Functional analysis of structural motifs in dicistroviruses

Virus Research 139 (2009) 137–147

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Functional analysis of structural motifs in dicistroviruses Nobuhiko Nakashima a,∗ , Toshio Uchiumi b a b

National Institute of Agrobiological Sciences, Owashi 1-2, Tsukuba, Ibaraki 305-8634, Japan Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181, Japan

a r t i c l e

i n f o

Article history: Available online 25 July 2008 Keywords: Internal ribosome entry site IRES Dicistroviridae Translation initiation Pseudoknot

a b s t r a c t The family Dicistroviridae is composed of positive-stranded RNA viruses which have monopartite genomes. These viruses carry genome-linked virus proteins (VPg) and poly (A) tails. The 5 untranslated region (UTR) is approximately 500 nucleotides and contains an internal ribosome entry site (IRES). These features resemble those of vertebrate picornaviruses, but dicistroviruses have other distinct characteristics. Picornaviruses have a single large open reading frame (ORF) encoding the capsid proteins at the 5 -end and the replicases at the 3 -end. In contrast, dicistroviruses have two nonoverlapping ORFs. The 5 -proximal ORF encodes the replicases and the 3 -proximal ORF encodes the capsid proteins. Usually, positive-stranded viruses which have capsid protein genes in the 3 part of the genome produce subgenomic RNA for synthesis of the capsid proteins, because abundant quantities of the capsid proteins are required for the viral replication cycle. In dicistroviruses, translation of the capsid proteins is controlled by an additional IRES. This IRES is located in the intergenic region (IGR) between the replicase and capsid coding regions, and mediates the initiation of translation for the capsid proteins. The IGR-IRES has a multiple stem-loop structure containing three pseudoknots. We describe the characteristics of dicistroviruses, including the RNA elements and viral proteins. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Although the family Picornaviridae is composed of vertebrate viruses, positive-stranded RNA viruses in invertebrates, which have properties similar to those of the picornaviruses, have frequently been referred to as picorna-like viruses. The similar characteristics include icosahedral virions with diameters of approximately 30 nm, three major capsid proteins of about 30 kDa, and monopartite genomes consisting of about 8–10 kb. Over 20 picorna-like viruses have been reported in insects (Christian and Scotti, 1998). Fulllength genome sequence analysis of these viruses has revealed that several of the picorna-like viruses in invertebrates have genomes that are organized differently than those of the picornaviruses. Viruses in this group have two nonoverlapping large open reading frames (ORFs) and have been classified into a new family, Dicistroviridae (Christian et al., 2005). The 5 untranslated region (UTR) in a number of groups of positive-stranded RNA viruses consists of several hundred nucleotides and includes a ribosome landing region for capindependent initiation of translation, which is called an internal

∗ Corresponding author. Tel.: +81 29 838 6166; fax: +81 29 838 6028. E-mail address: [email protected] (N. Nakashima). 0168-1702/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2008.06.006

ribosome entry site (IRES) element (Doudna and Sarnow, 2007; Martinez-Salas et al., 2008). The most outstanding characteristic of dicistroviruses is that they have two IRES elements, one for translation of the replicases (5 IRES) and the other for translation of the capsid proteins. The IRES for capsid translation is called the IGR (intergenic region)-IRES because it is located in the intergenic region between the replicase and capsid coding regions. IGR-IRESmediated initiation of translation does not use the canonical AUG triplet as the start codon but the tertiary structure of the upstream UTR determines its own initiation site. This unique IRES was identified in Plautia stali intestine virus (PSIV) (Sasaki et al., 1998; Sasaki and Nakashima, 1999, 2000), Rhopalosiphum padi virus (RhPV) (Domier et al., 2000) and cricket paralysis virus (CrPV) (Wilson et al., 2000a, 2000b). Compensatory mutation analysis was used to model the secondary structure of the IGR-IRES (Kanamori and Nakashima, 2001), and the three dimensional structure has now been resolved by cryo-electron microscopy (cryo-EM) and X-ray crystallography (Spahn et al., 2004; Schüler et al., 2006; Pfingsten et al., 2006; Costantino et al., 2008). Since several reviews containing descriptions of the initiation mechanism of these IGR-IRES elements have been published (Hellen and Sarnow, 2001; Jan et al., 2001; Jackson, 2005; Pisarev et al., 2005; Sarnow et al., 2005; Jan, 2006), this article aims to give an overview of the structural and functional features of the dicistroviruses.

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2. Typical structure of dicistroviral genome

conserved motifs in the deduced amino acid sequences encoded by the dicistrovirus ORF 1 have been reported.

