This journal is c The Royal Society of Chemistry 2013 Chem. Commun.

Cite this: DOI: 10.1039/c3cc40748a

Monitoring the HIV-1 integrase enzymatic activityusing atomic force microscopy in a 2LTR system†

Shlomit Guy,a Dvir Rotem,a Zvi Hayouka,a Ronen Gabizon,a Aviad Levin,b

Limor Zemel,a Abraham Loyter,b Danny Porath*ac and Assaf Friedler*ac

Integration of the HIV cDNA into the host chromosome is a key

event in the viral replication cycle. It is mediated by the viral

integrase (IN) enzyme, which is an attractive anti-HIV drug target.

Here we present the first AFM imaging of IN-mediated DNA

integration products in a two-LTR system.

The HIV-1 IN enzyme mediates the integration of the viral cDNAinto the host chromosome, which is a key event in the viralreplication cycle. The IN-mediated integration reaction proceedsin two steps: (1) 30-end processing, in which an IN dimercatalyses removal of the GT dinucleotide from each 30-end ofthe viral DNA and exposes the CA dinucleotide 30-hydroxylgroups.1,2 This reaction takes place in the cell cytoplasm;3 (2)strand transfer, in which the exposed oxygen atom at the 30-endsof the cleaved viral DNA attacks a phosphodiester bond in thetarget DNA.2,4 In the case of HIV, the sites of integration on thetwo target DNA strands are separated by 5 base pairs.1 This stepis mediated by an IN tetramer in the presence of the cellularco-factor LEDGF/p75 and occurs in the cell nucleus.5–7

Gel electrophoresis, DNA sequencing and enzyme-linked immu-nosorbent assay (ELISA) are commonly used for product analysisand quantitative determination of IN activity.8,9 Such methodsmeasure very large populations of molecules and therefore revealthe average behaviour of a system. Monitoring a process at thesingle molecule level enables observation of mechanistic detailsthat are masked when measuring large populations, e.g. distribu-tion of reactants, intermediates and products at any given timepoint along the reaction. Major tools, such as electron microscopyand atomic force microscopy (AFM), were developed and used inthe last two decades for direct visual imaging of single biological

molecules, such as DNA and proteins (e.g. ref. 10 and 11). Thesemethods enable us to observe the particular morphology of suchmolecules and interactions between them (e.g. ref. 12) and arebecoming more common in studying the fine details and mecha-nisms of enzymatic and biological reactions. Very few singlemolecule imaging studies of the HIV IN activity were reported. Inone study, a transmission electron microscope (TEM) was used forvisualizing in vitro integration products of the reaction.13 AFMimaging has advantages over TEM since it can be performed bothunder ambient conditions and in solution and there is no need forstaining of the samples. A study describing AFM imaging of INbinding to DNA was recently reported.14 Here we present the firstAFM imaging of IN-mediated integration products in a 2 longterminal repeat (LTR) system using an enzymatic assay thatcombines biochemical and single molecule techniques.

In vitro integration reaction of 750 bp dsDNA molecules, whichserved both as a donor and an acceptor, was carried out for 2 hoursat 37 1C. The DNA contained blunt-ended viral U3 and U5 LTRsequences in its termini. A sample was taken directly from thereaction mixture, diluted and adsorbed on a freshly cleaved micasubstrate for AFM imaging. The majority of the observed DNAmolecules were 250 nm long, which are unreacted molecules of the750 bp starting DNA (Fig. S1, ESI†). Only a few molecules seem tobe integration products (see below). This is not surprising, since theintegration reaction yield is very low. Up to 99% of reverse-transcribed cDNA molecules remain unintegrated within theinfected cells15 and the number of in vivo integration events isonly 1–2 per infected cell.16 We thus first used gel electrophoresis toseparate the reaction products and then followed the integratedand unreacted DNA separately using AFM.

To test the dependence of the integration products distributionon the reaction time, the in vitro integration reaction wasperformed at 37 1C and terminated at different time points byadding SDS. The DNA products were separated using agarose geland extracted for AFM imaging. Most of the DNA remainedunreacted (750 bp). The intensity of 1500 bp product bandsincreased with time up to 2 hours (Fig. 1). At longer reactiontimes, the product band intensity decreased and un-separatedDNA aggregates were formed. No bands of the product were

a Institute of Chemistry, The Hebrew University of Jerusalem, Givat Ram,

Jerusalem 91904, Israelb Department of Biological Chemistry, The Alexander Silberman Institute of Life

Sciences, The Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israelc The Harvey M. Kreuger Family Center for Nanosciene and Nanotechnology,

Safra Campus, The Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904,

Israel. E-mail: danny.porath@mail.huji.ac.il, assaf.friedler@mail.huji.ac.il

† Electronic supplementary information (ESI) available: Experimental details and5 figures. See DOI: 10.1039/c3cc40748a

Received 29th January 2013,Accepted 20th February 2013

DOI: 10.1039/c3cc40748a

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observed in all the negative controls: in the absence of IN and whenthe IN was inactivated by SDS before and during the reaction (Fig. 1).

