Virus Research 119 (2006) 16–28

Divergent IRES elements in invertebrates

Eric JanDepartment of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA

Available online 22 November 2005

Abstract

Viruses have evolved unique strategies and mechanisms to recruit ribosomes to ensure continued translation of their viral RNA during infection.The Dicistroviridae family of invertebrate viruses contains an unusual internal ribosome entry site (IRES), which can directly recruit ribosomesin the absence of initiation factors. Moreover, this IRES initiates translation at a non-AUG codon independent of an initiator Met-tRNA. Recentstudies have shown that the IRES mimicks a tRNA to interact with and manipulate the ribosome. The presence of this divergent IRES likelyallows translation of the dicistroviral RNA during infection when host translation is compromised. This review will explore the unique propertiesof this unprecedented mechanism of gene expression. Specific topics will examine structural components of the IRES, the mechanism of initiatingtranslation at non-AUG codons and the regulation of this IRES in vivo. The existence of this mechanism suggests that the repertoire of open readingframes in our genome may be greater than anticipated.©

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2005 Elsevier B.V. All rights reserved.

eywords: Internal ribosome entry site; Translation; Cricket paralysis virus; Ribosome

. Introduction

Viruses have evolved numerous strategies to efficiently andreferentially amplify their genome in the host cell. The virusas to evade the host antiviral response while at the same timeompete for specific host machinery required for proper pack-ging and expression of viral proteins. Translation of the viralNA is an integral part of the viral life cycle and viruses havevolved a number of ways of not only disrupting host proteinynthesis but also ensuring that the viral RNA can readily recruitibosomes. A common viral strategy is through the modifica-ion of host translation initiation factors, which prevents hostellular mRNAs from effectively recruiting ribosomes and lead-ng to increased cellular levels of free ribosomes. One of the

ost-studied examples is exemplified during poliovirus infec-ion; poliovirus encodes a protease, which cleaves the transla-ion initiation factor, eIF4G, resulting in inhibition of overallap-dependent translation (Gradi et al., 1998; Lamphear et al.,993, 1995). Conversely, poliovirus translation is unaffectedecause the 5′ untranslated region of the polioviral RNA con-ains an internal ribosome entry site (IRES) which can recruit

IRES does not require the full complement of initiation factorsto recruit the ribosome; therefore, the polioviral RNA can betranslated under conditions when certain initiation factors aremodified.

IRES elements are not only restricted to picornaviruses (i.e.poliovirus) but are also found in retroviruses and DNA virusessuch as HIV and herpes simplex viruses, respectively (for areview on IRESs, see Hellen and Sarnow, 2001). IRES ele-ments are also found in a growing list of cellular mRNAs suchas the oncogene, c-myc and the hypoxia-induced factor, VEGF.Presumably, the expression of proteins encoded by these IRES-containing mRNAs are important during cellular stress wheninitiation factors are modified and overall translation is inhibited.While there has been much work elucidating the mechanisms ofviral IRESs, the mechanism underlying cellular IRESs is forthe most part poorly understood. Considering that viral mech-anisms were most likely usurped from the host, understandinghow viral IRESs initiate translation will inevitably shed lightinto the mechanisms of cellular IRESs.

A very unusual IRES has been discovered in the invertebrateviral family, Dicistroviridae. This divergent IRES can recruit

ibosomes using the cleaved form of eIF4G and bypass theequirement for the cap binding protein, eIF4E. The polioviral

E-mail address: ejan@stanford.edu.

ribosomes in the absence of initiation factors, implying that theRNA elements within the IRES have all the properties to bind toand manipulate the ribosome. Moreover, initiation directed bythis IRES is at a non-AUG codon. The existence of this mech-anism suggests that there may be other viral and cellular RNAs

168-1702/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2005.10.011

E. Jan / Virus Research 119 (2006) 16–28 17

that can initiate translation in a manner similar to that of thisIRES. This review focuses on this novel mechanism of transla-tion initiation of these invertebrate IRESs.

2. Genomic structure of Dicistroviridae

The family Dicistroviridae (genus Cripavirus) (Mayo, 2002),also known as the Cricket paralysis virus-like viruses or picorna-like viruses, consists of 12 invertebrate members so far, includingthe insect viruses, Acute bee paralysis virus (ABPV) (Govan etal., 2000), Aphid lethal paralysis virus (ALPV) (Van Munsteret al., 2002), Black queen cell virus (BQCV) (Leat et al.,2000), Cricket paralysis virus (CrPV) (Wilson et al., 2000b),Drosophila C virus (DCV) (Johnson and Christian, 1998), Hime-tobi P virus (HiPV) (Nakashima et al., 1999), Kashmir beevirus (KBV) (de Miranda et al., 2004), Plautia stali intesti-nal virus (PSIV) (Sasaki et al., 1998), Rhopalosiphumpadi virus(RhPV) (Domier and McCoppin, 2003), Solenopsis invicta virus(SINV-1) (Valles et al., 2004), Triatoma virus (TrV) (Czibeneret al., 2000) and the non-insect invertebrate Taura syndromevirus (TSV) (Mari et al., 2002) (Table 1). Overall, the dicistro-viruses infect a range of insect cells from aphids and fire ants toDrosophila cells (Table 1). Whether each virus can infect a rangeof insect cells remains to be investigated, but it has been shownthat CrPV and DCV can infect a broad range of insect species andi1aisiMdta

n

Fig. 1. Comparison of genomic organization between Picornovirus and Dicistro-virus. The picornoviral RNA genome contains one large open reading framewhereas the dicistroviral RNA consists of two open reading frames (ORF1 andORF2). The picornoviral RNA encodes for the structural proteins (S) followedby the non-structural proteins (NS) whereas the dicistroviral ORF1 encodes forthe non-structural proteins (NS) and ORF2 encodes for the structural proteins(S). Non-structural proteins include but not limited to helicase (H), protease(P) and RNA-dependent RNA polymerase (R). Structural proteins include thecapsids (C). VpG denotes genome linked viral peptide, which is important forviral replication. Both genomes contain a poly A tail and an IRES within the 5′untranslated region. The dicistroviral RNA contains an IRES in the intergenicregion (IGR IRES).

approximately 9–10 kbp in length, contains a poly A tailand does not contain a 5′ 7-methylguanosine cap (Fig. 1).Instead, the 5′ end is bound by a viral VpG protein like othersingle-stranded RNA viruses such as picornaviruses (Kingand Moore, 1988). A number of studies have indicated that nosubgenomic RNA is produced during infection.