2.1. Similarity and dissimilarity with picornaviruses 2.4. Intergenic region The structure of the picornaviral genome is monocistronic, and it contains a polyprotein gene encoding the capsid proteins at the 5 -end and the replicases at the 3 -end (Fig. 1A). The 5 -end of the genome carries a covalently linked small viral protein (the genomelinked virus protein, VPg) which plays an important role in RNA replication (Nomoto et al., 1977; Paul, 2002). The length of the genomic RNA is in the range 7–9 kb and it includes a poly (A) tail. In contrast, the dicistroviral genome consists of 9–11 kb with two distinct ORFs. The 5 -proximal ORF encodes the replicases and the 3 -proximal ORF encodes the capsid proteins (Fig. 1B). To date, the whole genome sequences of 14 dicistroviruses, isolated from invertebrates including insects and shrimps, have been reported (Table 1). Because the presence of a VPg at the 5 -end was reported for DCV, CrPV, and PSIV (King and Moore, 1988; Nakashima and Shibuya, 2006), it is assumed that the dicistrovirus genome generally has a VPg peptide attached to the 5 -end. 2.2. 5 UTR The presence of an IRES element in the 5 UTR has been demonstrated for the dicistroviruses CrPV, RhPV, TrV and PSIV (Wilson et al., 2000b; Woolaway et al., 2001; Domier and McCoppin, 2003; Masoumi et al., 2003; Czibener et al., 2005; Shibuya and Nakashima, 2006). Features of this element are diversified among the dicistroviruses. For example, the 5 IRES of RhPV functions in insect, plant and mammalian translation systems (Royall et al., 2004), however that of CrPV functions in the rabbit reticulocyte lysate system but not in the wheat germ extract system (Wilson et al., 2000b), and that of PSIV functions in an insect translation system but not in the wheat and rabbit in vitro translation systems (Shibuya and Nakashima, 2006). Chemical and enzymatic structural analyses of RhPV and PSIV reveal that there is no structural conservation in the 5 IRES elements of the two viruses (Terenin et al., 2005; Shibuya and Nakashima, 2006). 2.3. ORF 1 ORF 1 of the dicistroviruses contains motifs for “picorna-like” replicases (i.e., helicase, protease and RNA-dependent RNA polymerase). This suggests that the replicases of dicistroviruses are analogous to those of the picornaviruses; however, there has been little progress thus far in the characterization of the dicistrovirus ORF1 products. The cleavage patterns for mature proteins encoded by the ORF1 are not fully understood, but some clues about protein processing have been reported. Firstly, some dicistroviruses, such as DCV, CrPV, and ABPV, possess a “picorna-like” 2A protease, including the NPGP motif, and the cleavage activity of some of these proteases has been demonstrated experimentally (Ryan et al., 2002; Luke et al., 2008). However, several other dicistroviruses do not have the 2A-like sequences, suggesting that dicistroviruses have various ways to produce mature proteins. Secondly, most dicistroviruses, except for ALPV, RhPV and HoCV-1, have repeated putative VPg coding sequences (Nakashima and Shibuya, 2006) between the helicase and protease genes (Fig. 1B). The maximum number of VPg repeats found thus far is six, in SINV-1. These repeated VPg coding sequences may be advantageous for viral replication, because these viruses can produce capsid proteins effectively by repressing the production of the replicases. Almost all other VPgcontaining viruses have single copies of the VPg coding region, although tandem repeated VPg sequences have been reported for foot and mouth disease virus (Forss and Schaller, 1982). No other

Dicistroviruses have another IRES in the intergenic region. This IGR-IRES is composed of approximately 200 nucleotides that fold into a complex structure containing multiple stem-loops and pseudoknots. The IGR-IRES regulates the translation of the structural protein precursor, although the IGR-IRES-mediated initiation of translation occurs in a phosphorylated condition of eIF2␣ (Thompson et al., 2001; Fernandez et al., 2002; see also Section 7). The presence of the IGR-IRES makes the viral genome dicistronic, and since its discovery viruses which have this genome organization have been assigned to the new family, Dicistroviridae (Christian et al., 2005). To date, two types of IGR-IRES element (Type I and Type II) are known (Fig. 1C). 2.5. ORF 2 Capsids in picornaviruses are composed of three major components, 1B (VP2), 1C (VP3), and 1D (VP1) (Fig. 1A), which have molecular masses of about 30 000, and a minor component, 1A (VP4) (Fig. 1A). The smallest capsid protein is cleaved from a precursor, 1AB (VP0). In dicistroviruses, the smallest capsid protein (VP4) (Nakashima et al., 1998) is cleaved from a precursor VP4–VP3 (VP0), and the precursor VP0 is located within the second segment of the capsid protein precursor (Fig. 1B; Sasaki et al., 1998). Since the deduced amino acid sequences surrounding the VP4–VP3 cleavage site are apparently distinct from those at the VP2–VP0 and VP3–VP1 junctions, cleavage of VP4 from VP0 is believed to occur concurrently with packaging of the viral RNA within the capsid. Indeed, X-ray crystallography of CrPV shows that the C-terminus of VP4 is very close to the N-terminus of VP3 (Tate et al., 1999), and the topology of capsid proteins is highly conserved between picornaviruses and dicistroviruses (Liljas et al., 2002). Usually, positive-stranded RNA viruses which have capsid coding sequences at their 3 -ends produce subgenomic RNA for capsid production (Strauss et al., 1996). However, in the case of dicistroviruses, subgenomic RNAs which have the expected sizes for capsid translation have not been detected in the tissues of infected hosts (Johnson and Christian, 1998; Sasaki et al., 1998, Moon et al., 1998; Nakashima et al., 1999; Wilson et al., 2000b). 2.6. 3 UTR The length of the 3 UTR in dicistroviruses varies from about 150 to 500 nucleotides. The function of the 3 UTR has not yet been studied in many of the dicistroviruses, although in other positivestranded viruses, the 3 UTR sequences are known to affect the control of IRES-mediated translation and RNA replication (Edgil and Harris, 2006). However, in the case of TrV, modulation of 5 -IRESand IGR-IRES-mediated translation by the 3 UTR was not detected (Czibener et al., 2005). 3. Initiation of translation in the absence of the AUG triplet in the dicistrovirus capsid genes 3.1. The canonical mechanism for eukaryotic translation initiation Usually the initiation of translation of eukaryotic mRNAs is dependent on the presence of the cap structure at the 5 -end of the mRNA. The cap structure is recognized by the eukaryotic initiation factor (eIF) 4E. eIF4E interacts with other eIFs to initiate the process of translation with the following steps: formation of the

