Translation Of Viral mRNAs Research Articles

Caliciviruses Differ in Their Functional Requirements for eIF4F Components

Two classes of viruses, namely members of the Potyviridae and Caliciviridae, use a novel mechanism for the initiation of protein synthesis that involves the interaction of translation initiation factors with a viral protein covalently linked to the viral RNA, known as VPg. The calicivirus VPg proteins can interact directly with the initiation factors eIF4E and eIF3. Translation initiation on feline calicivirus (FCV) RNA requires eIF4E because it is inhibited by recombinant 4E-BP1. However, to date, there have been no functional studies carried out with respect to norovirus translation initiation, because of a lack of a suitable source of VPg-linked viral RNA. We have now used the recently identified murine norovirus (MNV) as a model system for norovirus translation and have extended our previous studies with FCV RNA to examine the role of the other eIF4F components in translation initiation. We now demonstrate that, as with FCV, MNV VPg interacts directly with eIF4E, although, unlike FCV RNA, translation of MNV RNA is not sensitive to 4E-BP1, eIF4E depletion, or foot-and-mouth disease virus Lb protease-mediated cleavage of eIF4G. We also demonstrate that both FCV and MNV RNA translation require the RNA helicase component of the eIF4F complex, namely eIF4A, because translation was sensitive (albeit to different degrees) to a dominant negative form and to a small molecule inhibitor of eIF4A (hippuristanol). These results suggest that calicivirus RNAs differ with respect to their requirements for the components of the eIF4F translation initiation complex.

Rotavirus Nonstructural Protein NSP3 Is Not Required for Viral Protein Synthesis

Initiation is the rate-limiting step in protein synthesis and therefore an important target for regulation. For the initiation of translation of most cellular mRNAs, the cap structure at the 5' end is bound by the translation factor eukaryotic initiation factor 4E (eIF4E), while the poly(A) tail, at the 3' end, is recognized by the poly(A)-binding protein (PABP). eIF4G is a scaffold protein that brings together eIF4E and PABP, causing the circularization of the mRNA that is thought to be important for an efficient initiation of translation. Early in infection, rotaviruses take over the host translation machinery, causing a severe shutoff of cell protein synthesis. Rotavirus mRNAs lack a poly(A) tail but have instead a consensus sequence at their 3' ends that is bound by the viral nonstructural protein NSP3, which also interacts with eIF4GI, using the same region employed by PABP. It is widely believed that these interactions lead to the translation of rotaviral mRNAs, impairing at the same time the translation of cellular mRNAs. In this work, the expression of NSP3 in infected cells was knocked down using RNA interference. Unexpectedly, under these conditions the synthesis of viral proteins was not decreased, while the cellular protein synthesis was restored. Also, the yield of viral progeny increased, which correlated with an increased synthesis of viral RNA. Silencing the expression of eIF4GI further confirmed that the interaction between eIF4GI and NSP3 is not required for viral protein synthesis. These results indicate that NSP3 is neither required for the translation of viral mRNAs nor essential for virus replication in cell culture.

Initiation of Protein Synthesis by Hepatitis C Virus Is Refractory to Reduced eIF2 · GTP · Met-tRNAiMetTernary Complex Availability

A cornerstone of the antiviral interferon response is phosphorylation of eukaryotic initiation factor (eIF)2alpha. This limits the availability of eIF2.GTP.Met-tRNA(i)(Met) ternary complexes, reduces formation of 43S preinitiation complexes, and blocks viral (and most cellular) mRNA translation. However, many viruses have developed counterstrategies that circumvent this cellular response. Herein, we characterize a novel class of translation initiation inhibitors that block ternary complex formation and prevent the assembly of 43S preinitiation complexes. We find that translation driven by the HCV IRES is refractory to inhibition by these compounds at concentrations that effectively block cap-dependent translation in vitro and in vivo. Analysis of initiation complexes formed on the HCV IRES in the presence of inhibitor indicates that eIF2alpha and Met-tRNA(i)(Met) are present, defining a tactic used by HCV to evade part of the antiviral interferon response.

