-daily dolutegravir versus raltegravir in antiretroviral-naive adults withHIV-1 infection: 48 week results from the randomised, double-blind,non-inferiority SPRING-2 study. Lancet 381, 735–743 CrossRef Medline

136. Kobayashi, M., Yoshinaga, T., Seki, T., Wakasa-Morimoto, C., Brown,K. W., Ferris, R., Foster, S. A., Hazen, R. J., Miki, S., Suyama-Kagitani, A.,Kawauchi-Miki, S., Taishi, T., Kawasuji, T., Johns, B. A., Underwood,M. R., Garvey, E. P., Sato, A., and Fujiwara, T. (2011) In vitro antiretrovi-ral properties of S/GSK1349572, a next-generation HIV integrase inhib-itor. Antimicrob. Agents Chemother. 55, 813– 821 CrossRef Medline

137. Quashie, P. K., Mesplède, T., Han, Y.-S., Oliveira, M., Singhroy, D. N.,Fujiwara, T., Underwood, M. R., and Wainberg, M. A. (2012) Character-ization of the R263K mutation in HIV-1 integrase that confers low-levelresistance to the second-generation integrase strand transfer inhibitordolutegravir. J. Virol. 86, 2696 –2705 CrossRef Medline

138. Katlama, C., Soulié, C., Caby, F., Denis, A., Blanc, C., Schneider, L., Val-antin, M.-A., Tubiana, R., Kirstetter, M., Valdenassi, E., Nguyen, T., Pey-tavin, G., Calvez, V., and Marcelin, A.-G. (2016) Dolutegravir as mono-therapy in HIV-1-infected individuals with suppressed HIV viraemia. J.Antimicrob. Chemother. 71, 2646 –2650 CrossRef Medline

139. Rojas, J., Blanco, J. L., Marcos, M. A., Lonca, M., Tricas, A., Moreno, L.,Gonzalez-Cordon, A., Torres, B., Mallolas, J., Garcia, F., Gatell, J. M., andMartinez, E. (2016) Dolutegravir monotherapy in HIV-infected patientswith sustained viral suppression. J. Antimicrob. Chemother. 71,1975–1981 CrossRef Medline

140. Oldenbuettel, C., Wolf, E., Ritter, A., Noe, S., Heldwein, S., Pascucci, R.,Wiese, C., Von Krosigk, A., Jaegel-Guedes, E., Jaeger, H., Balogh, A.,Koegl, C., and Spinner, C. D. (2017) Dolutegravir monotherapy as treat-ment de-escalation in HIV-infected adults with virological control: Do-luMono cohort results. Antivir. Ther. 22, 169 –172 CrossRef Medline

141. Wijting, I., Rokx, C., Boucher, C., van Kampen, J., Pas, S., de Vries-Sluijs,T., Schurink, C., Bax, H., Derksen, M., Andrinopoulou, E. R., van derEnde, M., van Gorp, E., Nouwen, J., Verbon, A., Bierman, W., and Ri-jnders, B. (2017) Dolutegravir as maintenance monotherapy for HIV(DOMONO): a phase 2, randomised non-inferiority trial. Lancet HIV 4,e547-e554 CrossRef Medline

142. Blanco, J. L., Rojas, J., Paredes, R., Negredo, E., Mallolas, J., Casadella, M.,Clotet, B., Gatell, J. M., de Lazzari, E., Martinez, E., and DOLAM StudyTeam (2018) Dolutegravir-based maintenance monotherapy versus dualtherapy with lamivudine: a planned 24 week analysis of the DOLAMrandomized clinical trial. J. Antimicrob. Chemother. 73, 1965–1971CrossRef Medline

143. Aboud, M., Orkin, C., Podzamczer, D., Bogner, J. R., Baker, D., Khuong-Josses, M. A., Parks, D., Angelis, K., Kahl, L. P., Blair, E. A., Adkison, K.,Underwood, M., Matthews, J. E., Wynne, B., Vandermeulen, K., Gart-land, M., and Smith, K. (2019) Efficacy and safety of dolutegravir-rilpi-virine for maintenance of virological suppression in adults with HIV-1:100-week data from the randomised, open-label, phase 3 SWORD-1and SWORD-2 studies. Lancet HIV 10.1016/S2352-3018(19)30149-3CrossRef Medline

144. Wandeler, G., Buzzi, M., Anderegg, N., Sculier, D., BÈguelin, C., Egger,M., and Calmy, A. (2018) Virologic failure and HIV drug resistance onsimplified, dolutegravir-based maintenance therapy: systematic reviewand meta-analysis. Version 2. F1000Res 7, 1359 CrossRef Medline

145. Hightower, K. E., Wang, R., Deanda, F., Johns, B. A., Weaver, K., Shen, Y.,Tomberlin, G. H., Carter, H. L., 3rd, Broderick, T., Sigethy, S., Seki, T.,Kobayashi, M., and Underwood, M. R. (2011) Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravirand elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 in-tegrase-DNA complexes. Antimicrob. Agents Chemother. 55, 4552–4559CrossRef Medline

146. Malet, I., Subra, F., Charpentier, C., Collin, G., Descamps, D., Calvez, V.,Marcelin, A.-G., and Delelis, O. (2017) Mutations located outside the

integrase gene can confer resistance to HIV-1 integrase strand transferinhibitors. mBio 8, e00922-17 CrossRef Medline

147. Das, A. T., and Berkhout, B. (2018) How polypurine tract changes in theHIV-1 RNA genome can cause resistance against theintegrase inhibitordolutegravir. mBio 9, e00006-18 CrossRef Medline

148. Malet, I., Subra, F., Richetta, C., Charpentier, C., Collin, G., Descamps,D., Calvez, V., Marcelin, A.-G., and Delelis, O. (2018) Reply to Das andBerkhout, “How polypurine tract changes in the HIV-1 RNA genome cancause resistance against the integrase inhibitor dolutegravir”. mBio 9,e00623-18 Medline

149. Wijting, I. E. A., Lungu, C., Rijnders, B. J. A., van der Ende, M. E., Pham,H. T., Mesplede, T., Pas, S. D., Voermans, J. J. C., Schuurman, R., van deVijver, D. A. M. C., Boers, P. H. M., Gruters, R. A., Boucher, C. A. B., andvan Kampen, J. J. A. (2018) HIV-1 resistance dynamics in patients withvirologic failure to dolutegravir maintenance monotherapy. J. Infect. Dis.218, 688 – 697 CrossRef Medline