AFM imaging of DNA products, extracted from the 1500 bpband that was separated after two hours of incubation, showedtwo major integration products (Fig. 2): Y-shaped and linearmolecules (Fig. 2AII and AIII). The Y-shaped DNA molecules arethe products of strand transfer of one LTR to a random site alonganother DNA molecule.13 The Y-shaped DNA was the majorproduct, indicating that this is the main reaction that occurredduring the in vitro integration reaction. The linear molecules areprobably the products of one full integration event (see below).Additional products were observed in rare cases in samples takenfrom the 1500 bp band (Fig. S2, ESI†). These may correspond toY-shaped molecules followed by self-integration (Fig. S2B, ESI†),or intramolecular integration followed by strand transfer ofanother molecule (Fig. S2C, ESI†). The suggested mechanismsfor formation of these structures are shown in Fig. 2C.

Molecules of 2250 bp long DNA, corresponding to products oftwo consecutive integration events between three molecules, have

also been observed. AFM imaging showed branched moleculesthat are composed of three DNA segments, 250 nm long (750 bp)each (Fig. 2BII), and linear molecules, 750 nm long (Fig. 2BIII).None of these structures were observed in the AFM images of 750 bpDNA molecules that were not reacted with IN (Fig. S3, ESI†).

The direct observation of the in vitro integration productsenables dissecting the fine details and observing structures thatare difficult to characterize using other methods. The lineardouble and triple sized DNA observed here corresponds to fullintegration products, at least in part of the molecules. TheY-shaped structures are the result of a partial integrationreaction that contains a strand transfer of one LTR of onemolecule to a random site along another molecule. This mayreflect an intermediate stage in the reaction. When a change inthe DNA length may occur following infection due to in vivofragmentation, Y-shaped molecules may be produced in vivo.

We designed DNA molecules with different combinations ofLTRs at their termini (Fig. S4A, ESI†). In vitro integration reactionswere carried out for 2 hours at 37 1C and the products wereseparated by gel electrophoresis. Different integration efficiencies,quantified by the band intensity, were observed for the differentDNA molecules (Fig. S4B, ESI†). DNA containing only the U3sequence at one end was significantly less reactive than DNA withthe U5 sequence at one or two termini, and produced a very faint1500 bp band (Fig. S4B and C, ESI†). This observation is consistentwith previous biochemical studies.13,17 Based on AFM images of1500 bp DNA extracted from the gel (Fig. 3), we quantified theamount of Y-shaped and linear products (Fig. 3E). Products of thereaction in which the DNA contained only the U3 sequence at oneend were not included in the statistics because there were notenough molecules for significant statistical analysis. The integra-tion products of the different DNA molecules showed similar ratiosof Y-shaped and linear products: a reaction with DNA containingU3 and U5 sequences generated 84% of Y-shaped DNA. 80% ofY-shaped DNA was obtained in a reaction with DNA containing theU5 sequence at one terminus and 74% in a reaction with DNAcontaining the U5 sequence at both termini. It is possible that thedominance of the Y-shaped products is because IN was not able toefficiently catalyze full integration from blunt ended DNA mole-cules as used here, and using a 30 processed DNA sequence mayincrease the amount of linear products as a result of full integra-tion. It was shown that when using two 30-processed LTRs assubstrates, mimicking the 30 processed viral DNA, IN can performin vitro integration.18 In another experiment, a constructed 468 bpdonor DNA that contained 20 bp LTRs with 30-OH at the ends wasused. Full integration of this DNA into circular DNA by IN derivedfrom lysates of HIV-1 virions was demonstrated.19

Linear 2250 bp DNA products were observed only when theDNA with U3 and U5 LTRs was used (Fig. 2BIII), and not whenthe DNA containing only the U5 LTR was used (Fig. 3B). Thisstrengthens the possibility that the observed linear integrationproducts were formed through a full integration process thatrequires the presence of both U5 and U3 LTRs, as occurs in theknown in vivo integration mechanism.1–4

The cellular protein, Lens-Epithelium Derived Growth Factor(LEDGF), binds to IN and is essential for its in vivo activity bytethering it to the chromatin.20–22 Inhibiting the IN–LEDGF

Fig. 1 Gel electrophoresis separation of the in vitro integration reaction.