Intra cellular trafficking of influenza virus proteins

Influenza virus is a negative strand RNA virus and is one of the rare RNA viruses to replicate in the nucleus. The viral RNA is associated with 4 viral proteins to make ribonucleoprotein particles (RNPs). After cell entry the RNPs are dissociated from the viral matrix protein in the low pH of the endosome and are actively imported into the cell nucleus. After translation of viral mRNAs, the proteins necessary for the assembly of new RNPs (the nucleoprotein and the three subunits of the polymerase complex) are also imported into the nucleus. Apart from these four proteins, part of the newly made matrix protein is also imported and the NEP (nuclear export protein) enters the nucleus probably through diffusion. The nuclear localisation signals on all these viral proteins and their interaction with the cellular transport system are discussed. In the nucleus, the matrix protein binds to the newly assembled RNPs and NEP then binds to the matrix protein. NEP contains the nuclear export signal necessary for transport of the RNPs to the cytoplasm for the budding of new virus particles. There appears to be a intricate ballet in exposing and hiding nuclear transport signals which leads to a unidirectional transport of the RNPs to the nucleus at the start of the infection process and an opposite unidirectional export of RNPs at the end of the infection.

Analysis of a 3'-translation enhancer in a tombusvirus: a dynamic model for RNA-RNA interactions of mRNA termini.

Tomato bushy stunt virus is a (+)-strand RNA virus that is neither 5'-capped nor 3'-polyadenylated. Translation of viral proteins is instead mediated by an RNA element, the 3'-cap-independent translational enhancer (3'CITE), which is located in its 3' untranslated region (UTR). The 3'CITE is proposed to recruit the translational machinery to the viral message, while a long-distance RNA-RNA interaction between the 3'CITE and 5' UTR is thought to deliver the 43S ribosomal subunit to the 5' end of the viral mRNA. Here we provide the first evidence that the 5' UTR and 3'CITE interact physically. Mutational analysis showed that formation of this RNA-RNA interaction in vitro correlates well with efficient translation in vivo, thus supporting its functional relevance. Other analyses of the 3'CITE confirmed an overall Y-shaped RNA secondary structure and demonstrated the importance of numerous minor structural features for efficient translation of viral mRNAs. Functional studies on the role of the 5' UTR revealed that despite the absence of a cap structure, 43S subunits load at the very 5' end and scan in a 3' direction. These results indicate that the 5'-3' RNA-RNA interaction is likely disrupted by scanning ribosomal subunits and suggest a dynamic model for the interaction of mRNA termini during active translation.

Hijacking of the host-cell response and translational control during influenza virus infection

Influenza virus is a major public health problem with annual deaths in the US of 36,000 with pandemic outbreaks, such as in 1918, resulting in deaths exceeding 20 million worldwide. Recently, there is much concern over the introduction of highly pathogenic avian influenza H5N1 viruses into the human population. Influenza virus has evolved complex translational control strategies that utilize cap-dependent translation initiation mechanisms and involve the recruitment of both viral and host-cell proteins to preferentially synthesize viral proteins and prevent activation of antiviral responses. Influenza virus is a member of the Orthomyxoviridae family of negative-stranded, segmented RNA viruses and represents a particularly attractive model system as viral replication strategies are closely intertwined with normal cellular processes including the host defense and stress pathways. In this chapter, we review the parallels between translational control in influenza virus infected cells and in stressed cells with a focus on selective translation of viral mRNAs and the antagonism of the dsRNA and host antiviral responses. Moreover, we will discuss how the use of genomic technologies such as DNA microarrays and high through-put proteomics can be used to gain new insights into the control of protein synthesis during viral infection and provide a near comprehensive view of virus-host interactions.

Role of residues 121 to 124 of vesicular stomatitis virus matrix protein in virus assembly and virus-host interaction.

The recent solution of the crystal structure of a fragment of the vesicular stomatitis virus matrix (M) protein suggested that amino acids 121 to 124, located on a solvent-exposed loop of the protein, are important for M protein self-association and association with membranes. These residues were mutated from the hydrophobic AVLA sequence to the polar sequence DKQQ. Expression and purification of this mutant from bacteria showed that it was structurally stable and that the mutant M protein had self-association kinetics similar to those of the wild-type M protein. Analysis of the membrane association of M protein in the context of infection with isogenic recombinant viruses showed that both wild-type and mutant M proteins associated with membranes to the same extent. Virus expressing the mutant M protein did show an approximately threefold-lower binding affinity of M protein for nucleocapsid-M complexes. In contrast to the relatively minor effects of the M protein mutation on virus assembly, the mutant virus exhibited growth restriction in MDBK but not BHK cells, a slower induction of apoptosis, and lower viral-protein synthesis. Despite translating less viral protein, the mutant virus produced more viral mRNA, showing that the mutant virus could not effectively promote viral translation. These results demonstrate that the 121-to-124 region of the VSV M protein plays a minor role in virus assembly but is involved in virus-host interactions and VSV replication by augmenting viral-mRNA translation.