150. Van Duyne, R., Kuo, L. S., Pham, P., Fujii, K., and Freed, E. O. (2019)Mutations in the HIV-1 envelope glycoprotein can broadly rescue blocksat multiple steps in the virus replication cycle. Proc. Natl. Acad. Sci.U.S.A. 116, 9040 –9049 CrossRef Medline

151. McDonald, D., Wu, L., Bohks, S. M., KewalRamani, V. N., Unutmaz, D.,and Hope, T. J. (2003) Recruitment of HIV and its receptors to dendriticcell-T cell junctions. Science 300, 1295–1297 CrossRef Medline

152. Law, K. M., Satija, N., Esposito, A. M., and Chen, B. K. (2016) Cell-to-cellspread of HIV and viral pathogenesis. Adv. Virus Res. 95, 43– 85 CrossRefMedline

153. Sigal, A., Kim, J. T., Balazs, A. B., Dekel, E., Mayo, A., Milo, R., andBaltimore, D. (2011) Cell-to-cell spread of HIV permits ongoing replica-tion despite antiretroviral therapy. Nature 477, 95–98 CrossRef Medline

154. Agosto, L. M., Zhong, P., Munro, J., and Mothes, W. (2014) Highly activeantiretroviral therapies are effective against HIV-1 cell-to-cell transmis-sion. PLoS Pathog. 10, e1003982 CrossRef Medline

155. Andrews, C. D., Spreen, W. R., Mohri, H., Moss, L., Ford, S., Gettie, A.,Russell-Lodrigue, K., Bohm, R. P., Cheng-Mayer, C., Hong, Z., Markow-itz, M., and Ho, D. D. (2014) Long-acting integrase inhibitor protectsmacaques from intrarectal simian/human immunodeficiency virus. Sci-ence 343, 1151–1154 CrossRef Medline

156. Andrews, C. D., Yueh, Y. L., Spreen, W. R., St Bernard, L., Boente-Car-rera, M., Rodriguez, K., Gettie, A., Russell-Lodrigue, K., Blanchard, J.,Ford, S., Mohri, H., Cheng-Mayer, C., Hong, Z., Ho, D. D., and Markow-itz, M. (2015) A long-acting integrase inhibitor protects female macaquesfrom repeated high-dose intravaginal SHIV challenge. Sci. Transl. Med.7, 270 –274

157. Radzio, J., Spreen, W., Yueh, Y. L., Mitchell, J., Jenkins, L., García-Lerma,J. G., and Heneine, W. (2015) The long-acting integrase inhibitorGSK744 protects macaques from repeated intravaginal SHIV challenge.Sci. Transl. Med. 7, 270ra5 CrossRef Medline

158. Singh, K., Sarafianos, S. G., and Sönnerborg, A. (2019) Long-acting anti-HIV drugs targeting HIV-1 reverse transcriptase and integrase. Pharma-ceuticals (Basel) 12, E62 CrossRef Medline

159. Radzio-Basu, J., Council, O., Cong, M.-E., Ruone, S., Newton, A., Wei, X.,Mitchell, J., Ellis, S., Petropoulos, C. J., Huang, W., Spreen, W., Heneine,W., and García-Lerma, J. G. (2019) Drug resistance emergence in ma-caques administered cabotegravir long-acting for pre-exposure prophy-laxis during acute SHIV infection. Nat. Commun. 10, 2005 CrossRefMedline

160. Smith, S. J., Zhao, X. Z., Burke, T. R., Jr., and Hughes, S. H. (2018) Effica-cies of cabotegravir and bictegravir against drug-resistant HIV-1 inte-grase mutants. Retrovirology 15, 37 CrossRef Medline

161. Sillman, B., Bade, A. N., Dash, P. K., Bhargavan, B., Kocher, T., Mathews,S., Su, H., Kanmogne, G. D., Poluektova, L. Y., Gorantla, S., McMillan, J.,Gautam, N., Alnouti, Y., Edagwa, B., and Gendelman, H. E. (2018) Cre-ation of a long-acting nanoformulated dolutegravir. Nat. Commun. 9,443 CrossRef Medline

162. Mandal, S., Prathipati, P. K., Belshan, M., and Destache, C. J. (2019) Apotential long-acting bictegravir loaded nano-drug delivery system forHIV-1 infection: a proof-of-concept study. Antiviral Res. 167, 83– 88CrossRef Medline

JBC REVIEWS: HIV integrase mechanisms and inhibition

J. Biol. Chem. (2019) 294(41) 15137–15157 15153

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

163. Zhang, W. W., Cheung, P. K., Oliveira, N., Robbins, M. A., Harrigan, P. R.,and Shahid, A. (2018) Accumulation of multiple mutations in vivo con-fers cross-resistance to new and existing integrase inhibitors. J. Infect.Dis. 218, 1773–1776 CrossRef Medline

164. Tsantrizos, Y. S., Boes, M., Brochu, C., Fenwick, C., Malenfant, E.,Mason, S., and Pesant, M. (November 22, 2007) Inhibitors of humanimmunodeficiency virus replication. International Patent NumberWO/2001/131350

165. Fenwick, C. W., Tremblay, S., Wardrop, E., Bethell, R., Coulomb, R.,Elston, R., Faucher, A.-M., Mason, S., Simoneau, B., Tsantrizos, Y., andYoakim, C. (2011) Resistance studies with HIV-1 non-catalytic site inte-grase inhibitors. Antivir. Ther. 16, Suppl. 1, A9

166. Fader, L. D., Malenfant, E., Parisien, M., Carson, R., Bilodeau, F., Landry,S., Pesant, M., Brochu, C., Morin, S., Chabot, C., Halmos, T., Bousquet,Y., Bailey, M. D., Kawai, S. H., Coulombe, R., et al. (2014) Discovery of BI224436, a noncatalytic site integrase inhibitor (NCINI) of HIV-1. ACSMed. Chem. Lett. 5, 422– 427 CrossRef Medline

167. Christ, F., Voet, A., Marchand, A., Nicolet, S., Desimmie, B. A., March-and, D., Bardiot, D., Van der Veken, N. J., Van Remoortel, B., Strelkov,S. V., De Maeyer, M., Chaltin, P., and Debyser, Z. (2010) Rational designof small-molecule inhibitors of the LEDGF/p75-integrase interactionand HIV replication. Nat. Chem. Biol. 6, 442– 448 CrossRef Medline

168. Kessl, J. J., Jena, N., Koh, Y., Taskent-Sezgin, H., Slaughter, A., Feng, L., deSilva, S., Wu, L., Le Grice, S. F. J., Engelman, A., Fuchs, J. R., and Kvar-atskhelia, M. (2012) Multimode, cooperative mechanism of action ofallosteric HIV-1 integrase inhibitors. J. Biol. Chem. 287, 16801–16811CrossRef Medline