Originally, the dicistroviruses were considered to be insecthomologs of the mammalian picornaviruses owing to severalcharacteristics: they are RNA viruses, have similar physico-chemical properties (Scotti, 1985), produce only a few structuralproteins and are small in size (Jousset et al., 1977; Moore et al.,1980; Plus et al., 1978). Moreover, the dicistroviruses encodeenzymes similar to that of the picornaviruses such as the heli-case, the RNA-dependent RNA polymerase (RdRp) and a viral

TT

D Length of intergenic region (nt) Initiation codon of IGR IRES

A 213 GCC (ala)A 175 GCU (ala)B 211 GCU (ala)C 192 GCU (ala)D 188 GCU (ala)H 175 GCA (ala)K 213 GCU (ala)P g) 186 CAA (gin)RST ypan

Chaga

T

L e beenf on thel

n Drosophila tissue culture cells (Plus et al., 1978; Reinganum,975). Understanding the biology of these viruses is importants some of these viruses have a large impact in agriculture. Fornstance, TSV is responsible for mortalities of farmed penaeidhrimp and has had a negative impact in the shrimp farmingndustry (Lightner and Redman, 1998; Overstreet et al., 1997).

oreover, at least one of these viruses affects human health andisease: TrV infects Triatoma infestans, the insect vector forhe human pathogen Trypanosoma cruzi, which is the causativegent of Chagas disease (Ronderos and Schnack, 1987).

The dicistroviruses are positive-sense, single-stranded,on-enveloped RNA viruses (Fig. 1). The viral genome is

able 1he Dicistroviridae family and properties of the IGRs

icistrovirus Host

cute bee paralysis virus (ABPV) Honeybee, bumblebeesphid lethal paralysis virus (ALPV) Aphidslack queen cell virus (BQCV) Beesricket paralysis virus (CrPV) Wide range of insect cellsrosophila C virus (DCV) Wide range of insect cellsimetobi P virus (HiPV) Laodelphax surauellus (plant hopper)ashmir bee virus (KBV) Honeybeeslautia stali intestine virus (PSIV) Plautia stali (brown-winged green buhopalosiphum padi virus (RhPV) Aphidsolenopsis invicta virus (SINV-1) Solenopsis invicta (fire ants)riatoma virus (TrV) Triautma infestans (insect vector of Tr

cruzi, which is the causative agent ofdisease in humans)

aura syndrome virus (TSV) Penaeid shrimp

ist of dicistroviruses discovered so far with their host and the systems that havrom the stop codon of ORF1 to the predicted initiation codon of ORF2, basedisted in Fig. 2.

533 GCA (ala)205 GCU (ala)

osomas

209 GCU (ala)

209 GCU (ala)

tested to support viral infection. The length of the intergenic region is definedIGR structure predictions in Fig. 2. The accession numbers for each virus are

18 E. Jan / Virus Research 119 (2006) 16–28

3C-like protease (Fig. 1). Finally, electron micrographs of DCVindicate that these viruses are similar in size and shape to picor-naviruses (Moore and Tinsley, 1982). It was proposed that theCricket paralysis virus-like viruses were picorna-like viruses(Christian et al., 2000).

Sequencing of the RNA genomes of the dicistroviruses revealtwo main differences in genomic organization from the picor-naviruses (Fig. 1). First, whereas the picornaviral genome con-tains a single open reading frame (ORF) coding for one largepolypeptide, the dicistroviruses contain two ORFs (Fig. 1).The 5′ ORF (ORF1) encodes the viral non-structural proteinsincluding the helicase, protease and the RdRp. The 3′ ORF(ORF2) encodes the viral structural proteins. Like the picor-naviruses, the ORFs are translated as a long polyprotein, whichis then proteolytically cleaved by the viral protease. Second,the linear organization of the structural and non-structural pro-teins is different between the picornaviruses and dicistroviruses(Fig. 1). Within the large ORF of picornaviruses, the structuralproteins are translated first, followed by the non-structural pro-teins, resulting in an equimolar expression of the non-structuraland structural proteins. In contrast, the Dicistroviridae ORF1encodes the non-structural proteins followed by the structuralproteins encoded by ORF2. During CrPV and DCV infection,the expression of the structural proteins is in molar excess overthat of the non-structural proteins, indicating that the expressionof the two ORFs is regulated by different mechanisms (Mooree

w15cRIItWwrwmDemiorect14cparp

Separating the two ORFs is the intergenic region (IGR),which ranges from 175 to 533 nucleotides (Table 1). It has beenshown that the IGRs of PSIV and CrPV contain an IRES that caninitiate translation at a non-AUG codon and does not require theinitiator Met-tRNAi. Moreover, this IRES appears to be diver-gent from all other IRESs and initiates translation via a novelmechanism. The rest of the review focuses on the mechanism ofthese divergent IGR IRES.

3. Structure of the IGR IRES

The first indication that translation of ORF2 was by anunusual mechanism came from N-terminal sequencing of thestructural proteins, which revealed that the N-terminal aminoacid of ORF2 was not methionine. For example, the N-terminalamino acid of DCV ORF2 polyprotein was alanine encoded by aGCU codon (Johnson and Christian, 1998). Furthermore, usingPSIV as a model, Nakashima and co-workers found that theIGR contained an IRES and that the first amino acid of the PSIVORF2 was glutamine encoded by a CAA, not an AUG codon(Sasaki and Nakashima, 1999). The authors elegantly showedthat methionyl-peptidases did not remove a putative amino-terminal methionine, confirming that the first amino acid of theORF2 was indeed glutamine (Sasaki and Nakashima, 2000).