Synthesis, Processing, and Composition of the Virion-associated HTLV-1 Reverse Transcriptase

It is not known whether the low infectivity and low virion-associated polymerase activity of human T-cell lymphotropic virus type-1 (HTLV-1) are due to the quantity or quality of the reverse transcriptase (RT), because the protein has not yet been fully characterized. We have developed anti-RT antibodies and constructed HTLV-1 expression plasmids that express truncated or hemagglutinin-tagged Pol polyproteins to examine the maturation and composition of HTLV-1 RT. We detected virion-associated proteins corresponding to RT-integrase (IN) (pr98) and RT (p62) as well as smaller proteins containing the polymerase (p49) or RNase H domains. We have identified the amino acid sequences in the Pol polyprotein that are cleaved by HTLV-1 protease to yield RT and IN. We have also identified the cleavage sites within RT that give rise to the p49 polymerase fragment. Immunoprecipitation of an epitope-tagged p62 subunit coprecipitated p49, indicating that the HTLV-1 RT complex can exist as a p62/p49 heterodimer analogous to the RT of HIV-1 (p66/p51).

Eukaryotic Translation Initiation Factor 4E Availability Controls the Switch between Cap-Dependent andInternal Ribosomal Entry Site-Mediated Translation

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5' end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.

Phosphorylation and dephosphorylation events that regulate viral mRNA translation

As they are completely dependent upon the protein synthesis machinery resident in the cells of their host to translate their mRNAs, it is imperative that viruses are able to effectively manipulate the elaborate cellular regulatory network that controls translation. Indeed, this exquisite dependence on host functions has made viral models attractive systems to explore translational regulatory mechanisms operative in eukaryotic cells. Central among these are an intricate array of phosphorylation and dephosphorylation events that have far reaching consequences on the activity of cellular translation factors. Not only do these modulate the activity of a given factor, but they can also determine if the translation of host proteins persists in infected cells, the efficiency with which viral mRNAs are translated and the outcome of a systemic host anti-viral response. In this review, we discuss how various viruses manipulate the phosphorylation state of key cellular translation factors, illustrating the critical nature these interactions play in virus replication, pathogenesis and innate host defense.

Unrip, a factor implicated in cap-independent translation, associates with the cytosolic SMN complex and influences its intracellular localization

Spliceosomal Uridine-rich small ribonucleo protein (U snRNP) assembly is an active process mediated by the macromolecular survival motor neuron (SMN) complex. This complex contains the SMN protein and six additional proteins, named Gemin2-7, according to their localization to nuclear structures termed gems. Here, we provide biochemical evidence for the existence of another, yet atypical, SMN complex component, termed unr-interacting protein (unrip). This abundant factor has been previously shown to form a complex with unr, a protein implicated in cap-independent translation of cellular and viral mRNA. We show that unrip is integrated into a complex with unr or with the SMN complex in vivo in a mutually exclusive manner. In the latter case, unrip is recruited to the active SMN complex via a stable interaction with Gemin7. However, unlike SMN and Gemins, unrip localizes predominantly to the cytoplasm and is absent from gems/Cajal bodies. Interestingly, RNAi-induced reduction of unrip protein levels leads to enhanced accumulation of SMN in the nucleus as evident by the increased formation of nuclear gems/Cajal bodies. Our data identify unrip as the first component of the U snRNP assembly machinery that associates with the SMN complex in a compartment-specific way. We speculate that unrip plays a crucial role in the intracellular distribution of the SMN complex.

UNR translation can be driven by an IRES element that is negatively regulated by polypyrimidine tract binding protein

Upstream of N-ras (Unr) has been described as an internal initiation trans-acting factor (ITAF) in the cap-independent translation of some particular viral and cellular mRNAs. Two factors led us to hypothesize that the UNR 5′-untranslated region (5′-UTR) may contain an internal ribosome entry site (IRES). The first was the requirement for persisting Unr expression under conditions that correlate with cap-independent translation. The other was the observation that the primary UNR transcript contains a 447 nt long 5′-UTR including two upstream AUGs that may restrict translation initiation via cap-dependent ribosome scanning. Here we report that the UNR 5′-UTR allows IRES-dependent translation, as revealed by a dicistronic reporter assay. Various controls ruled out the contribution of leaky scanning, cryptic promoter sequences or RNA processing events to the ability of the UNR 5′-UTR to mediate internal initiation of translation. Ultraviolet cross-linking analysis and RNA affinity chromatography revealed the binding of polypyrimidine tract binding protein (PTB) to the UNR IRES, requiring a pyrimidine-rich region (nucleotides 335–355). Whereas overexpression of PTB in several cell lines inhibited UNR IRES activity and UNR protein expression, depletion of endogenous PTB using RNAi increased UNR IRES activity. Moreover, a mutant version of the UNR IRES lacking the PTB binding site was more efficient at directing IRES-mediated translation. In conclusion, our results demonstrate that translation of the ITAF Unr can itself be regulated by an IRES that is downregulated by PTB.