169. Balakrishnan, M., Yant, S. R., Tsai, L., O’Sullivan, C., Bam, R. A., Tsai, A.,Niedziela-Majka, A., Stray, K. M., Sakowicz, R., and Cihlar, T. (2013)Non-catalytic site HIV-1 integrase inhibitors disrupt core maturationand induce a reverse transcription block in target cells. PLoS One 8,e74163 CrossRef Medline

170. Le Rouzic, E., Bonnard, D., Chasset, S., Bruneau, J.-M., Chevreuil, F., LeStrat, F., Nguyen, J., Beauvoir, R., Amadori, C., Brias, J., Vomscheid, S.,Eiler, S., Lévy, N., Delelis, O., Deprez, E., et al. (2013) Dual inhibition ofHIV-1 replication by integrase-LEDGF allosteric inhibitors is predomi-nant at the post-integration stage. Retrovirology 10, 144 CrossRefMedline

171. Sharma, A., Slaughter, A., Jena, N., Feng, L., Kessl, J. J., Fadel, H. J., Malani,N., Male, F., Wu, L., Poeschla, E., Bushman, F. D., Fuchs, J. R., and Kvar-atskhelia, M. (2014) A new class of multimerization selective inhibitors ofHIV-1 integrase. PLoS Pathog. 10, e1004171 CrossRef Medline

172. Schroder, A. R. W., Shinn, P., Chen, H., Berry, C., Ecker, J. R., and Bush-man, F. (2002) HIV-1 integration in the human genome favors activegenes and local hotspots. Cell 110, 521–529 CrossRef Medline

173. Sowd, G. A., Serrao, E., Wang, H., Wang, W., Fadel, H. J., Poeschla, E. M.,and Engelman, A. N. (2016) A critical role for alternative polyadenylationfactor CPSF6 in targeting HIV-1 integration to transcriptionally activechromatin. Proc. Natl. Acad. Sci. U.S.A. 113, E1054 –E1063 CrossRefMedline

174. Achuthan, V., Perreira, J. M., Sowd, G. A., Puray-Chavez, M., McDougall,W. M., Paulucci-Holthauzen, A., Wu, X., Fadel, H. J., Poeschla, E. M.,Multani, A. S., Hughes, S. H., Sarafianos, S. G., Brass, A. L., and Engelman,A. N. (2018) Capsid-CPSF6 interaction licenses nuclear HIV-1 traffick-ing to sites of viral DNA integration. Cell Host Microbe 24, 392– 404.e8CrossRef Medline

175. Nishizawa, Y., Usukura, J., Singh, D. P., Chylack, L. T., Jr., and Shinohara,T. (2001) Spatial and temporal dynamics of two alternatively splicedregulatory factors, lens epithelium-derived growth factor (ledgf/p75) andp52, in the nucleus. Cell Tissue Res. 305, 107–114 CrossRef Medline

176. Maertens, G., Cherepanov, P., Pluymers, W., Busschots, K., De Clercq, E.,Debyser, Z., and Engelborghs, Y. (2003) LEDGF/p75 is essential for nu-clear and chromosomal targeting of HIV-1 integrase in human cells.J. Biol. Chem. 278, 33528 –33539 CrossRef Medline

177. Ge, H., Si, Y., and Roeder, R. G. (1998) Isolation of cDNAs encoding noveltranscription coactivators p52 and p75 reveals an alternate regulatorymechanism of transcriptional activation. EMBO J. 17, 6723– 6729CrossRef Medline

178. Pradeepa, M. M., Sutherland, H. G., Ule, J., Grimes, G. R., and Bickmore,W. A. (2012) Psip1/Ledgf p52 binds methylated histone H3K36 andsplicing factors and contributes to the regulation of alternative splicing.PLoS Genet. 8, e1002717 CrossRef Medline

179. Eidahl, J. O., Crowe, B. L., North, J. A., McKee, C. J., Shkriabai, N., Feng,L., Plumb, M., Graham, R. L., Gorelick, R. J., Hess, S., Poirier, M. G.,Foster, M. P., and Kvaratskhelia, M. (2013) Structural basis for high-affinity binding of LEDGF PWWP to mononucleosomes. Nucleic AcidsRes. 41, 3924 –3936 CrossRef Medline

180. van Nuland, R., van Schaik, F. M., Simonis, M., van Heesch, S., Cuppen,E., Boelens, R., Timmers, H. M., and van Ingen, H. (2013) NucleosomalDNA binding drives the recognition of H3K36-methylated nucleosomesby the PSIP1-PWWP domain. Epigenetics Chromatin 6, 12 CrossRefMedline

181. Cherepanov, P., Devroe, E., Silver, P. A., and Engelman, A. (2004) Iden-tification of an evolutionarily-conserved domain in LEDGF/p75 thatbinds HIV-1 integrase. J. Biol. Chem. 279, 48883– 48892 CrossRefMedline

182. Cherepanov, P., Sun, Z.-Y. J., Rahman, S., Maertens, G., Wagner, G., andEngelman, A. (2005) Solution structure of the HIV-1 integrase-bindingdomain in LEDGF/p75. Nat. Struct. Mol. Biol. 12, 526 –532 CrossRefMedline

183. Dyda, F., Hickman, A. B., Jenkins, T. M., Engelman, A., Craigie, R., andDavies, D. R. (1994) Crystal structure of the catalytic domain of HIV-1integrase: similarity to other polynucleotidyl transferases. Science 266,1981–1986 CrossRef Medline

184. Cherepanov, P., Ambrosio, A. L. B., Rahman, S., Ellenberger, T., andEngelman, A. (2005) From the cover: structural basis for the recognitionbetween HIV-1 integrase and transcriptional coactivator p75. Proc. Natl.Acad. Sci. U.S.A. 102, 17308 –17313 CrossRef Medline

185. Hare, S., Shun, M. C., Gupta, S. S., Valkov, E., Engelman, A., and Che-repanov, P. (2009) A novel co-crystal structure affords the design ofgain-of-function lentiviral integrase mutants in the presence of modifiedPSIP1/LEDGF/p75. PLoS Pathog. 5, e1000259 CrossRef Medline

186. Hayouka, Z., Rosenbluh, J., Levin, A., Loya, S., Lebendiker, M., Veprint-sev, D., Kotler, M., Hizi, A., Loyter, A., and Friedler, A. (2007) InhibitingHIV-1 integrase by shifting its oligomerization equilibrium. Proc. Natl.Acad. Sci. U.S.A. 104, 8316 – 8321 CrossRef Medline