Fig. 2 AFM imaging of integration products. In vitro integration reaction wasperformed with DNA molecules containing U3 and U5 viral LTR sequences attheir termini and separated on 1% agarose gel. (A) AFM imaging of integratedDNA extracted from the 1500 bp band (frame in Fig. 1). (B) AFM imaging ofintegrated DNA extracted from the 2250 bp band. Close-up views are in frames.The branch lengths are indicated in (A)II and (B)II. (C) The suggested mechanismsfor the formation of the different integration products. (a) Full integration. (b)Y-shaped molecules generated by strand transfer of one LTR to a random site inanother molecule. (c) Y-shape followed by self-integration. (d) Self-integration.(e) Self-integration followed by strand transfer of another molecule.

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interaction is a target for developing IN inhibitors.23,24 In our lab, wedeveloped the LEDGF 361–370 peptide, derived from the IN-bindingloop of LEDGF, which inhibited IN catalytic activity in vitro and incells and HIV-1 replication in cells and in a mouse model.25–27 Weperformed in vitro integration reaction in the presence of LEDGFand LEDGF 361–370. The incubation time was 2 hours at 37 1C andDNA with viral U3 and U5 LTRs was used in all cases. No 1500 bpband was observed in the presence of the peptide (Fig. S5A, ESI†),indicating that LEDGF 361–370 inhibited the IN activity, consistentwith previous reports.26,27 When the LEDGF protein was added tothe reaction mixture, the 1500 bp band intensity increased (Fig. S5B,ESI†) compared to the reaction in the absence of this protein,indicating that the IN enzymatic activity was indeed stimulated.28,29

Second integration events were observed as indicated by thepresence of a 2250 bp band, three times the length of the initialDNA molecules. The products of 1500 and 2250 bp DNA wereextracted from the gel containing the products of the integrationreaction in the presence of LEDGF (Fig. S5C, ESI†). AFM imagingshowed linear and branched DNA molecules, as observed in theother reactions (Fig. 2). Out of 60 molecules, 17% of the integratedproducts were linear. This suggests that LEDGF stimulated the INenzymatic activity without affecting the product distribution.

In summary, we combined a biochemical enzymatic assaywith a single molecule technique, and the in vitro integrationproducts were visualized using AFM. Full integration productswere observed as linear 1500 bp DNA and partial integrationproducts were observed as 1500 bp Y-shaped DNA, which werethe predominant products. Linear 2250 bp products were alsoobserved as a result of two consecutive integration events. Whilethe previous AFM study by Kotova et al.14 showed complexes ofIN with a short DNA segment, our study is the first that reportsAFM imaging of products of the integration of DNA with twoLTRs and includes the observation of full integration products.

AFM enables further studies in this field due to its abilities toovercome electron microscopy limitations and obtain 3D infor-mation, to detect intermediate products present in low abun-dance, and to image objects and processes directly in a solutionenvironment. This enzymatic assay may also serve to study themode of action of IN inhibitors, and develop improved com-pounds based on its mechanistic insight.

AF was supported by a starting grant from the EuropeanResearch Council under the European Community’s SeventhFramework Programme (FP7/2007-2013)/ERC Grant agreementno. 203413. AF and DP were supported by the Minerva Centerfor Bio-Hybrid complex systems.

Notes and references1 R. Craigie, J. Biol. Chem., 2001, 276, 23213.2 B. Van Maele, K. Busschots, L. Vandekerckhove, F. Christ and

Z. Debyser, Trends Biochem. Sci., 2006, 31, 98–105.3 C. M. Farnet and W. A. Haseltine, Proc. Natl. Acad. Sci. U. S. A., 1990,

87, 4164.4 A. Engelman, K. Mizuuchi and R. Craigie, Cell, 1991, 67, 1211–1221.5 J. C. H. Chen, J. Krucinski, L. J. W. Miercke, J. S. Finer-Moore,

A. H. Tang, A. D. Leavitt and R. M. Stroud, Proc. Natl. Acad. Sci. U. S. A.,2000, 97, 8233.

6 D. Esposito and R. Craigie, Adv. Virus Res., 1999, 52, 319–324, 324a,324b, 325–333.

7 K. E. Yoder and F. D. Bushman, J. Virol., 2000, 74, 11191.8 R. Craigie, Nucleic Acids Res., 1991, 19, 2729.9 Y. Hwang, D. Rhodes and F. Bushman, Nucleic Acids Res., 2000, 28,