Regulation of Translation by Ribosome Shunting through Phosphotyrosine-Dependent Coupling of Adenovirus Protein 100k to Viral mRNAs

Adenovirus simultaneously inhibits cap-dependent host cell mRNA translation while promoting the translation of its late viral mRNAs during infection. Studies previously demonstrated that tyrosine kinase activity plays a central role in the control of late adenovirus protein synthesis. The tyrosine kinase inhibitor genistein decreases late viral mRNA translation and prevents viral inhibition of cellular protein synthesis. Adenovirus protein 100k blocks cellular mRNA translation by disrupting the cap-initiation complex and promotes viral mRNA translation through an alternate mechanism known as ribosome shunting. 100k protein interaction with initiation factor eIF4G and the viral 5' noncoding region on viral late mRNAs, known as the tripartite leader, are both essential for ribosome shunting. We show that adenovirus protein 100k promotes ribosome shunting in a tyrosine phosphorylation-dependent manner. The primary sites of phosphorylated tyrosine on protein 100k were mapped and mutated, and two key sites are shown to be essential for protein 100k to promote ribosome shunting. Mutation of the two tyrosine phosphorylation sites in 100k protein does not impair interaction with initiation factor 4G, but it severely reduces association of 100k with tripartite leader mRNAs. 100k protein therefore promotes ribosome shunting and selective translation of viral mRNAs by binding specifically to the adenovirus tripartite leader in a phosphotyrosine-dependent manner.

Virus-Associated RNA I–Deleted Adenovirus, a Potential Oncolytic Agent Targeting EBV-Associated Tumors

Given the growing number of tumor types recognizably associated with EBV infection, it is critically important that therapeutic strategies are developed to treat such tumors. Replication-selective oncolytic adenoviruses represent a promising new platform for anticancer therapy. Virus-associated I (VAI) RNAs of adenoviruses are required for efficient translation of viral mRNAs. When the VAI gene is deleted, adenovirus replication is impeded in most cells (including HEK 293 cells). EBV-encoded small RNA1 is uniformly expressed in most EBV-associated human tumors and can functionally substitute for the VAI RNAs of adenovirus. It enables replication to proceed through complementation of VAI-deletion mutants. We hypothesized that VAI-deleted adenovirus would selectively replicate in EBV-positive tumor cells due to the presence of EBV-encoded small RNA1 with no (or poor) replication in normal or EBV-negative tumor cells. In this report, we show that high levels of replication occurred in the VAI-deleted mutant in the EBV-positive tumor cells compared with low (or negligible) levels in EBV-negative and normal human primary cells. Correspondingly, high toxicity levels were observed in EBV-positive tumor cells but not in EBV-negative tumor or normal human primary cells. In vivo, VAI-deleted adenovirus showed superior antitumoral efficacy to wild-type adenovirus in EBV-positive tumor xenografts, with lower hepatotoxicity than wild-type adenovirus. Our data suggest that VAI-deleted adenovirus is a promising replication-selective oncolytic virus with targeting specificity for EBV-associated tumors.

Early and late HHV-6 gene transcripts in multiple sclerosis lesions and normal appearing white matter

Multiple sclerosis is an inflammatory demyelinating disease of the CNS, the aetiology of which is believed to have both genetic and environmental components. We have investigated one of the candidate viruses for the environmental component of multiple sclerosis, the neurotropic human herpesvirus 6 (HHV-6). Utilizing fluorescent in situ hybridization (FISH) techniques, we have examined human post-mortem tissues for the presence of immediate early and late viral gene expression in multiple sclerosis patient normal appearing white matter (NAWM), lesional tissue and normal control brain samples. HHV-6 gene transcription was detected in all tissue samples and was restricted to oligodendrocytes, as determined by double mRNA FISH analysis. Quantitative analysis of viral mRNA expression indicated that both NAWM and lesional multiple sclerosis samples exhibited significantly higher levels of HHV-6 expression compared with the normal control samples. Lesional samples exhibited the highest levels of viral gene expression, with NAWM exhibiting an intermediate level between lesional and control tissues. Immunofluorescence against early and late HHV-6 proteins verified active translation of HHV-6 viral mRNA in oligodendrocytes. Southern blot analysis of nested polymerase chain reactions using extracted genomic DNA and cDNA confirmed the presence of the HHV-6 genome in all individuals, with the active expression profile mirroring the FISH results. The frequent high level of HHV-6 infection in multiple sclerosis samples suggests a possible role in pathogenesis.