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Fig. 1. Comparison of genome organization between picornaviruses (A) and dicistroviruses (B). In both cases, the viral RNA genome includes a poly (A) tail [(A)n] at the 3 -end and a genome-linked virus protein (VPg) at the 5 -end (closed circle). The picornaviruses are monocistronic, while the dicistroviruses have two cistrons, indicated in (B) by open reading frames (ORFs) 1 and 2. Protein coding regions are indicated by open boxes and the internal ribosome entry sites (IRES) are indicated by black boxes. The dicistrovirus has an additional IRES in the intergenic region (IGR-IRES). (C) Two typical structures (Type I and Type II), each with three domains (Domains 1, 2 and 3), have been identified for the IGR-IRES elements of dicistroviruses. Circles represent nucleotides and dots mark base-pair interactions in the secondary structure. The Type II IGR-IRES has an additional stem-loop structure in Domain 3. Depending on the type of IGR-IRES element, partially conserved nucleotides occur in Domain 1. Sites of pseudoknots are indicated by PK I, PK II and PK III. Viruses with each type of structure are shown below the diagram. The abbreviations of the viruses and accession numbers of the viral sequences are shown in Table 1.

Table 1 Hosts and nucleotide positions of IGR-IRES elements in sequenced dicistroviruses Virus (Abbreviation)

Host

Accession Number

Nucleotide position of IGR-IRES

Reference

Acute bee paralysis virus (ABPV) Aphid lethal paralysis virus (ALPV) Black queen-cell virus (BQCV) Cricket paralysis virus (CrPV) Drosophila C virus (DCV) Himetobi P virus (HiPV) Homalodisca coagulata virus-1 (HoCV-1) Israeli acute paralysis virus (IAPV) Kashmir bee virus (KBV) Plautia stali intestine virus (PSIV) Rhopalosiphum padi virus (RhPV) Solenopsis invicta virus-1 (SINV-1) Taura syndrome virus (TSV) Triatoma virus (TrV)

Honeybee Aphid Honeybee Cricket Fruitfly Planthopper Leafhopper Honeybee Honeybee Stinkbug Aphid Fireant Penaeid shrimp Triatomine bug

AF150629 AF536531 AF183905 AF218039 AF014388 AB017037 DQ288865 EF219380 AY275710 AB006531 AF022937 AY634314 AF277675 AF178440

6340–6538 6639–6822 5647–5836 6029–6216 6078–6266 6286–6472 5802–5989 6419–6617 6428–6629 6005–6192 6935–7109 4223–4422 6761–6952 5925–6111

Govan et al., 2000 van Munster et al., 2002 Leat et al., 2000 Wilson et al., 2000b Johnson and Christian, 1998 Nakashima et al., 1999 Hunnicutt et al., 2006 Maori et al., 2007 de Miranda et al., 2004 Sasaki et al., 1998 Moon et al., 1998 Valles et al., 2004 Mari et al., 2002 Czibener et al., 2000