Similarly, work on the CrPV IGR showed that it also con-tained an IRES and the first amino acid was an alanine encodedbatettoSeti(

bitWtdm2tiairaFcpw

t al., 1980, 1981; Wilson et al., 2000b).Indeed, it was discovered that translation of the two ORFs

as dictated by two distinct IRESs (Sasaki and Nakashima,999; Wilson et al., 2000b). One of the IRESs is within the′UTR and initiates translation of ORF1 at the predicted AUGodon (Royall et al., 2004; Woolaway et al., 2001). Using thehPV 5′UTR IRES as a model, deletion analysis of the 5′UTR

RES demonstrated there were no defined boundaries of theRES, which was similar to what was observed with many ofhe cellular IRESs (Huez et al., 1998; Stoneley et al., 1998;

oolaway et al., 2001). This suggests that multiple domainsithin the IRES can recruit the ribosome through different or

edundant pathways and/or factors. Using reporter constructs, itas shown that the RhPV 5′UTR IRES is functional in mam-alian, plant and insect cells and in in vitro systems such asrosophila, rabbit reticulocyte and wheat germ extracts (Royall

t al., 2004; Woolaway et al., 2001). This is unusual becauseost IRESs are only functional in their respective systems. For

nstance, the EMCV IRES and many of the cellular IRESs arenly functional in mammalian systems and are functional inabbit reticulocyte lysates only when supplemented with HeLaxtracts (Hellen and Sarnow, 2001). Given that the RhPV IRESan initiate translation in a number of systems, it suggests thathe mechanism of the 5′UTR IRES may be conserved. Of the2 family members, 11 of them have long 5′UTRs ranging from17 to 964 nt, which is a characteristic that is not conducive foranonical scanning mode of translation. Given this property, it isrobable, like the RhPV 5′UTR, that the other family memberslso have an IRES in their 5′UTR. Further studies of the factorequirement for this 5′UTR IRES will shed light into its unusualroperties.

y a GCU codon (Wilson et al., 2000b). Mutagenesis approachesnd N-terminal sequencing of the ORF2 polyprotein confirmedhat alanine was the first amino acid and not methionine (Wilsont al., 2000b). Moreover, to further confirm that an initiator Met-RNAi was not incorporated, IGR IRES translation was tested inhe presence of the drug, edeine. Edeine inhibits the recognitionf the AUG start codon by scanning 40S subunits (Kozak andhatkin, 1978). CrPV IGR IRES translation was insensitive todeine, suggesting that IGR IRES translation does not requirehe recognition of an AUG codon for initiation, further support-ng the idea that the IRES does not initiate with a methionineWilson et al., 2000a).

Mutagenesis analysis examined the exact initiation site andegan to dissect the secondary structure of the IRES. These stud-es confirmed that the triplet preceding the initiation codon ofhe IGR IRES was not decoded (Sasaki and Nakashima, 2000;

ilson et al., 2000a,b). For example, in CrPV IGR studies,he CCU triplet that precedes the GCU alanine codon was notecoded but was instead important in basepairing with an ele-ent upstream of the CCU triplet (Fig. 2A and B) (Wilson et al.,

000a,b). This basepairing formed a pseudoknot-like structure,ermed Pseudoknot I or PKI (Fig. 2A and B). A conservative def-nition of a pseudoknot is any basepairing within a stem loop tonother region of the RNA. Disruption of the basepairing inhib-ted IRES activity and making the compensatory mutations thatestored the basepairing and pseudoknot structure rescued IRESctivity (Kanamori and Nakashima, 2001; Wilson et al., 2000b).or added proof, the CCU triplet was mutated to a UAG stopodon rendering this triplet unable to be decoded. If the com-ensatory mutations were introduced to restore the basepairingithin the pseudoknot, IRES activity was restored with the UAG

E. Jan / Virus Research 119 (2006) 16–28 19

Fig. 2. Comparison of IGRs of Discistroviridae family (A) Alignment of IGRs. The IGRs are grouped into two classes I and II. Some of the predicted basepairingwere adapted from (Nishiyama et al., 2003). Predicted basepairing and inverted repeat complementary sequences in stem loops are highlighted by arrows and arecolor-coded (purple, dark green, light blue and yellow). Basepairing that form pseudoknots, PKI, PKII and PKIII, are bracketed and color-coded in blue, red andgreen, respectively. Conserved nucleotides are in bold and boxed. The accession numbers for each virus are: PSIV: AB006531, HiPV: AB017037, DCV: AF014388,CrPV: AF218039, TrV: AF178440, BQCV: AF183905, RhPV: AF022937, ALPV: AF536531, SINV-1: AY634314, KBV: AY275710, TSV: AF178440, ABPV:AF150629. Nucleotide positions in viral genomes are shown at left. Underlined triplets indicate the stop codon for ORF2. (B) Diagram of representative classesI and II IGR IRES structures, CrPV IGR IRES and SINV-1 IGR IRES structures, respectively. Predicted basepairing within stem loops are color-coded using thelegend in Fig. 2A. Also, pseudoknot basepairing are shown in blue (PKI), red (PKII) and green (PKIII). Bold lettering indicates conserved nucleotides at least withineach class of IGR IRESs. The initiation alanine codon, which codes for the first amino acid of ORF2 for CrPV and SINV-1 IGR IRESs, is adjacent to PKI. P and Arepresent the triplet that is in the ribosomal P and A site when 40S and 80S ribosomes are assembled on the IGR IRES. Shown are nucleotides 6026–6222 of theCrPV IGR and nucleotides 4220–4404 of the SINV-1 IGR, based on their viral genomic positions.

20 E. Jan / Virus Research 119 (2006) 16–28

stop codon in place of the CCU triplet (Wilson et al., 2000b).These experiments further demonstrated that translation of theCrPV IGR IRES initiated at the GCU alanine codon. Moreover,this indicated that the pseudoknot structure was important forIRES activity.

Using this type of mutagenesis and compensatory mutagen-esis approach, a model of the IGR IRES was proposed (Fig. 2Aand B) (Kanamori and Nakashima, 2001). The IGR IRESs forCrPV and PSIV were later confirmed by structural probing usingchemical and enzymatic approaches (Jan and Sarnow, 2002;Nishiyama et al., 2003). The IGR IRES formed a complexoverlapping triple pseudoknot RNA structure (PKI, PKII andPKIII), with all of the pseudoknots being important for IRESactivity (Fig. 2A and B) (Jan and Sarnow, 2002; Kanamori andNakashima, 2001). The IGR was unusual in that it lacked thehigh GC content that is normally observed with complex RNAstructures. For most of the IGR IRESs, the entire IGR is devotedto the predicted stem loops and pseudoknot structures of theIRES, which range from 180 to 210 nt in length (Table 1). How-ever, for some such as HiPV, TrV and ALPV, one of the predictedstem loops overlaps with ORF1, raising the question of howthese IGR IRESs fold correctly when ribosomes are translat-ing through ORF1. Also of interest is the RhPV IGR, which ismuch longer (533 nt) than the predicted IGR IRES structure. Ithas been shown that the full length RhPV IGR contains IRESactivity (Table 1) (Domier and McCoppin, 2003; Woolaway etamd

tiPsDtftbpa(ntJswai

IICIcIi

Sarnow, 2005; Hatakeyama et al., 2004). A second differencebetween the two classes of IRES is that there are distinct Bulgeregion consensus sequences for each IGR IRES class. This isin contrast with the conserved nucleotides within SL1 and SL2that are conserved between classes. A third difference is theconsensus sequence downstream of SL1, UUAC, which is onlypresent in class I IRESs. Finally, it appears that the invertedrepeats that form SL2 are shorter in class II compared to class I.The significance of these differences between the two classes ofIGR IRESs is not known. It will be interesting to explore whetherswapping domains between the two classes of IGR IRESs hasan effect on IRES activity and/or viral yield.