187. Tsiang, M., Jones, G. S., Hung, M., Samuel, D., Novikov, N., Mukund, S.,Brendza, K. M., Niedziela-Majka, A., Jin, D., Liu, X., Mitchell, M., Sako-wicz, R., and Geleziunas, R. (2011) Dithiothreitol causes HIV-1 integrasedimer dissociation while agents interacting with the integrase dimer in-terface promote dimer formation. Biochemistry 50, 1567–1581 CrossRefMedline

188. Turlure, F., Maertens, G., Rahman, S., Cherepanov, P., and Engelman, A.(2006) A tripartite DNA-binding element, comprised of the nuclear lo-calization signal and two AT-hook motifs, mediates the association ofLEDGF/p75 with chromatin in vivo. Nucleic Acids Res. 34, 1653–1665CrossRef Medline

189. Llano, M., Vanegas, M., Fregoso, O., Saenz, D., Chung, S., Peretz, M., andPoeschla, E. M. (2004) LEDGF/p75 determines cellular trafficking of di-verse lentiviral but not murine oncoretroviral integrase proteins and is acomponent of functional lentiviral preintegration complexes. J. Virol. 78,9524 –9537 CrossRef Medline

190. Busschots, K., Vercammen, J., Emiliani, S., Benarous, R., Engelborghs, Y.,Christ, F., and Debyser, Z. (2005) The interaction of LEDGF/p75 withintegrase is lentivirus-specific and promotes DNA binding. J. Biol. Chem.280, 17841–17847 CrossRef Medline

191. Cherepanov, P. (2007) LEDGF/p75 interacts with divergent lentiviralintegrases and modulates their enzymatic activity in vitro. Nucleic AcidsRes. 35, 113–124 CrossRef Medline

192. Llano, M., Saenz, D. T., Meehan, A., Wongthida, P., Peretz, M., Walker,W. H., Teo, W., and Poeschla, E. M. (2006) An essential role for LEDGF/p75 in HIV integration. Science 314, 461– 464 CrossRef Medline

193. Vandekerckhove, L., Christ, F., Van Maele, B., De Rijck, J., Gijsbers, R.,Van den Haute, C., Witvrouw, M., and Debyser, Z. (2006) Transient andstable knockdown of the integrase cofactor LEDGF/p75 reveals its role in

JBC REVIEWS: HIV integrase mechanisms and inhibition

15154 J. Biol. Chem. (2019) 294(41) 15137–15157

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

the replication cycle of human immunodeficiency virus. J. Virol. 80,1886 –1896 CrossRef Medline

194. Marshall, H. M., Ronen, K., Berry, C., Llano, M., Sutherland, H., Saenz,D., Bickmore, W., Poeschla, E., and Bushman, F. D. (2007) Role of PSIP1/LEDGF/p75 in lentiviral infectivity and integration targeting. PLoS One2, e1340 CrossRef Medline

195. Shun, M.-C., Raghavendra, N. K., Vandegraaff, N., Daigle, J. E., Hughes,S., Kellam, P., Cherepanov, P., and Engelman, A. (2007) LEDGF/p75functions downstream from preintegration complex formation to effectgene-specific HIV-1 integration. Genes Dev. 21, 1767–1778 CrossRefMedline

196. Schrijvers, R., De Rijck, J., Demeulemeester, J., Adachi, N., Vets, S.,Ronen, K., Christ, F., Bushman, F. D., Debyser, Z., and Gijsbers, R. (2012)LEDGF/p75-independent HIV-1 replication demonstrates a role forHRP-2 and remains sensitive to inhibition by LEDGINs. PLoS Pathog. 8,e1002558 CrossRef Medline

197. Wang, H., Jurado, K. A., Wu, X., Shun, M. C., Li, X., Ferris, A. L., Smith,S. J., Patel, P. A., Fuchs, J. R., Cherepanov, P., Kvaratskhelia, M., Hughes,S. H., and Engelman, A. (2012) HRP2 determines the efficiency and spec-ificity of HIV-1 integration in LEDGF/p75 knockout cells but does notcontribute to the antiviral activity of a potent LEDGF/p75-binding siteintegrase inhibitor. Nucleic Acids Res. 40, 11518 –11530 CrossRefMedline

198. De Rijck, J., Vandekerckhove, L., Gijsbers, R., Hombrouck, A., Hendrix, J.,Vercammen, J., Engelborghs, Y., Christ, F., and Debyser, Z. (2006) Over-expression of the lens epithelium-derived growth factor/p75 integrasebinding domain inhibits human immunodeficiency virus replication.J. Virol. 80, 11498 –11509 CrossRef Medline

199. Meehan, A. M., Saenz, D. T., Morrison, J., Hu, C., Peretz, M., and Po-eschla, E. M. (2011) LEDGF dominant interference proteins demonstrateprenuclear exposure of HIV-1 integrase and synergize with LEDGF de-pletion to destroy viral infectivity. J. Virol. 85, 3570 –3583 CrossRefMedline

200. Fader, L. D., Bailey, M., Beaulieu, E., Bilodeau, F., Bonneau, P., Bousquet,Y., Carson, R. J., Chabot, C., Coulombe, R., Duan, J., Fenwick, C., Gar-neau, M., Halmos, T., Jakalian, A., James, C., et al. (2016) Aligning po-tency and pharmacokinetic properties for pyridine-based NCINIs. ACSMed. Chem. Lett. 7, 797– 801 CrossRef Medline

201. Amadori, C., van der Velden, Y. U., Bonnard, D., Orlov, I., van Bel, N., LeRouzic, E., Miralles, L., Brias, J., Chevreuil, F., Spehner, D., Chasset, S.,Ledoussal, B., Mayr, L., Moreau, F., García, F., et al. (2017) The HIV-1integrase-LEDGF allosteric inhibitor MUT-A: resistance profile, impair-ment of virus maturation and infectivity but without influence on RNApackaging or virus immunoreactivity. Retrovirology 14, 50 CrossRefMedline

202. Tsiang, M., Jones, G. S., Niedziela-Majka, A., Kan, E., Lansdon, E. B.,Huang, W., Hung, M., Samuel, D., Novikov, N., Xu, Y., Mitchell, M., Guo,H., Babaoglu, K., Liu, X., Geleziunas, R., and Sakowicz, R. (2012) Newclass of HIV-1 integrase (IN) inhibitors with a dual mode of action. J. Biol.Chem. 287, 21189 –21203 CrossRef Medline