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pre-initiation complex, ribosome scanning for the initiator AUG, joining of the 60S ribosome subunit and dissociation of the initiation factors, and translocation towards polypeptide elongation (for review see Pestova et al., 2007). Since the mRNA cap has an important role in the initiation of translation, this mechanism is called cap-dependent initiation. In contrast, cap-independent internal initiation is known in viruses and several cellular mRNAs (for review see Hellen and Sarnow, 2001). Internal initiation of translation was first reported for picornaviruses, poliovirus and encephalomyocarditis virus (Jang et al., 1988; Pelletier and Sonenberg, 1988). These viruses have long 5 UTRs. Since the initiator AUG for the viral protein gene is located several hundreds of nucleotides downstream of the 5 -end, normal ribosome scanning is unlikely to occur. Instead of ribosome scanning, the 5 UTR of picornaviruses forms a tertiary structure that enables the ribosome to enter at an internal site on the mRNA. This region is called the IRES and this mode of translation initiation is called IRES-mediated initiation of translation. It was believed that the first amino acid in polypeptides which are synthesized by ribosomes in native conditions must be methionine. The reasoning was that there are two kinds of Met-tRNAs with distinct functional roles. One type is the elongator Met-tRNA, which incorporates methionines into the internal part of the polypeptide chain, and the other is the initiator Met-tRNA, which incorporates methionine as the first amino acid of the polypeptide chain. Only the initiator Met-tRNAMet can form a ternary complex with eIF2 and GTP, and within this complex, it is located at the P site of the 40S ribosome. For this reason, it was thought that the initial amino acid in native protein synthesis can only be methionine (Drabkin and RajBhandary, 1998). Transfer RNAs which have anticodon triplets for other than the AUG triplet are all elongator tRNAs, and they cannot form this initiation complex. Although several cases of nonAUG translation initiation codons have been reported, the first amino acids in the encoded polypeptides are still methionine. This mismatching occurs through the formation of “wobble base-pairs” between the anticodon triplet of the initiator Met-tRNA and the non-AUG initiation codon, thus allowing initiation at the non-AUG triplet. In these cases, it is known that mutation from the non-AUG triplet to the AUG triplet improves translational efficiency. 3.2. Identification of the translation initiation site for the capsid proteins of dicistroviruses N-terminal amino acid sequence analysis of the PSIV capsid proteins revealed that the 5 -end of the coding region for the capsid precursor is located at nucleotide position 6193 of the viral genome. However, there is no AUG initiation codon in that region. Preliminary in vitro translation experiments suggested that the intergenic region, upstream of the PSIV capsid coding sequence, is required for translation, but that the upstream ORF 1 is not necessary. In addition, translation of the capsid protein precursor occurred independently of a cap, indicating that the capsid proteins of PSIV are produced by IRES-mediated translation (Sasaki and Nakashima, 1999). At that time, the genome sequences of Drosophila C virus (DCV) (Johnson and Christian, 1998), Rhopalosiphum padi virus (Moon et al., 1998), and himetobi P virus (HiPV) (Nakashima et al., 1999) were also available. These four viruses share unique features in their capsid gene expression, including no initiator AUG triplet, and no subgenomic RNA production. These facts suggested that some invertebrate viruses must have unusual protein expression systems. To identify the translation initiation site for the PSIV capsid proteins, termination codons were introduced upstream of the 5 -end of the capsid coding region. This experiment indicated that the triplet CUU (6190–6192), which is just upstream of the triplet CAA

(6193–6195), is the initiation triplet. This CAA triplet is at the 5 -end of the capsid coding region. Frame shift analyses confirmed that the CUU triplet is the place where the reading frame for capsid protein translation is determined in PSIV (Sasaki and Nakashima, 1999). However, this CUU triplet, which is 2 nucleotides different from the canonical AUG, is not recognized by scanning ribosomes. Furthermore, mutation of the CUU to AUG decreased the efficiency of PSIV capsid gene translation (Sasaki and Nakashima, 1999). These results revealed that base-pair interactions between the CUU triplet and the anticodon triplet of the initiator Met-tRNA are not necessary for translation of the PSIV capsid proteins. 3.3. Identification of three pseudoknots in the IGR Usually, viral IRES elements are rich in multiple base-pair interactions. In picornaviruses, the presence of many stem-loops has been reported. In addition, in hepatitis C virus, a pseudoknot structure was found near the translation initiation site. Because of these observations, attention was focused on the flanking sequences at the 5 -end of the capsid coding regions in PSIV, HiPV, DCV, and RhPV. Inverted repeat sequences that might be involved in pseudoknots were found in these regions (Fig. 2A), although a part of the inverted repeat in PSIV is located in a stem region in a computational prediction of its IRES secondary structure (Fig. 2B). Pseudoknots are structural elements of RNA that have diverse functions in various biological processes (Brierley et al., 2007). They are formed by base-pairing between a loop sequence in a stem-loop structure, and complementary nucleotides elsewhere in the RNA. The first example of a pseudoknot to be identified was a tRNA-like structure in the 3 UTR of a plant virus, and pseudoknots were later found in many RNA viruses, including the IRES element of hepatitis C virus. Although computer programs such as Mfold and Pfold have been developed to predict the secondary structures of RNAs, the prediction of pseudoknots is difficult (Baird et al., 2006; Brierley et al., 2007). Several programs have been developed for the automatic prediction for RNA pseudoknots. However, computer predictions for RNA pseudoknots are not reliable without manual and experimental verification. Therefore, compensatory mutation analysis was used in combination with the Mfold prediction (Mathews et al., 1999; Zuker, 2003) to elucidate the secondary structure of the PSIV IGR-IRES. In vitro translation analysis with compensatory mutations indicated that the inverted repeat sequence shown in Fig. 2A and the stem-loop VI structure are necessary for capsid protein translation in PSIV. These results indicate that the pseudoknot PK I is necessary for function (Sasaki and Nakashima, 2000). Translation initiation at sites other than an AUG triplet have also been reported for the capsid proteins of RhPV and CrPV (Domier et al., 2000; Wilson et al., 2000a), thus this pseudoknot was also predicted to form in these viruses. Deletion assays indicated that additional upstream regions in the PSIV intergenic region were needed for translation, prompting the analysis of predicted secondary structures in the upstream regions. However, pseudoknots are difficult to predict because they involve base-pair interactions between very short complementary sequences. In the case of PK I, interactions occur between 5 nucleotides in the loop region of stem-loop VI and 5 nucleotides located immediately upstream of the 5 terminus of the capsid coding region (Fig. 2A and B). When a computational search is performed, there are too many possible interactions between such short nucleotide sequences. However, the positions of sequences involved in functional interactions should be conserved among the viruses. To facilitate finding these conserved positions, the 200 base region upstream of stem-loop VI was compared between PSIV, DCV, and HiPV (Kanamori and Nakashima,