Although there are differences between the two classes ofIGR IRESs, all of the IRES structures are predicted to initiateat non-AUG codons (Cevallos and Sarnow, 2005; Hatakeyamaet al., 2004). All but one IGR IRES are predicted to initiatetranslation at an alanine codon (Table 1). An interesting questionthat arises from this comparison is whether the alanine codon isessential for IGR IRES-mediated translation? To address this,a comprehensive analysis was performed where the initiationcodon of the PSIV IGR IRES was replaced with codons for theother 19 amino acids (Shibuya et al., 2003). All mutant IRESshad some translation activity, indicating that the initiation codonwas not absolutely essential for IGR IRES-mediated translation.Interestingly, while certain amino acids had no effect on IRESactivity, some had an inhibitory effect. The reason for this isucupw

4

rsiaf4aeteAmGb2

torahb

l., 2001). It remains to be investigated whether other RNA ele-ents within the RhPV IGR may affect IRES activity, perhaps

uring the course of infection.Comparing the IGRs of the Dicistroviridae family revealed

hat they are predicted to form a similar RNA structure, form-ng the three overlapping pseudoknot structures, PKI, PKII andKIII, stem loops, SL1 and SL2, and a Bulge region. Fig. 2Ahows an alignment of the IGRs of the 12 members of theicistroviridae family. Although the overall RNA structure of

he IGR IRESs was conserved, it was surprising to find that veryew nucleotides were conserved. This suggested that it is nothe primary sequence of the IGR that dictated IRES function,ut the three-dimensional shape of the predicted stem loop andseudoknot structures. The few nucleotides that are conservedre mostly located in loop regions located within stem loop 1SL1) and 2 (SL2) (Fig. 2A and B). Mutations of these conserveducleotides inhibited IRES activity and in some cases, appearedo inhibit binding to 40S subunits (Costantino and Kieft, 2005;an and Sarnow, 2002; Nishiyama et al., 2003). Given the con-ervation of these loop nucleotides and the fact that nucleotidesithin loop regions are often important for RNA–protein inter-

ctions, it is most likely that these conserved loop regions aremportant for interactions with the ribosome.

A closer inspection of the IGRs revealed that the predictedGR IRES structures can be grouped into two classes, I andI (Fig. 2A and B). Class I IGR IRESs are exemplified by therPV IGR IRES and SINV-1 IGR IRES is a prototypical class

I IGR IRES (Fig. 2B). One main difference between the twolasses is an extra stem loop (SL3) within PKI of class II IGRRES (Fig. 2 A). It has been shown using the TSV IGR that thentegrity of SL3 was important for IRES activity (Cevallos and

nknown. It is possible tertiary interactions exist between theodon and the IGR IRES that have so far not been revealedsing current structural probing techniques. Alternatively, it isossible that the incoming aminoacylated tRNA may interactith the IRES and affect IRES function.

. Mechanism of the IGR IRES

Normally, a number of canonical initiation factors areequired to recruit 40S subunits to an mRNA, whereby the 40Subunit then scans the 5′UTR until an appropriate AUG codons located (Hershey and Merrick, 2000). In contrast, IRESs suchs EMCV and HCV IRESs only require a subset of initiationactors to recruit the 40S subunit. To recruit and position the0S subunit at the initiation codon, the EMCV IRES requiresll canonical initiation factors except the cap binding protein,IF4E (Pestova et al., 1996). The HCV IRES can bind directlyo 40S subunits and only requires eIF3 and the ternary complex,IF2/GTP/Met-tRNAi, to properly position the ribosome at theUG start codon (Pestova et al., 1998). Following 40S recruit-ent, a number of initiation factors such as eIF5 and eIF5B andTP hydrolysis are required for 60S subunits to join and assem-le 80S ribosomes (Hershey and Merrick, 2000; Pestova et al.,000).

The IGR IRES mediates translation initiation by a mechanismhat is very distinct from canonical translation initiation andther IRES-mediated translation initiation. Remarkably, 80Sibosomes can assemble on the IGR IRES using purified 40Snd 60S subunits in the absence of initiation factors and GTPydrolysis, all of which indicate a novel pathway of 80S assem-ly (Wilson et al., 2000a). Two pathways can be envisioned for

E. Jan / Virus Research 119 (2006) 16–28 21

80S assembly on the IGR IRES. In one pathway, 40S subunitsbind the IRES followed by 60S joining. In the second pathway,preformed 80S ribosomes bind directly to the IGR IRES. Sup-porting the first scenario, it has been found that 40S subunits canbind to the IGR IRES in the absence of initiation factors (Wilsonet al., 2000a). Binding constants range between 2 and 20 nM,indicating a tight specific interaction between the IGR IRES

and the 40S subunit and is similar to the range to that of HCVIRES/40S subunit binding (Costantino and Kieft, 2005; Jan andSarnow, 2002; Kieft et al., 2001; Otto et al., 2002). Moreover,it has also been shown that 60S subunits can join to prebound40S/IGR IRES complexes, which suggests that 80S assembleon the IGR IRES by first binding to 40S subunit followed by60S joining (Jan et al., 2003). However, the data do not formally

FIata

ig. 3. Model of CrPV IGR IRES-mediated translation. 80S ribosomes can assembleRES followed by 60S joining in the absence of initiation factors. (B) Alternatively, pre assembled on the IGR IRES, the CCU triplet occupies the ribosomal P site and thehe A site. (D) Delivery of Ala-tRNA to the ribosomal A site is mediated by elongatind GDP, EF2 and GTP hydrolysis then catalyzes the translocation of the ala-tRNA f

on the IGR IRES via two pathways: (A) 40S subunits can bind directly to IGRreformed 80S ribosomes can bind to the IGR IRES. (C) When 80S ribosomesGCU alanine codon, which encodes for the first amino acid of ORF2, occupies

on factor, EF1A and GTP. (E) Following GTP hydrolysis and release of EF1Arom the A site to the P site.