203. Fenwick, C., Amad, M., Bailey, M. D., Bethell, R., Bös, M., Bonneau, P.,Cordingley, M., Coulombe, R., Duan, J., Edwards, P., Fader, L. D., Fau-cher, A. M., Garneau, M., Jakalian, A., Kawai, S., et al. (2014) Preclinicalprofile of BI 224436, a novel HIV-1 non-catalytic-site integrase inhibitor.Antimicrob. Agents Chemother. 58, 3233–3244 CrossRef Medline

204. Gupta, K., Brady, T., Dyer, B. M., Malani, N., Hwang, Y., Male, F., Nolte,R. T., Wang, L., Velthuisen, E., Jeffrey, J., Van Duyne, G. D., and Bush-man, F. D. (2014) Allosteric inhibition of human immunodeficiency virusintegrase: late block during viral replication and abnormal multimeriza-tion involving specific protein domains. J. Biol. Chem. 289, 20477–20488CrossRef Medline

205. Wilson, T. A., Koneru, P. C., Rebensburg, S. V., Lindenberger, J. J., Kobe,M. J., Cockroft, N. T., Adu-Ampratwum, D., Larue, R. C., Kvaratskhelia,M., and Fuchs, J. R. (2019) An isoquinoline scaffold as a novel class ofallosteric HIV-1 integrase inhibitors. ACS Med. Chem. Lett. 10, 215–220CrossRef Medline

206. Christ, F., Shaw, S., Demeulemeester, J., Desimmie, B. A., Marchand, A.,Butler, S., Smets, W., Chaltin, P., Westby, M., Debyser, Z., and Pickford,

C. (2012) Small molecule inhibitors of the LEDGF/p75 binding site ofintegrase (LEDGINs) block HIV replication and modulate integrase mul-timerization. Antimicrob. Agents Chemother. 56, 4365– 4374 CrossRefMedline

207. Peese, K. M., Allard, C. W., Connolly, T., Johnson, B. L., Li, C., Patel, M.,Sorensen, M. E., Walker, M. A., Meanwell, N. A., McAuliffe, B., Minas-sian, B., Krystal, M., Parker, D. D., Lewis, H. A., Kish, K., Zhang, P., Nolte,R. T., Simmermacher, J., Jenkins, S., Cianci, C., and Naidu, B. N. (2019)5,6,7,8-Tetrahydro-1,6-naphthyridine derivatives as potent HIV-1-inte-grase-allosteric-site inhibitors. J. Med. Chem. 62, 1348 –1361 CrossRefMedline

208. Demeulemeester, J., Chaltin, P., Marchand, A., De Maeyer, M., Debyser,Z., and Christ, F. (2014) LEDGINs, non-catalytic site inhibitors of HIV-1integrase: a patent review (2006 –2014). Expert Opin. Ther. Pat. 24,609 – 632 CrossRef Medline

209. Desimmie, B. A., Schrijvers, R., Demeulemeester, J., Borrenberghs, D.,Weydert, C., Thys, W., Vets, S., Van Remoortel, B., Hofkens, J., De Rijck,J., Hendrix, J., Bannert, N., Gijsbers, R., Christ, F., and Debyser, Z. (2013)LEDGINs inhibit late stage HIV-1 replication by modulating integrasemultimerization in the virions. Retrovirology 10, 57 CrossRef Medline

210. Slaughter, A., Jurado, K. A., Deng, N., Feng, L., Kessl, J. J., Shkriabai, N.,Larue, R. C., Fadel, H. J., Patel, P. A., Jena, N., Fuchs, J. R., Poeschla, E.,Levy, R. M., Engelman, A., and Kvaratskhelia, M. (2014) The mechanismof H171T resistance reveals the importance of N�-protonated His171 forthe binding of allosteric inhibitor BI-D to HIV-1 integrase. Retrovirology11, 100 CrossRef Medline

211. Feng, L., Sharma, A., Slaughter, A., Jena, N., Koh, Y., Shkriabai, N., Larue,R. C., Patel, P. A., Mitsuya, H., Kessl, J. J., Engelman, A., Fuchs, J. R., andKvaratskhelia, M. (2013) The A128T resistance mutation reveals aber-rant protein multimerization as the primary mechanism of action ofallosteric HIV-1 integrase inhibitors. J. Biol. Chem. 288, 15813–15820CrossRef Medline

212. Gupta, K., Turkki, V., Sherrill-Mix, S., Hwang, Y., Eilers, G., Taylor, L.,McDanal, C., Wang, P., Temelkoff, D., Nolte, R. T., Velthuisen, E., Jeffrey,J., Van Duyne, G. D., and Bushman, F. D. (2016) Structural basis forinhibitor-induced aggregation of HIV integrase. PLoS Biol. 14, e1002584CrossRef Medline

213. Deng, N., Hoyte, A., Mansour, Y. E., Mohamed, M. S., Fuchs, J. R., Engel-man, A. N., Kvaratskhelia, M., and Levy, R. (2016) Allosteric HIV-1 inte-grase inhibitors promote aberrant protein multimerization by directlymediating inter-subunit interactions: structural and thermodynamicmodeling studies. Protein Sci. 25, 1911–1917 CrossRef Medline

214. Koneru, P. C., Francis, A. C., Deng, N., Rebensburg, S. V., Hoyte, A. C.,Lindenberger, J., Adu-Ampratwum, D., Larue, R. C., Wempe, M. F., En-gelman, A. N., Lyumkis, D., Fuchs, J. R., Levy, R. M., Melikyan, G. B., andKvaratskhelia, M. (2019) HIV-1 integrase tetramers are the antiviral tar-get of pyridine-based allosteric integrase inhibitors. eLife 8, e46344CrossRef Medline

215. Bonnard, D., Le Rouzic, E., Eiler, S., Amadori, C., Orlov, I., Bruneau,J.-M., Brias, J., Barbion, J., Chevreuil, F., Spehner, D., Chasset, S., Ledous-sal, B., Moreau, F., Saïb, A., Klaholz, B. P., et al. (2018) Structure-functionanalyses unravel distinct effects of allosteric inhibitors of HIV-1 integraseon viral maturation and integration. J. Biol. Chem. 293, 6172– 6186CrossRef Medline

216. Engelman, A., Englund, G., Orenstein, J. M., Martin, M. A., and Craigie,R. (1995) Multiple effects of mutations in human immunodeficiency vi-rus type 1 integrase on viral replication. J. Virol. 69, 2729 –2736 Medline

217. Engelman, A. (1999) In vivo analysis of retroviral integrase structure andfunction. Adv. Virus Res. 52, 411– 426 CrossRef Medline

218. Engelman, A. (2011) The pleiotropic nature of human immunodefi-ciency virus integrase mutations. in HIV-1 Integrase: Mechanism andInhibitor Design (Neamati, N. ed) pp. 67– 81, John Wiley & Sons, Inc.,Hoboken, NJ