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Fig. 2. Construction of secondary structure models of the dicistrovirus IGR-IRES elements. (A) Inverted repeat sequences, conserved at the 5 -ends of capsid coding regions, indicated where to begin analysis for the mechanism of capsid translation. (B) Mfold-predicted secondary structure of the IGR-IRES region of the PSIV capsid coding region, containing 7 stem-loop structures (I–VII). To confirm the functional importance of the predicted stem structures, compensatory mutations were introduced in the shaded regions (Kanamori and Nakashima, 2001). Stems shown in blue were not important for capsid protein translation, while those shaded in gray were needed for translation. Nucleotides that interact in pseudoknots (PK I, PK II and PK III) are linked by lines. (C) Secondary structure of the IGR-IRES of PSIV. The reliability of the model was checked by structure probing analysis using RNA modification reagents (Nishiyama et al., 2003). Nucleotides that were indicated to be single-stranded by the probing experiments are marked with closed circles. Nucleotides that were protected from modification by the reagents in the presence of the 40S ribosome are marked in yellow. Asterisks represent nucleotide interactions that form pseudoknots and dots represent base-pair interaction for stems.

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Table 2 Summary of the possible base-pair interactionsa , detected by computer, that were used to find pseudoknot (PK) II in the intergenic regions of PSIV, DCV, and HiPV

Up to 37 possible interactions were identified, but only four or five are shown for each virus. (a) The number of residues in possible base-pair interactions was set at 5–15. (b) Reversed nucleotide sequences corresponding to nucleotides 6149–5950 in PSIV, 6223–6024 in DCV and 6249–6230 in HiPV were analyzed. (c) Nucleotide positions 4–8 (gcucc) in the 6149–5950 PSIV sequence corresponds with an inverted repeat sequence at position 111–107 (cgagg). Cells shown in bold type indicate the conserved interactions for PK II.

2001). Their nucleotide sequences were reversed, and inverted segments in the region were identified computationally. Table 2 shows a summary of the search results. The PSIV, DCV, and HiPV sequences had 37, 37, and 30 possible interactions in the 200-nucleotide regions (Table 2). Among these interactions, the positions of three interactions, which are shown in bold type in Table 2, are conserved in the three viruses. The locations of these conserved potential interactions suggested that they are probably functional. Therefore, mutational analyses were carried out using PSIV, and the interaction was confirmed and named PK II (Kanamori and Nakashima, 2001). The sequences of more viruses with similar genome organizations have been published, including ABPV, BQCV, CrPV and TrV (Table 1), and all of these viruses have the PK II interaction (Kanamori and Nakashima, 2001). The analysis described above demonstrated that if the terminal position of an RNA sequence of interest is defined, then a minimum of three sample sequences can give us information about the positions of functional interactions. After the two pseudoknots PK I and PK II had been identified, the secondary structure of the IGR-IRES in PSIV was examined by compensatory mutations. Most of the predicted stem-loop structures appeared to be necessary for IGR-IRES-mediated translation, but the bottom stem of stem-loop IV (shown in Fig. 2B) appeared to be unnecessary. This led to the identification of the third pseudoknot, PK III, because the base-pair interactions of nucleotides located this region are conserved in other viruses which have genome organizations similar to PSIV. The functionality of PK III was confirmed by compensatory mutational analysis (Kanamori and Nakashima, 2001).