22 E. Jan / Virus Research 119 (2006) 16–28

exclude the possibility that preformed 80s ribosomes can stillbind the IRES. To address this possibility, Pestova and Hellenhave provided evidence that preassembled 80S ribosomes canbind to the IRES (Pestova et al., 2004). In these experiments,80S ribosomes can bind to the IGR IRES in the presence ofeIFl, eIFlA and eIF3, which added together, prevent 40S and60S joining if the 80S ribosomes had dissociated (Pestova etal., 2004). Thus, it appears that 80S ribosomes can assemble onthe IGR IRES by both pathways (Fig. 3). These studies raisenew and exciting questions. How does the IGR IRES recruitribosomes in vivo during viral infection? Also, how does theIGR IRES compete for 40S and 60S subunits that may be boundto initiation factors that prevent 80S assembly? Related to this,what is the status of initiation factors during infection? A furtherunderstanding of this novel pathway of 80S assembly will pro-vide insight into how viral IRESs compete for ribosomes duringinfection.

To better understand how the IGR IRES can bind directlyto 40S subunits, Kieft and co-workers used hydroxyl radicalprobing to examine the structure of the IGR IRES in its freeversus 40S bound state. They showed that the IGR IRES foldsinto a tightly packed structure that excludes solvent and thatthis preformed compact architecture is necessary to bind 40Ssubunits (Costantino and Kieft, 2005). This is in agreement withfootprinting studies showing that the IGR IRES structure doesnot change significantly upon binding to 40S and 80S ribosomes(HideI21IRsatR

dttttttiswoFiTwn

given that the A is +1 (Pestova et al., 1998). However, if a 40Sis bound to an RNA with no aminoacylated tRNA in the P-site,a toeprint will occur 13–14 nucleotides away (Pestova et al.,1998).

Using purified 40S and 60S subunits, 40S and 80S ribosomeassembly on the CrPV IGR IRES produced a toeprint 13–14nucleotides from the first C of the CCU proline triplet (Jan etal., 2003; Pestova and Hellen, 2003; Wilson et al., 2000a). Thus,the CCU triplet is situated in the P site of the ribosome and thedownstream GCU alanine triplet, which codes for the first aminoacid, is in the A site of the ribosome (Fig. 2B). This indicatedthat translation initiated by the IGR IRES from the A site ofthe ribosome (Fig. 3). Moreover, the data suggested that theIGR IRES binds to and positions the ribosome directly at theinitiation site. At this point, the working model was that the IGRIRES forms an RNA structure that occupies the P site of theribosome to initiate translation from the A site of the ribosome(Wilson et al., 2000a).

Because the IRES initiates translation from the A site of theribosome, the IRES undergoes an unusual first translocation stepwhereby the ribosome has to translocate without peptide bondformation (Wilson et al., 2000a). Precedence for this type oftranslocation has been observed in prokaryotic systems: translo-cation only requires a tRNA in the P site and a truncated tRNAconsisting of the anticodon stem loop in the A site (Joseph andNoller, 1998). This suggests that peptide formation is not anaaam

fIeeseoso

rt2sga2escta8a2t

Jan and Sarnow, 2002; Nishiyama et al., 2003). In contrast, theCV IRES adopts an extended RNA structure, which results

n regions of local compact structures and leading to a moreynamic and conformationally flexible free RNA state (Kieftt al., 1999). Once bound to 40S subunits and eIF3, the HCVRES remains in an extended architecture (Kieft et al., 1999,001; Kolupaeva et al., 2000; Pestova et al., 1998; Sizova et al.,998; Spahn et al., 2001b). Thus, unlike the IGR IRES, the HCVRES uses a strategy to be conformationally flexible in the freeNA state to allow local compact RNA structures to form and

ubsequently bind distinct regions of the 40S subunit and eIF3 inn extended architecture (Costantino and Kieft, 2005). Overall,hese studies reveal how two different viral IRESs use distinctNA folding strategies to recruit the ribosome.

Because the IGR IRES can bind to 40S and 80S ribosomesirectly, it suggests that the IGR IRES contains specific elementshat interact with and manipulate the ribosome. To begin to bet-er understand and dissect the IGR elements that are requiredo recruit the ribosome, toeprinting analyses were undertakeno address where the ribosome binds on the IGR IRES. In thisechnique, a DNA oligonucleotide is annealed to an RNA 3′ tohe area of interest, reverse transcriptase is added and cDNAs synthesized. If the reverse transcriptase encounters a highlytructured RNA element or a protein bound to the RNA, itill lead to an arrested cDNA product, or toeprint. The sizef this cDNA product can be determined on a sequencing gel.rom the location of the toeprint, we can count upstream and

nfer the triplets that are in the P and A site of the ribosome.his is possible because it has been determined that a ribosomeith a Met-tRNAi in the P site will produce a toeprint 15–16ucleotides downstream from the A of the AUG in the P site,

bsolute prerequisite for translocation and all that is requiredre specific tRNA interactions with the ribosome. These inter-ctions may be mimicked by the IGR IRES, thus allowing it toanipulate the ribosome to promote translocation.An interesting question raised by this mechanism is what

actors are required for the first translocation step on the IGRRES? Normally, during elongation, two factors are required:longation factors eEFlA and eEF2 (Merrick and Nyborg, 2000).EF1A/GTP delivers the cognate aminoacylated-tRNA to the Aite of the ribosome. Following GTP hydrolysis and release ofEF1A/GDP, peptidyl tranfer from the P site to the A site tRNAccurs via the intrinsic peptidyl transferase activity of the ribo-ome. eEF2 and GTP hydrolysis then catalyzes the translocationf the peptidyl-tRNA from the A site to the P site.

Using toeprinting analysis to monitor the movement of aibosome bound to the IGR IRES, it was shown that the firstranslocation step required both elongation factors (Jan et al.,003; Pestova and Hellen, 2003) (Fig. 3). Furthermore, it washown that translocation can be monitored for several elon-ation cycles by adding the subsequent aminoacylated tRNAsnd that translocation occurred with high stringency (Jan et al.,003; Pestova and Hellen, 2003). Finally, it was shown by twoxperiments that peptide bond formation occurred in this recon-tituted system. In one experiment, Met-puromycin peptidesould be synthesized driven by the CrPV IGR IRES providedhat the GCU codon was replaced with the AUG codon (Pestovand Hellen, 2003). In another experiment, incubating prebound0S/IGR IRES complexes with purified elongation factors andminoacylated tRNAs resulted in peptide production (Jan et al.,003). It appears that all that is required for IGR IRES-mediatedranslation are purified 40S and 60S subunits, eEF2, eEF1A

E. Jan / Virus Research 119 (2006) 16–28 23

and aminoacylated-tRNAs. Development of this reconstitutedsystem allows for studies of not only IGR IRES-mediated trans-lation initiation but also of events downstream of initiation suchas the regulation of elongation and termination.