219. Madison, M. K., Lawson, D. Q., Elliott, J., Ozantürk, A. N., Koneru, P. C.,Townsend, D., Errando, M., Kvaratskhelia, M., and Kutluay, S. B. (2017)Allosteric HIV-1 integrase inhibitors lead to premature degradationof the viral RNA genome and integrase in target cells. J. Virol. 91,e00821-17 Medline

JBC REVIEWS: HIV integrase mechanisms and inhibition

J. Biol. Chem. (2019) 294(41) 15137–15157 15155

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

220. Fadel, H. J., Morrison, J. H., Saenz, D. T., Fuchs, J. R., Kvaratskhelia, M.,Ekker, S. C., and Poeschla, E. M. (2014) TALEN knockout of the PSIP1gene in human cells: analyses of HIV-1 replication and allosteric inte-grase inhibitor mechanism. J. Virol. 88, 9704 –9717 CrossRef Medline

221. Feng, L., Dharmarajan, V., Serrao, E., Hoyte, A., Larue, R. C., Slaughter,A., Sharma, A., Plumb, M. R., Kessl, J. J., Fuchs, J. R., Bushman, F. D.,Engelman, A. N., Griffin, P. R., and Kvaratskhelia, M. (2016) The com-petitive interplay between allosteric HIV-1 integrase inhibitor BI/D andLEDGF/p75 during the early stage of HIV-1 replication adversely affectsinhibitor potency. ACS Chem. Biol. 11, 1313–1321 CrossRef Medline

222. Vranckx, L. S., Demeulemeester, J., Saleh, S., Boll, A., Vansant, G., Schri-jvers, R., Weydert, C., Battivelli, E., Verdin, E., Cereseto, A., Christ, F.,Gijsbers, R., and Debyser, Z. (2016) LEDGIN-mediated inhibition ofintegrase–LEDGF/p75 interaction reduces reactivation of residual latentHIV. EBioMedicine 8, 248 –264 CrossRef Medline

223. Maldarelli, F., Wu, X., Su, L., Simonetti, F. R., Shao, W., Hill, S., Spindler,J., Ferris, A. L., Mellors, J. W., Kearney, M. F., Coffin, J. M., and Hughes,S. H. (2014) Specific HIV integration sites are linked to clonal expansionand persistence of infected cells. Science 345, 179 –183 CrossRef Medline

224. Wagner, T. A., McLaughlin, S., Garg, K., Cheung, C. Y. K., Larsen, B. B.,Styrchak, S., Huang, H. C., Edlefsen, P. T., Mullins, J. I., and Frenkel, L. M.(2014) Proliferation of cells with HIV integrated into cancer genes con-tributes to persistent infection. Science 345, 570 –573 CrossRef Medline

225. Wang, Z., Gurule, E. E., Brennan, T. P., Gerold, J. M., Kwon, K. J., Hos-mane, N. N., Kumar, M. R., Beg, S. A., Capoferri, A. A., Ray, S. C., Ho,Y.-C., Hill, A. L., Siliciano, J. D., and Siliciano, R. F. (2018) Expandedcellular clones carrying replication-competent HIV-1 persist, wax, andwane. Proc. Natl. Acad. Sci. U.S.A. 115, E2575–E2584 CrossRef Medline

226. Hughes, S. H., and Coffin, J. M. (2016) What integration sites tell us aboutHIV persistence. Cell Host Microbe 19, 588 –598 CrossRef Medline

227. Singh, P. K., Plumb, M. R., Ferris, A. L., Iben, J. R., Wu, X., Fadel, H. J.,Luke, B. T., Esnault, C., Poeschla, E. M., Hughes, S. H., Kvaratskhelia, M.,and Levin, H. L. (2015) LEDGF/p75 interacts with mRNA splicing factorsand targets HIV-1 integration to highly spliced genes. Genes Dev. 29,2287–2297 CrossRef Medline

228. Debyser, Z., Vansant, G., Bruggemans, A., Janssens, J., and Christ, F. (2018)Insight in HIV integration site selection provides a block-and-lock strategyfor a functional cure of HIV infection. Viruses 11, E12 CrossRef Medline

229. Gérard, A., Ségéral, E., Naughtin, M., Abdouni, A., Charmeteau, B.,Cheynier, R., Rain, J.-C., and Emiliani, S. (2015) The integrase cofactorLEDGF/p75 associates with Iws1 and Spt6 for postintegration silencingof HIV-1 gene expression in latently infected cells. Cell Host Microbe 17,107–117 CrossRef Medline

230. Liu, H., Wu, X., Xiao, H., Conway, J. A., and Kappes, J. C. (1997) Incor-poration of functional human immunodeficiency virus type 1 integraseinto virions independent of the Gag-Pol precursor protein. J. Virol. 71,7704 –7710 Medline

231. de Béthune, M.-P. (2010) Non-nucleoside reverse transcriptase inhibi-tors (NNRTIs), their discovery, development, and use in the treatment ofHIV-1 infection: a review of the last 20 years (1989 –2009). Antivir. Res.85, 75–90 CrossRef Medline

232. Akiyama, H., Ishimatsu, M., Miura, T., Hayami, M., and Ido, E. (2008)Construction and infection of a new simian/human immunodeficiencychimeric virus (SHIV) containing the integrase gene of the human im-munodeficiency virus type 1 genome and analysis of its adaptation tomonkey cells. Microbes Infect. 10, 531–539 CrossRef Medline

233. Wu, X., Liu, H., Xiao, H., Conway, J. A., Hehl, E., Kalpana, G. V., Prasad,V., and Kappes, J. C. (1999) Human immunodeficiency virus type 1 inte-grase protein promotes reverse transcription through specific interac-tions with the nucleoprotein reverse transcription complex. J. Virol. 73,2126 –2135 Medline

234. Zhu, K., Dobard, C., and Chow, S. A. (2004) Requirement for integraseduring reverse transcription of human immunodeficiency virus type 1and the effect of cysteine mutations of integrase on its interactions withreverse transcriptase. J. Virol. 78, 5045–5055 CrossRef Medline

235. Herschhorn, A., Oz-Gleenberg, I., and Hizi, A. (2008) Quantitative anal-ysis of the interactions between HIV-1 integrase and retroviral reversetranscriptases. Biochem. J. 412, 163–170 CrossRef Medline