3.4. Additional stem-loop in the Type II IGR-IRES The complete genome sequence of ABPV was reported in 2000, but the 5 terminus of the capsid precursor coding region remained unknown. Alignment of intergenic regions indicated that the 5 half of the ABPV intergenic region was similar to those of PSIV, DCV, HiPV, but the 3 half of the region did not resemble those of the other viruses, and in particular, PK I was not identified. This problem was not resolved until the 5 -end of the capsid coding region of TSV was mapped (Mari et al., 2002). Because TSV has a longer IGR sequence, it is possible for an additional stem-loop to form in the region of Domain 3 (Nishiyama et al., 2003). This additional stem-loop was shown to be necessary for the translation of the capsid ORF (Hatakeyama et al., 2004; Cevallos and Sarnow, 2005). An in-frame AUG triplet that is located a few codons upstream of the 5 -end of the capsid precursor gene does not function as an initiator, but the base-pair interactions in the PK I are responsible for translation. These experiments showed that TSV does also have an IGR-IRES, but its structure is significantly different from those of other dicistroviruses that had been characterized at that time. The intergenic regions of ABPV, KBV, IAPV, and SINV-1 have very similar structures to that of TSV (Fig. 3, Type II). The function of the additional stem-loop in this class of IRES has not been determined, but it has been suggested that the stem-loop could help release the 80S complex from the ribosome, or act as a repressor of 80S ribosome formation (Pfingsten et al., 2007). 4. Functions of conserved structural elements in the IGR-IRES An alignment of the nucleotide sequences of the IGR-IRES elements shows that short nucleotide segments are conserved in several regions (Sasaki and Nakashima, 1999), and that these conserved nucleotide sequences are located in the loop regions of the structure model (Fig. 1C). The effects of mutations in these conserved sequences on translational efficiency have been documented for PSIV and CrPV (Nishiyama et al., 2003; Jan and Sarnow, 2002). Enzymatic and chemical structural probing analyses gave information on the location of the solvent side of the IRES (Nishiyama et al., 2003; Jan and Sarnow, 2002). Furthermore, footprint analyses were used to identify the nucleotides which interact with ribosomes (Nishiyama et al., 2003; Jan and Sarnow, 2002). By these approaches, the functions of each of the IGR-IRES domains, (Domains 1, 2, and 3) were analyzed. 4.1. Domain 1 The nucleotide sequences involved in PK II are not conserved, however the two bulges (marked in yellow in Fig. 1C) are well conserved among the dicistroviruses, although the sequences differ between Type I and Type II IGR-IRES elements. Footprint analysis indicated that residue A6014 in Domain 1 of PSIV (see Fig. 2C) is not protected by the 40S subunit, but is protected by the coexistence of the 40S and 60S subunits, suggesting that when the IRES–40S complex is formed, A6014 is located at the inter-subunit face (Nishiyama et al., 2003). Indeed, mutations to this part of the PSIV IRES block stable 60S subunit binding (Pfingsten et al., 2006). A cryo-EM reconstructed image also shows that the corresponding residue in CrPV (A6039) is a candidate for interacting with the 60S ribosome protein L1, which is located at the inter-subunit face close to the E site (Schüler et al., 2006). 4.2. Domain 2 The most conserved region in the IGR-IRES is Domain 2. The AUUU loop is absolutely conserved in all 14 dicistroviruses that

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Fig. 3. Secondary structure models for the IGR-IRES elements of 14 dicistroviruses. Red asterisks and dots indicate nucleotide interactions in pseudoknots and stems, respectively. Regions that correspond between viruses are indicated by colors. The positions of the first and last nucleotides in the IGR-IRES elements are shown. The abbreviations of the viruses and accession numbers of the viral sequences are shown in Table 1. TSV, SINV-1, ABPV, KBV, and IAPV have the Type II IGR-IRES.

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have been sequenced to date. Mutations in this loop decrease the efficiency of IGR-IRES-mediated translation, and the affinity of the element for ribosome binding (Jan and Sarnow, 2002; Nishiyama et al., 2003). Domain 2 has another highly conserved loop sequence, CAGCC, although some of the viruses have slight variations in this loop (Fig. 3). Mutations into this CAGCC loop also decrease translational efficiency and ribosome binding affinity. A synthesized Domain 2 RNA segment binds to the 40S subunit but not to the 60S subunit. In addition, the 40S ribosome, when bound to the Domain 2 segment, cannot form an 80S ribosome (Nishiyama et al., 2003). This suggests that the primary role of Domain 2 is in binding to the 40S subunit, and that a conformational change in Domain 2 when bound to the 40S subunit would be required for the 80S ribosome assembly. 4.3. Domain 3 Domain 3 contains PK I, which determines the reading frame for capsid translation. Domain 3 does not have strong interaction with the 40S subunit (Jan and Sarnow, 2002; Nishiyama et al., 2003; Pestova et al., 2004), though Domain 3 is in contact with 40S in the cryo-EM reconstructions (Spahn et al., 2004; Schüler et al., 2006). IGR-IRES-mediated translation depends on the structure of the folded RNA, and specific interactions between nucleotides in Domain 3 are critical for function. When exogenous sequences are used to replace the native viral coding sequence, such constructs frequently disturb the structure of Domain 3, and translation of the exogenous sequences are inhibited by this structural distortion (Shibuya et al., 2003, 2004). This implies that downstream nucleotides in the coding sequence are important for effective IGRIRES-mediated translation. The activation of eEF2-dependent GTPase activity of the 80S ribosome by binding of PSIV IGR-IRES suggested that a part of IGRIRES including Domain 3 mimics the P/E state tRNA, which activates the GTPase (Yamamoto et al., 2007). A model of the docked structure of the CrPV Domain 3 with the 40S ribosome also explains that the position of Domain 3 resembles that of the hypothesized P/E hybrid state of tRNA (Costantino et al., 2008). This state would facilitate the beginning of an elongation reaction for peptide synthesis, in the absence of the first peptidyl tRNA at the P site, during IGR-IRES-mediated translation. 5. Ribosome binding and functions of pseudoknots in the IGR-IRES The IGR-IRES RNA of CrPV binds to the 40S ribosomal subunit in the absence of initiation factors (Wilson et al., 2000a). Since a majority of the structural elements in the IGR-IRES are conserved, the function of each pseudoknot (PK I, PK II, and PK III) in ribosome binding was analyzed using mutated IRES elements from PSIV. Mutations which disrupted the PK I or PK II interactions slightly decreased binding affinity to the ribosome. However, mutations disrupting the base-pair interactions of PK III seriously decreased binding affinity to the ribosome (Nishiyama et al., 2003). The importance of PK III for binding with the ribosome has also been shown in CrPV (Jan and Sarnow, 2002). These results suggest that PK III is the most important IGR-IRES structure for ribosome binding. In secondary structure models, PK III is located in the region where stem-loops IV and V are connected. Stem-loops IV and V contain the most conserved loop sequences, i.e., the AUUU and CAGCC loops, although HiPV, TrV and HoCV-1 have varied loop sequences in the region corresponding to the CAGCC loop (Figs. 2B, C and 3). This suggests that these loop sequences probably have important roles in the interaction between the IGR-IRES and the ribosome. Indeed, mutations in these loop sequences and footprint analyses using