A summary model for CrPV IGR IRES-mediated translationis depicted in Fig. 3. The IGR IRES can bind to 40S subunitsfollowed by 60S joining. Alternatively, the preformed 80S ribo-somes can bind directly to the IGR IRES. Here, the IGR IRESoccupies the P site of the ribosome and positions the ribosomesuch that GCU alanine codon is in the A site. Following deliveryof an Ala-tRNA to the ribosomal A site by eEF1A/GTP, eEF2and GTP hydrolysis catalyzes the translocation of the Ala-tRNAfrom the A site to the P site. At this point, the IGR IRES hasset the ribosome into elongation mode and elongation proceedsnormally.

5. Ribosome interactions with the IGR IRES

Because the IGR IRES can bind directly to 40S subunits andassemble 80S ribosomes, a simple hypothesis is that the RNAelements within the IGR IRES are interacting with specific com-ponents of the ribosome to manipulate its activity. Given therelatively small size of the IRES (−180 nt) and the compact-ness of the IRES, it is likely that every part of the IRES playsan important role in binding and manipulating the ribosome.Several mutations were generated within the IGR IRES andIaesrIotssI2wwai(miaftt

PteehaI

mapped the position of the IGR IRES relative to the position ofeIF1 bound to the 40S (Pestova et al., 2004). These experimentsshowed that the CCU triplet, as predicted, is located in the Psite of the ribosome and that the regions of PKII and PKIII aresolvent exposed and are likely in the E site of the ribosome.These experiments are all in agreement with the hypothesis thatthe IRES occupies the mRNA cleft to initiate translation fromthe A site of the ribosome.

The interactions of the IGR IRES and the ribosome weremore closely examined by cryo-electron microscopy (cryo-EM)reconstructions. IGR IRES/40S and IGR IRES/80S complexeswere resolved at 20 and 17.5 A resolution, respectively (Fig. 4)(Spahn et al., 2004b). The IGR IRES occupies the E, P andpartially the A site of the ribosome, which are in the mRNA cleftnormally occupied by the E, P and A site tRNAs. Moreover, itappears that the IGR IRES interacts with helixes 18 and 30 ofthe small rRNA and ribosomal protein S5, regions where tRNAsnormally interact with the ribosome (Cate et al., 1999; Culveret al., 1999). On the 60S side, the IGR IRES also interacts withregions that normally interact with the tRNAs, most notably withribosomal protein LI and the central protuberance area, regionswhere the P and E site tRNAs normally interact with the 60Ssubunit (Fig. 5B). Thus, the IGR IRES is mimicking tRNAs tointeract with and manipulate the ribosome.

From this work, it appears that the IGR IRES interacts withthe conserved core of the ribosome, the mRNA channel (Fig. 4AaIcsabon

r(abrtmtofo(cr

smcrtsf

RES activity was assessed (Costantino and Kieft, 2005; Jannd Sarnow, 2002; Kanamori and Nakashima, 2001; Nishiyamat al., 2003). Some of the mutations disrupted the pseudoknottructures and some altered the nucleotides within loop or Bulgeegions. Overall, all mutations resulted in a loss of IRES activity.t is possible that the mutations may be inhibiting recruitmentf the 40S subunit or assembly of 80S ribosome, positioning ofhe ribosome on the IGR IRES or inhibition of an event down-tream such as translocation. It was found that mutations ofpecific elements within the IRES inhibited distinct aspects ofRES function (Costantino and Kieft, 2005; Jan and Sarnow,002; Nishiyama et al., 2003). Pseudoknots, PKII and PKIII,ere primarily responsible for binding to the ribosome and PKIas important for positioning the ribosome such that the GCU

lanine codon is in the A site (Fig. 2B). Moreover, mutationsn the loop region of SL1 appeared to inhibit ribosome bindingCostantino and Kieft, 2005). Overall, the fact that these ele-ents are acting independently and that the IRES can directly

nteract with the ribosome suggest that these elements are inter-cting with the ribosome to affect distinct aspects of ribosomeunction. Understanding how these elements are interacting withhe ribosome will shed light into how the viral IRES manipulateshe ribosome.

To determine how the IRES is interacting with the ribosome,estova and Hellen used site directed hydroxyl radical probing

o determine the position of the IRES on the ribosome (Pestovat al., 2004). Previously, the authors determined the position ofIF1 on the 40S subunit by directed cleavage of the 18S rRNA byydroxyl radicals generated on the surface of eIF1 (Lomakin etl., 2003). Using this technique, and given the fact that the IGRRES and eIF1 can bind simultaneously to 40S subunits, they

nd F) (Spahn et al., 2004b). This may explain why the IGRRES is functional in a number of eukaryotic systems. This is inontrast to the HCV IRES, which can also bind directly to 40Subunits, but is only functional in mammalian systems (Otto etl., 2002; Pestova et al., 1998). This is because the HCV IRESinds mostly on the variant solvent side of the 40S subunit andnly part of the IRES, domain II, reaches into the mRNA cleftear the E site of the ribosome (Spahn et al., 2001b).

The IGR IRES also induces conformational changes in theibosome upon binding to 40S subunits and upon 80S assemblySpahn et al., 2004b). Upon binding to the IGR IRES, thereppear to be connections induced on the solvent side of the 40Setween the head and the body (between helixes 34 and 18 ofRNA) near the entrance of the mRNA (Fig. 4B). It was proposedhat these “latch” interactions may help thread the incoming

RNA into the mRNA cleft (Spahn et al., 2001b). Interestingly,his latch interaction is also observed in cryo-EM reconstructionsf HCV IRES/40S complexes (Spahn et al., 2001b). Despite theact that the IGR IRES and HCV IRES bind to distinct regionsf the 40S, they both induce similar conformational changesSpahn et al., 2004b). This suggests that these conformationalhanges are intrinsic to ribosome function and that they mayepresent changes that occur normally during translation.