236. Wilkinson, T. A., Januszyk, K., Phillips, M. L., Tekeste, S. S., Zhang, M.,Miller, J. T., Le Grice, S. F. J., Clubb, R. T., and Chow, S. A. (2009)Identifying and characterizing a functional HIV-1 reverse transcriptase-binding site on integrase. J. Biol. Chem. 284, 7931–7939 CrossRefMedline

237. Tekeste, S. S., Wilkinson, T. A., Weiner, E. M., Xu, X., Miller, J. T., LeGrice, S. F. J., Clubb, R. T., and Chow, S. A. (2015) Interaction betweenreverse transcriptase and integrase is required for reverse transcriptionduring HIV-1 replication. J. Virol. 89, 12058 –12069 CrossRef Medline

238. Burns, C. C., Gleason, L. M., Mozaffarian, A., Giachetti, C., Carr, J. K., andOverbaugh, J. (2002) Sequence variability of the integrase protein from adiverse collection of HIV type 1 isolates representing several subtypes.AIDS Res. Hum. Retroviruses 18, 1031–1041 CrossRef Medline

239. Rhee, S.-Y., Liu, T. F., Kiuchi, M., Zioni, R., Gifford, R. J., Holmes, S. P.,and Shafer, R. W. (2008) Natural variation of HIV-1 group M integrase:implications for a new class of antiretroviral inhibitors. Retrovirology 5,74 CrossRef Medline

240. Malet, I., Soulie, C., Tchertanov, L., Derache, A., Amellal, B., Traore, O.,Simon, A., Katlama, C., Mouscadet, J.-F., Calvez, V., and Marcelin, A.-G.(2008) Structural effects of amino acid variations between B andCRF02-AG HIV-1 integrases. J. Med. Virol. 80, 754 –761 CrossRefMedline

241. Low, A., Prada, N., Topper, M., Vaida, F., Castor, D., Mohri, H., Hazuda,D., Muesing, M., and Markowitz, M. (2009) Natural polymorphisms ofhuman immunodeficiency virus type 1 integrase and inherent suscepti-bilities to a panel of integrase inhibitors. Antimicrob. Agents Chemother.53, 4275– 4282 CrossRef Medline

242. Ceccherini-Silberstein, F., Malet, I., D’Arrigo, R., Antinori, A., Marcelin,A. G., and Perno, C. F. (2009) Characterization and structural analysis ofHIV-1 integrase conservation. AIDS Rev. 11, 17–29 Medline

243. Hoyte, A. C., Jamin, A. V., Koneru, P. C., Kobe, M. J., Larue, R. C., Fuchs,J. R., Engelman, A. N., and Kvaratskhelia, M. (2017) Resistance to pyri-dine-based inhibitor KF116 reveals an unexpected role of integrase inHIV-1 Gag-Pol polyprotein proteolytic processing. J. Biol. Chem. 292,19814 –19825 CrossRef Medline

244. Shen, L., Peterson, S., Sedaghat, A. R., McMahon, M. A., Callender, M.,Zhang, H., Zhou, Y., Pitt, E., Anderson, K. S., Acosta, E. P., and Siliciano,R. F. (2008) Dose-response curve slope sets class-specific limits on inhib-itory potential of anti-HIV drugs. Nat. Med. 14, 762–766 CrossRefMedline

245. Rabi, S. A., Laird, G. M., Durand, C. M., Laskey, S., Shan, L., Bailey, J. R.,Chioma, S., Moore, R. D., and Siliciano, R. F. (2013) Multi-step inhibitionexplains HIV-1 protease inhibitor pharmacodynamics and resistance.J. Clin. Invest. 123, 3848 –3860 CrossRef Medline

246. Briones, M. S., Dobard, C. W., and Chow, S. A. (2010) Role of humanimmunodeficiency virus type 1 integrase in uncoating of the viral core.J. Virol. 84, 5181–5190 CrossRef Medline

247. Gallay, P., Hope, T., Chin, D., and Trono, D. (1997) HIV-1 infection ofnondividing cells through the recognition of integrase by the importin/karyopherin pathway. Proc. Natl. Acad. Sci. U.S.A. 94, 9825–9830CrossRef Medline

248. Bouyac-Bertoia, M., Dvorin, J. D., Fouchier, R. A. M., Jenkins, Y., Meyer,B. E., Wu, L. I., Emerman, M., and Malim, M. H. (2001) HIV-1 infectionrequires a functional integrase NLS. Mol. Cell 7, 1025–1035 CrossRefMedline

249. Fassati, A., Gorlich, D., Harrison, I., Zaytseva, L., and Mingot, J. M. (2003)Nuclear import of HIV-1 intracellular reverse transcription complexes ismediated by importin 7. EMBO J. 22, 3675–3685 CrossRef Medline

250. Armon-Omer, A., Graessmann, A., and Loyter, A. (2004) A syntheticpeptide bearing the HIV-1 integrase 161–173 amino acid residues medi-ates active nuclear import and binding to importin �: characterization ofa functional nuclear localization signal. J. Mol. Biol. 336, 1117–1128CrossRef Medline

251. Hearps, A. C., Wagstaff, K. M., Piller, S. C., and Jans, D. A. (2008) TheN-terminal basic domain of the HIV-1 matrix protein does not contain aconventional nuclear localization sequence but is required for DNAbinding and protein self-association. Biochemistry 47, 2199 –2210CrossRef Medline

JBC REVIEWS: HIV integrase mechanisms and inhibition

15156 J. Biol. Chem. (2019) 294(41) 15137–15157

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

252. Ao, Z., Danappa Jayappa, K., Wang, B., Zheng, Y., Kung, S., Rassart, E.,Depping, R., Kohler, M., Cohen, E. A., and Yao, X. (2010) Importin �3interacts with HIV-1 integrase and contributes to HIV-1 nuclear importand replication. J. Virol. 84, 8650 – 8663 CrossRef Medline

253. Jayappa, K. D., Ao, Z., Yang, M., Wang, J., and Yao, X. (2011) Identifica-tion of critical motifs within HIV-1 integrase required for importin �3interaction and viral cDNA nuclear import. J. Mol. Biol. 410, 847– 862CrossRef Medline

254. Ao, Z., Huang, G., Yao, H., Xu, Z., Labine, M., Cochrane, A. W., and Yao,X. (2007) Interaction of human immunodeficiency virus type 1 integrasewith cellular nuclear import receptor importin 7 and its impact on viralreplication. J. Biol. Chem. 282, 13456 –13467 CrossRef Medline

255. Allouch, A., and Cereseto, A. (2011) Identification of cellular factorsbinding to acetylated HIV-1 integrase. Amino Acids 41, 1137–1145CrossRef Medline