dimethyl sulfate (DMS) and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT) suggested that these loop sequences are important for translation and ribosome binding (Nishiyama et al., 2003; Jan and Sarnow, 2002). Domains 1 and 2 of the IGR-IRES have been shown to be folded compactly in a way that positions these loops correctly for ribosome interaction (Costantino and Kieft, 2005). PK II appears to be a junction between the ribosome binding domains (Domains 1 and 2) and the reading frame determinant, Domain 3. Binding analyses using a full-length IGR-IRES and a truncated IRES in which Domain 3 was deleted, indicated that Domain 3 competes with the poly (U) and the anticodon stem-loop of tRNAPhe on the 80S ribosome (Nishiyama et al., 2003). Therefore, PK I does not have major contribution to the binding of the IGR-IRES to the 40S ribosome, but PK I determines both the reading frame and the site where peptide synthesis starts. Because the IGR-IRES of PSIV does not bind to the 60S subunit, the sites on the 40S subunit where it interacts with the PSIV IGRIRES were examined. Footprinting experiments using the chemical reagents DMS and CMCT showed that the 18S rRNA within the 40S subunit does not bind to the IGR-IRES (Nishiyama et al., 2007). However, an interaction between the IRES and a ribosomal protein was suggested by the following cross-linking experiment. Since Domain 3 does not bind to the 40S subunit (Jan and Sarnow, 2002; Nishiyama et al., 2003; Pestova et al., 2004), an RNA sequence containing only Domains 1 and 2, labeled with the nucleotide analog 4-thiouridine, was used for the analysis. This nucleotide analog is commercially available and makes a zero-length cross-link with interacting molecules. We detected an interaction between rpS25 and the conserved AUUU loop of the PSIV IGR-IRES (Nishiyama et al., 2007). However, recent data obtained by cryo-EM showed that the yeast 80S ribosomal rpS5 interacts with the AUUU loop of CrPV IGR-IRES, and the CAGCC loop interacts with an unknown protein on a small subunit (Schüler et al., 2006). This discrepancy may be explained by differences in experimental conditions. The cross-linking study with the 4-thiouridine-labeled PSIV Domain 1–2 was performed using a complex with the isolated 40S subunit, whereas the cryo-EM analysis was carried out using the CrPV IRES and the 80S ribosome. It has been proposed that the interaction between the IGR-IRES and the 40S subunit is changed by the joining of the 60S subunit to form the 80S ribosome (Spahn et al., 2004). In addition, evidence from protein–protein cross-linking experiments suggests that rpS25 and rpS5 are neighboring proteins in the 40S subunit of rat liver (Uchiumi et al., 1981), when the universal designations of these proteins are used (McConkey et al., 1979). It is therefore possible that the AUUU loop first makes contact with rpS25, which is presumably located at a position close to the exit channel of the mRNA around the E site, and when the 60S subunit joins to the 40S subunit, the AUUU loop may detach from rpS25 and make contact with the neighboring rpS5. 6. First aminoacyl tRNA entry during IGR-IRES-mediated translation The IGR-IRES-mediated initiation of translation requires no initiation factor (Pestova and Hellen, 2003; Jan et al., 2003). In fact, binding of the first aminoacyl tRNA to the ribosome and its translocation to the P site occur via the actions of only the elongation factors eEF1A and eEF2 (Yamamoto et al., 2007). This indicates that IGR-IRES-mediated translation starts in an elongation mode. However, the IGR-IRES-mediated binding of the first aminoacyl tRNA and its translocation are unusual, because these occur in the absence of a tRNA at the P site. This unusual mechanism is driven by the binding of the IGR-IRES to the ribosome. In this process, a part of the IRES structure, including Domain 3, seems to mimic the tRNA at