When 80S ribosomes are assembled on the IGR IRES, thetalk region of the 60S subunit, which consists of the riboso-al P proteins, becomes extended and can now be resolved by

ryo-EM (Fig. 5B) (Spahn et al., 2004b). Normally, the stalkegion is flexible and is not resolved by cryo-EM reconstruc-ions (Agrawal et al., 1999; Gomez-Lorenzo et al., 2000). Thetalk region of the ribosome has been shown to be importantor recruitment and stimulation of GTPase activation on elonga-

24 E. Jan / Virus Research 119 (2006) 16–28

Fig. 4. Cryo-EM maps of vacant HeLa 40S (C and D) and 80S (E) and 40S/CrPV IGR IRES (A and B) and 80S/CrPV IGR IRES complexes (F). View of 40S shownfrom intersubunit face (A and C) and from the solvent side (B and D). (A–D) The 40S subunit is painted yellow and the density corresponding to the IGR IRES inpink. Landmarks for the 40S subunit are the following: b, body; bk, beak; h, head; pt, platform; and sh, shoulder. The entry and exit channels for the mRNA (Frank etal., 2000; Yusupova et al., 2001) are designated “entry” and “exit.” The position of five 18S rRNA helices (hl6, hl8, h30 and h34) and of protein rpS5 are indicated, asidentified by comparison with a cryo-EM map of the yeast 80S ribosome (Spahn et al., 2001a). The IGR IRES occupies the E, P and A sites of the ribosome (orangearrows). Upon binding to the IGR IRES, the 40S undergoes conformational changes: view from the inter-subunit face, the head rotates relative to the body givinga different angle of the beak and the view from the solvent side, connections or “latch” interactions are produced near the entry of the mRNA channel. (E and F)Vacant 80S and 80S/CrPV IGR IRES complexes. The ribosomal 40S subunit is painted yellow, the 60S subunit blue and the CrPV IRES pink. View of 80S ribosomefrom the side showing the entrance of the mRNA channel. Landmarks for the 60S subunit are the following: CP, central protuberance; L1, L1 protuberance; SB, stalkbase; and SRL, sarcin–ricin loop. Fig. 4(A–D) are courtesy from Christian M.T. Spahn. Fig. 4E and F are “reprinted from Spahn et al. (2004b), with permission fromElsevier.”.

tion factors (Diaconu et al., 2005). Interestingly, the stalk is alsoresolved when yeast 80S ribosomes bind to elongation factor,EF2, and when E. coli 70S ribosomes bind to EF-G (Fig. 5Dand E) (Agrawal et al., 1999; Gomez-Lorenzo et al., 2000).

Thus, it appears that the IGR IRES is inducing the same confor-mational change as an elongation factor. The IGR IRES maybe priming the ribosome for the next step in translation bysetting the ribosome in elongation mode, possibly be facilitat-

E. Jan / Virus Research 119 (2006) 16–28 25

Fig. 5. Cryo-EM reconstructions of 60S subunits from (A) empty 80S ribosomes, (B) 80S/CrPV IGR IRES complexes, (C) empty yeast 80S ribosomes, (D) EF2bound yeast 80S (adapted from Gomez-Lorenzo et al., 2000) and 50S subunits from (E) EF-G bound E. coli 70S (adapted from Agrawal et al., 1999). Views of largesubunit (blue) bound to CrPV IGR IRES (pink) or elongation factor (red). Note the extended stalk (St), shown by arrows, present in reconstructions of 80S/CrPV IGRIRES complexes, EF2 bound yeast 80S and EF-G bound E. coli 70S but not empty 80S or 70S ribosomes. Fig. 5A and B are “reprinted from Spahn et al. (2004b),with permission from Elsevier.” Fig. 5C–E are “reprinted from Gomez-Lorenzo et al. (2000), with permission from EMBO J.”.

ing the delivery of the next aminoacylated tRNA or elongationfactor.

Given that IGR IRES is interacting with the ribosome bymimicking tRNA binding in the mRNA channel, future stud-ies of understanding how elements within the IGR IRES induceribosomal conformational changes will not only shed light intohow a minimal RNA element is manipulating the ribosomebut will also provide insight into how tRNAs normally interactwith the ribosome. Furthermore, it has become more appreci-ated that tRNAs may have an active role in interacting with theribosome and mediating translation. For instance, it has beenshown that mutations in the ribose backbone in the acceptorstem of the tRNA that normally occupies the ribosomal E site,can inhibit translocation (Feinberg and Joseph, 2001; Joseph,2003). Because the IGR IRES is also interacting with the E site,it is most likely that elements within the IRES are mimicking thetRNA–ribosomal E site interaction to mediate the translocationevent. Further studies may elucidate whether these interactionsare important for aminoacylated tRNA delivery to the A siteand/or mediate the translocation event by inducing conforma-tional changes (e.g. extension of the stalk) on the ribosome.

6. Regulation of IGR IRES-mediated translation

Given that the IGR IRES can assemble 80S ribosomes inde-pendent of initiation factors, it is predicted that the IGR IRES isf

initiation factors are limiting. For the most part, the regulationof translational efficiency of an mRNA is controlled at the levelof initiation through the modification or cleavage of initiationfactors (Dever, 2002; Mathews et al., 2000). For instance, sev-eral initiation factors are phosphorylated or sequestered leadingto inhibition of 40S recruitment or recognition of the AUGcodon. From the in vitro data described above, it can be pos-tulated that the IGR IRES does not require the ternary com-plex eIF2/GTP/Met-tRNAi, which is normally required to bringthe Met-tRNAi to the 40S subunit (Dever, 2002; Hershey andMerrick, 2000; Hinnebusch, 2000). To address this, IGR IRESactivity was tested under cellular stresses that induce eIF2�phosphorylation, which leads to an overall decrease in the poolof the ternary complex (Hinnebusch, 2000).

eIF2� is one of three subunits of eIF2 and is a major target oftranslational control (for review, see Hinnebusch, 2000). Dur-ing 40S scanning and recognition of the AUG start codon, GTPon eIF2 is hydrolyzed and eIF2/GDP is released from the 40Ssubunit. eIF2/GDP is converted to eIF2/GTP via the guanidinenucleotide exchange factor, eIF2B, allowing eIF2 to once againbind initiator Met-tRNA. Under a number of cellular stresses aswell as viral infection, eIF2� kinases are activated leading tophosphorylation of eIF2�. This phosphorylated form of eIF2�now can bind tightly to eIF2B, becoming a competitive inhibitorand sequesters eIF2B. Since the overall levels of eIF2� is inmolar excess over eIF2B, small increases in the phosphorylationo

unctional under a number of cellular stresses when functional f eIF2� will lead to a significant inhibition of eIF2B activity

26 E. Jan / Virus Research 119 (2006) 16–28

(Oldfield et al., 1994). Consequently, eIF2 can no longer be recy-cled and bind another initiator Met-tRNAi, leading to a decreasein overall translation.