4884–4892.10 H. Cohen, T. Sapir, N. Borovok, T. Molotsky, R. Di Felice,

A. B. Kotlyar and D. Porath, Nano Lett., 2007, 7, 981–986.11 A. Heyman, I. Medalsy, O. Bet Or, O. Dgany, M. Gottlieb, D. Porath

and O. Shoseyov, Angew. Chem., Int. Ed., 2009, 48, 9290–9294.12 F. Moreno-Herrero, L. Holtzer, D. A. Koster, S. Shuman, C. Dekker

and N. H. Dekker, Nucleic Acids Res., 2005, 33, 5945–5953.13 P. Cherepanov, D. Surratt, J. Toelen, W. Pluymers, J. Griffith, E. De

Clercq and Z. Debyser, Nucleic Acids Res., 1999, 27, 2202–2210.14 S. Kotova, M. Li, E. K. Dimitriadis and R. Craigie, J. Mol. Biol., 2010,

399, 491–500.15 T. W. Chun, L. Carruth, D. Finzi, X. Shen, J. A. DiGiuseppe, H. Taylor,

M. Hermankova, K. Chadwick, J. Margolick and T. C. Quinn, Nature,1997, 387, 183–188.

16 S. L. Butler, M. S. T. Hansen and F. D. Bushman, Nat. Med., 2001, 7,631–633.

17 E. Brin and J. Leis, J. Biol. Chem., 2002, 277, 10938–10948.18 A. Faure, C. Calmels, C. Desjobert, M. Castroviejo, A. Caumont-Sarcos,

L. Tarrago-Litvak, S. Litvak and V. Parissi, Nucleic Acids Res., 2005, 33, 977.19 G. Goodarzi, G. J. Im, K. Brackmann and D. Grandgenett, J. Virol.,

1995, 69, 6090.20 M. Llano, D. T. Saenz, A. Meehan, P. Wongthida, M. Peretz, W. H.

Walker, W. Teo and E. M. Poeschla, Science, 2006, 314, 461.21 L. Vandekerckhove, F. Christ, B. Van Maele, J. De Rijck, R. Gijsbers,

C. Van den Haute, M. Witvrouw and Z. Debyser, J. Virol., 2006, 80, 1886.22 G. Maertens, P. Cherepanov, W. Pluymers, K. Busschots, E. De Clercq,

Z. Debyser and Y. Engelborghs, J. Biol. Chem., 2003, 278, 33528–33539.23 M. Maes, A. Loyter and A. Friedler, FEBS J., 2012, 279, 2795–2809.24 F. Christ, A. Voet, A. Marchand, S. Nicolet, B. A. Desimmie,

D. Marchand, D. Bardiot, N. J. Van der Veken, B. Van Remoorteland S. V. Strelkov, Nat. Chem. Biol., 2010, 6, 442–448.

25 Z. Hayouka, A. Levin, M. Maes, E. Hadas, D. E. Shalev, D. J. Volsky,A. Loyter and A. Friedler, Biochem. Biophys. Res. Commun., 2010, 394,260–265.

26 Z. Hayouka, J. Rosenbluh, A. Levin, S. Loya, M. Lebendiker,D. Veprintsev, M. Kotler, A. Hizi, A. Loyter and A. Friedler, Proc.Natl. Acad. Sci. U. S. A., 2007, 104, 8316–8321.

27 A. Levin, H. Benyamini, Z. Hayouka, A. Friedler and A. Loyter, FEBSJ., 2011, 278, 316–330.

28 P. Cherepanov, Nucleic Acids Res., 2007, 35, 113–124.29 M. McNeely, J. Hendrix, K. Busschots, E. Boons, A. Deleersnijder,

M. Gerard, F. Christ and Z. Debyser, J. Mol. Biol., 2011, 410, 811–830.

Fig. 3 AFM imaging of integration reactions with DNA containing different combi-nations of LTRs. (A) DNA molecules containing a U5 sequence at one terminus.Products were extracted from the 1500 bp band in the gel. (B) DNA moleculescontaining a U5 sequence at one terminus. Products were extracted from the 2250 bpband. (C) DNA molecules containing a U5 sequence at both termini. Products wereextracted from the 1500 bp band. (D) DNA molecules containing a U3 sequence atone end. Products were extracted from the 1500 bp band. Close-up views areindicated by the white squares. (E) 1500 bp Y-shaped vs. linear product percentagedepending on the LTR sequence. The average percentage was calculated based on atleast 200 molecules from 3 independent experiments. The error bars are the standarddeviation. For each DNA molecule, the sum of Y-shaped and linear molecules is 100%.Each AFM image represents at least 15 different scanned areas of 2–4 mm2 each.

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