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the P site. It is noteworthy that IRES binding to the ribosome at the P/E site, as suggested in cryo-EM data (Schüler et al., 2006), stimulates the eEF2-dependent GTPase activity 3-fold (Yamamoto et al., 2007). This result suggests that IRES binding affects the binding site of the elongation factor in an allosteric manner. This view is supported by a finding by cryo-EM that IRES binding largely changes the conformational ordering of the P protein stalk located in the GTPase-associated center of the 60S subunit (Spahn et al., 2004). It is likely that IGR-IRES-induced conformational modulation of the 40S and 60S subunits is responsible for the enhancement of eEF2 action and for binding of the first aminoacyl tRNA to the ribosome in the absence of an initiation factor. 7. Strategies against host cells in dicistroviruses The 2C-like helicase, 3C-like protease, and 3D-like RNAdependent RNA polymerase are quite well conserved among the dicistroviruses, however, the 5 -end of ORF 1 varies among these viruses. Recently, DCV was reported to have a dsRNA binding motif in the 5 region of ORF 1, but such a motif was not found in the corresponded region of CrPV, which is considered to be the closest relative of DCV (van Rij et al., 2006). However, viral suppressors of the host RNAi system have been identified within 150 codons from the 5 -end of ORF 1 in CrPV (Wang et al., 2006). In addition, sequence similarity to inhibitor of apoptosis proteins is found in the 5 part of ORF 1 of TSV (Mari et al., 2002). These observations indicate the development of distinct mechanisms for escape from the hosts’ innate immunity, and suggest that the N-terminal part of the ORF1 may be a hot spot for the evolutionary ‘arms race’ between the viruses and their hosts (Cherry and Silverman, 2006). Distinct features in the 5 IRES elements of dicistroviruses (mentioned in Section 2.2) may be involved in these survival strategies. The efficiency of translation initiation in cells is affected by the phosphorylation of eIF2␣. Because eIF2-GTP-tRNAiMet complexes are required for the canonical initiation of translation, IGR-IRESmediated translation must compete with these complexes in order to interact with the ribosome. The IGR-IRES of CrPV functions poorly in wild-type yeast cells, but functions efficiently when the cells express GCN2 mutations that result in enhanced phosphorylation of eIF2␣ (Thompson et al., 2001). The importance of eIF2␣ phosphorylation on IGR-IRES-mediated translation has also been demonstrated in a mammalian cell system (Fernandez et al., 2002). Since dicistroviruses use IGR-IRES-mediated translation for their capsid production, the viruses must control the host mechanisms that trigger phosphorylation of eIF2␣ during dicistroviral infection. However, these mechanisms have not yet been clarified. Genomewide screening for host genes responsible for viral infection is being done in Drosophila (Cherry et al., 2005). Such projects will shed light on these questions in the future. 8. Perspective Experiments with reconstituted translation systems have shown that there is no requirement for eIFs in the IGR-IRESmediated initiation of translation (Pestova and Hellen, 2003, 2005; Jan et al., 2003). This has led to new studies involving the functional analysis of factors affecting translation (Humphreys et al., 2005; Sato et al., 2007), and many discoveries are expected. Four different species of dicistroviruses have been found in honeybees (Table 1). The presence of multiple viral species in a singe host suggests that there are likely to be many as yet undiscovered species of dicistroviruses. Recent metagenomic analyses using seawater samples have indicated that some marine viruses also have dicistronic genome organizations (Culley et al., 2006, 2007; Suttle,

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2007). Such observations imply that there are probably other IGRIRES-like RNA structures, hidden in undiscovered viral genomes. Acknowledgements We would like to thank J. Sasaki, Y. Kanamori, Y. Hatakeyama, T. Nishiyama, N. Shibuya and H. Yamamoto for their cooperation in the PSIV work. References Baird, S.D., Turcotte, M., Korneluk, R.G., Holcik, M., 2006. Searching IRES. RNA 12, 1755–1785. Brierley, I., Pennell, S., Gilbert, R.J., 2007. Viral RNA pseudoknots: versatile motifs in gene expression and replication. Nat. Rev. Microbiol. 5, 598–610. Cevallos, R.C., Sarnow, P., 2005. Factor-independent assembly of elongationcompetent ribosomes by an internal ribosome entry site located in an RNA virus that infects penaeid shrimp. J. Virol. 79, 677–683. Cherry, S., Doukas, T., Armknecht, S., Whelan, S., Wang, H., Sarnow, P., Perrimon, N., 2005. Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition. Genes Dev. 19, 445–452. 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