It was shown that IGR IRES-mediated translation is activeduring ER stress and amino acid starvation when eIF2� is phos-phorylated (Fernandez et al., 2002; Wilson et al., 2000a). ERstress induced by the unfolded protein response leads to theactivation of the eIF2� kinase, PERK and a rapid inhibitionof translation (Schroder and Kaufman, 2005). Similarly, aminoacid starvation activates the eIF2� kinase, GCN2, also lead-ing to an inhibition of translation (Hinnebusch, 2000). Duringthese stresses, the inhibition of protein synthesis resulted in anincreased pool of free 40S and 60S subunits for which the IGRIRES can now efficiently compete. In agreement with this, itwas shown that in yeast, IGR IRES-mediated translation is onlyactive when the levels of eIF2 or Met-tRNAi is lowered, presum-ably due to an increase in the pool of free ribosomes (Thompsonet al., 2001). It remains to be seen how the IGR IRES can specif-ically compete for the free 40S or 80S ribosomes in the cell.Also, it will be interesting to see whether the IGR IRES is activeduring other cellular stresses that induce eIF2� phosphoryla-tion (e.g. hypoxia) or during cellular stresses that modify otherinitiation factors such as rapamycin treatment, which leads tosequestration of the cap binding protein, eIF4E. Understandingthe conditions under which IGR IRES-mediated translation isactive will lead to a better idea of how and when the IGR IRESc

psCbWsPlIlcirtoetciMe

saI2gam

We would predict that these cellular mRNAs would be trans-lated during situations when the IGR IRES is functional, suchas during viral infection, cellular stresses and especially wheneIF2� or other initiation factors are modified.

7. Conclusions

Viruses have multifaceted approaches to recruit ribosomesto their genomes. The Discsitroviridae family of viruses hasevolved an unprecedented mechanism of ribosome recruitmentthat ensures continued translation of ORF2 during viral infectionwhen overall host translation is inhibited. Unlike the scanningmode of translation of mRNAs and other IRES-mediated mech-anisms of translation initiation, the IGR IRES mimicks a tRNAto bind in the ribosomal mRNA channel and set the ribosome inan elongation mode. Moreover, the IGR IRES actively inducesribosome conformational changes, which are likely intrinsicto ribosome function and translation. Indeed, it appears thatthe IGR IRES is inducing similar ribosome conformationalchanges as compared to ribosomes undergoing translation nor-mally (Agrawal et al., 1999; Gomez-Lorenzo et al., 2000; Spahnet al., 2004a). Thus, studies with the IGR IRES will not onlyshed light into how viral IRESs recruit ribosomes but also howtRNAs may normally interact with the ribosome. Future studiesinclude identifying the interactions of the IRES with the ribo-sRsItv

Iocfisr

toiiisint

A

co

an effectively compete for ribosomes.An interesting question is how does the IGR IRES com-

ete for ribosomes during viral infection? Previous studies havehown that overall host protein synthesis is inhibited duringrPV infection, concomitant with massive translation of ORF2y the IGR IRES (Moore et al., 1980, 1981; Wilson et al., 2000b).hat initiation factors may be modified to inhibit host protein

ynthesis and at the same time allow for IGR IRES translation?reliminary experiments have shown that eIF2� is phosphory-

ated during CrPV infection when ORF2 is translated by theGR IRES (Martin Bushell, Eric Jan and Peter Sarnow, unpub-ished data). This is consistent with the idea that the IGR IRESan compete for free ribosomes when host protein synthesis isnhibited. However, it remains to be seen how the IGR IRES canecruit 40S and 80S ribosomes in the presence of other initia-ion factors. A recent in vitro study has shown that 80S assemblyn the IGR IRES is inhibited by the addition of purified eIF3,IF1 and eIF1A (Pestova et al., 2004). The status or nature ofhese initiation factors during viral infection is unknown. Furtherharacterization of the status of initiation factors will shed lightnto the mechanism of how host protein synthesis is inhibited.

oreover, it remains to be seen whether the phosphorylation ofIF2� is caused by or a consequence of viral infection.

Finally, because the IGR IRES is functional in a number ofystems including mammalian cells, yeast, wheat germ extracts,nd rabbit reticulocyte lysates, it suggests that the mechanism ofGR IRES-mediated translation is conserved (Fernandez et al.,002; Thompson et al., 2001; Wilson et al., 2000b). Moreover,iven that the dicistroviruses most likely usurped this mech-nism from the host machinery, we hypothesize that cellularRNAs exist that can initiate translation by this mechanism.

omal proteins and/or rRNA, inevitably revealing the minimalNA elements responsible for interacting and inducing ribo-

ome conformational changes. Ultimately, we can engineer anRES where we can manipulate the expression of certain pro-eins, which are required for cellular survival or death duringiral infection or cell stress.

Another unexplored area of IGR IRESs is the regulation ofGR IRES-mediated translation during viral infection. Someutstanding questions are: how does the IGR IRES directlyompete for and recruit ribosomes in the presence of initiationactors? What is the status of functional initiation factors duringnfection? Overall, these questions will lead to a better under-tanding of how viral IRESs, in general, are able to hijack theibosome and ensure translation of the viral RNA.

Finally, an exciting area of study is emerging from research onhe IGR IRES. Because this IGR IRES can function in a varietyf systems, it is likely that similar mechanisms of translationnitiation are used by some cellular mRNAs. Thus, investigationnto the prevalence and significance of IGR IRES-like translationnitiation is likely to reveal cellular mRNAs translated undertress, as seen for the IGR IRES during viral infection. Moremportantly, it is possible that future work will reveal novel,on-AUG initiated open reading frames, thus greatly expandinghe repertoire of open reading frames in our genome.

cknowledgements

I am very grateful to Kara Norman and Randy Cevallos forritical reading and discussions. E.J. is supported by DRG-1630f the Damon Runyon Cancer Research Foundation.

E. Jan / Virus Research 119 (2006) 16–28 27

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