256. Jager, S., Cimermancic, P., Gulbahce, N., Johnson, J. R., McGovern, K. E.,Clarke, S. C., Shales, M., Mercenne, G., Pache, L., Li, K., Hernandez, H.,Jang, G. M., Roth, S. L., Akiva, E., Marlett, J., et al. (2011) Global landscapeof HIV-human protein complexes. Nature 481, 365–370 CrossRefMedline

257. Christ, F., Thys, W., De Rijck, J., Gijsbers, R., Albanese, A., Arosio, D.,Emiliani, S., Rain, J. C., Benarous, R., Cereseto, A., and Debyser, Z. (2008)Transportin-SR2 imports HIV into the nucleus. Curr. Biol. 18,1192–1202 CrossRef Medline

258. Yamashita, M., and Emerman, M. (2004) Capsid is a dominant determi-nant of retrovirus infectivity in nondividing cells. J. Virol. 78, 5670 –5678CrossRef Medline

259. Petit, C., Schwartz, O., and Mammano, F. (2000) The karyophilic prop-erties of human immunodeficiency virus type 1 integrase are not re-quired for nuclear import of proviral DNA. J. Virol. 74, 7119 –7126CrossRef Medline

260. Limón, A., Devroe, E., Lu, R., Ghory, H. Z., Silver, P. A., and Engelman, A.(2002) Nuclear localization of human immunodeficiency virus type 1preintegration complexes (PICs): V165A and R166A are pleiotropic in-tegrase mutants primarily defective for integration, not PIC nuclear im-port. J. Virol. 76, 10598 –10607 CrossRef Medline

261. Dvorin, J. D., Bell, P., Maul, G. G., Yamashita, M., Emerman, M., andMalim, M. H. (2002) Reassessment of the roles of integrase and the cen-tral DNA flap in human immunodeficiency virus type 1 nuclear import.J. Virol. 76, 12087–12096 CrossRef Medline

262. Lu, R., Limón, A., Devroe, E., Silver, P. A., Cherepanov, P., and Engelman,A. (2004) Class II integrase mutants with changes in putative nuclearlocalization signals are primarily blocked at a post-nuclear entry step ofhuman immunodeficiency virus type 1 replication. J. Virol. 78,12735–12746 CrossRef Medline

263. Krishnan, L., Matreyek, K. A., Oztop, I., Lee, K., Tipper, C. H., Li, X., Dar,M. J., Kewalramani, V. N., and Engelman, A. (2010) The requirement forcellular transportin 3 (TNPO3 or TRN-SR2) during infection maps tohuman immunodeficiency virus type 1 capsid and not integrase. J. Virol.84, 397– 406 CrossRef Medline

264. Cribier, A., Segeral, E., Delelis, O., Parissi, V., Simon, A., Ruff, M., Ben-arous, R., and Emiliani, S. (2011) Mutations affecting interaction of inte-grase with TNPO3 do not prevent HIV-1 cDNA nuclear import. Retro-virology 8, 104 CrossRef Medline

265. De Iaco, A., and Luban, J. (2011) Inhibition of HIV-1 infection by TNPO3depletion is determined by capsid and detectable after viral cDNA entersthe nucleus. Retrovirology 8, 98 CrossRef Medline

266. De Houwer, S., Demeulemeester, J., Thys, W., Rocha, S., Dirix, L., Gijs-bers, R., Christ, F., and Debyser, Z. (2014) The HIV-1 integrase mutantR263A/K264A is 2-fold defective for TRN-SR2 binding and viral nuclearimport. J. Biol. Chem. 289, 25351–25361 CrossRef Medline

267. Maertens, G. N., Cook, N. J., Wang, W., Hare, S., Gupta, S. S., Öztop, I.,Lee, K., Pye, V. E., Cosnefroy, O., Snijders, A. P., KewalRamani, V. N.,Fassati, A., Engelman, A., and Cherepanov, P. (2014) Structural basis fornuclear import of splicing factors by human Transportin 3. Proc. Natl.Acad. Sci. U.S.A. 111, 2728 –2733 CrossRef Medline

268. Demeulemeester, J., Blokken, J., De Houwer, S., Dirix, L., Klaassen, H.,Marchand, A., Chaltin, P., Christ, F., and Debyser, Z. (2018) Inhibitors ofthe integrase–transportin-SR2 interaction block HIV nuclear import.Retrovirology 15, 5 CrossRef Medline

269. Wagstaff, K. M., Headey, S., Telwatte, S., Tyssen, D., Hearps, A. C.,Thomas, D. R., Tachedjian, G., and Jans, D. A. (2019) Molecular dissec-tion of an inhibitor targeting the HIV integrase dependent preintegrationcomplex nuclear import. Cell. Microbiol. 21, e12953 CrossRef Medline

270. Burlein, C., Wang, C., Xu, M., Bhatt, T., Stahlhut, M., Ou, Y., Adam, G. C.,Heath, J., Klein, D. J., Sanders, J., Narayan, K., Abeywickrema, P., Heo, M. R.,Carroll, S. S., Grobler, J. A., et al. (2017) Discovery of a distinct chemical andmechanistic class of allosteric HIV-1 integrase inhibitors with antiretroviralactivity. ACS Chem. Biol. 12, 2858–2865 CrossRef Medline

271. Kessl, J. J., Eidahl, J. O., Shkriabai, N., Zhao, Z., McKee, C. J., Hess, S.,Burke, T. R., Jr., and Kvaratskhelia, M. (2009) An allosteric mechanismfor inhibiting HIV-1 integrase with a small molecule. Mol. Pharmacol.76, 824 – 832 CrossRef Medline

272. Fassati, A., and Goff, S. P. (2001) Characterization of intracellular reversetranscription complexes of human immunodeficiency virus type 1. J. Vi-rol. 75, 3626 –3635 CrossRef Medline

273. Yu, S. F., Baldwin, D. N., Gwynn, S. R., Yendapalli, S., and Linial, M. L.(1996) Human foamy virus replication: a pathway distinct from that ofretroviruses and hepadnaviruses. Science 271, 1579 –1582 CrossRefMedline

JBC REVIEWS: HIV integrase mechanisms and inhibition

J. Biol. Chem. (2019) 294(41) 15137–15157 15157

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Alan N. Engelmaninhibition

Multifaceted HIV integrase functionalities and therapeutic strategies for their

doi: 10.1074/jbc.REV119.006901 originally published online August 29, 20192019, 294:15137-15157.J. Biol. Chem. 

  10.1074/jbc.REV119.006901Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/294/41/15137.full.html#ref-list-1

This article cites 268 references, 112 of which can be accessed free at

by guest on October 11, 2019

http://ww

w.jbc.org/

Dow

nloaded from