Proteomics of herpes simplex virus infected cell protein 27: association with translation initiation factors

The herpes simplex virus (HSV) immediate early ICP27 protein plays an essential role in stimulating viral early and late gene expression. ICP27 appears to be multifunctional in that it has been reported to stimulate viral late gene transcription, polyadenylation site usage, and RNA export. We report here on proteomic studies involving immunoprecipitation of ICP27 and mass spectrometric identification of co-precipitated proteins. These studies show an association of ICP27 with the cellular translation initiation factors poly A binding protein (PABP), eukaryotic initiation factor 3 (eIF3), and eukaryotic initiation factor 4G (eIF4G) in infected cells. Immunoprecipitation-western blot studies confirmed these associations. Finally, purified MBP-tagged ICP27 (MBP-27) can interact with eIF3 subunits p47 and p116 in vitro. These results suggest that ICP27 may also play a role in stimulating translation of certain viral and host mRNAs and/or in inhibiting host mRNA translation.

Determinants of apical loop–internal loop RNA–RNA interactions involving the HCV IRES

Domain II of the hepatitis C virus internal ribosome entry site is a major RNA structure involved in the viral mRNA translation. It comprises four different structural domains. We performed in vitro selection against the apical loop of the domain II and we identified RNA aptamers folding as an imperfect hairpin with an internal loop of interacting with the apical loop of the domain II. This RNA–RNA interaction creates apical loop–internal loop complex. The aptamer binds the target with an apparent K d of 35 nM. In this study, the main structural elements of the target and the aptamer involved in the formation of the complex are characterized by mutation, deletion, and RNase probing analysis. We demonstrate that a complementary loop flanked by G,C rich upper and lower stems are crucial for such RNA–RNA interactions.

608. VAI Deleted Adenovirus as an Oncolytic Agent Targeting Human EBV-Associated Tumours

The EBV genome is frequently present in Burkitt's lymphomas, B-Cell lymphomas of immunocompromised patients, nasopharyngeal carcinomas, Hodgkin's disease, T-cell lymphomas, natural killer cell leukemia/lymphoma, smooth-muscle tumors and gastric cancer. More recently, there have been scattered reports linking EBV with conventional epithelial cancers of other primary sites including breast cancers, lung cancers, prostate cancers, liver cancers, colon cancers and lymphoepithelioma-like carcinoma of the esophagus. Given the ever-growing number of tumor types associated with EBV infection, it is therefore critically important to develop therapeutic strategies to treat EBV-associated tumors. Replication-selective oncolytic adenovirus is a promising new platform for anti-cancer therapy. Data from numerous clinical trials have demonstrated several attenuated oncolytic viral mutants to be safe and to replicate selectively in tumor cells with significant anti-tumor potency. Virus associated (VA) 1 and 2 RNA are two low molecular weight adenoviral RNAs that are synthesized late during the viral replication cycle and are required for efficient translation of late viral mRNAs. When the VA1 gene is deleted, replication is impeded in most cells (including HEK 293 cells). The Epstein Barr Virus (EBV) genome encodes two small, non-polyadenylated nuclear RNAs (EBER 1 and 2) that are uniformly expressed in most EBV-associated human tumors. EBER 1 and 2 can functionally substitute for the VA1 RNAs of adenovirus, enabling replication to proceed normally through complementation of VA1 deletion mutants. We hypothesized that VA1-deleted adenovirus would selectively replicate in EBV-positive tumor cells due to complementation by EBER 1, with poor or no replication in normal cells and EBV-negative tumor cells. Our data demonstrate that cytotoxicity of VA1 RNA deletion mutants was restricted to EBV-positive tumor cells (e.g. c666, AGS-EBV, Raji, Jiyoye). Cytopathic effects were much lower in EBV-negative tumor cells (Hep-2, AGS-ATCC, Panc-1). The replication of the VA1 deletion mutant virus was 14–25 times higher in EBV-positive tumor cells than in EBV-negative tumor cells and comparable with wild type adenovirus. The VA1 mutant did not replicate in normal cells in culture such as normal human bronchial epithelial cells (NHBE), and replicated poorly in prostate epithelial cells (PrEC). In vivo, VAI deleted adenovirus showed the same efficacy as wild type adenovirus in xenograft models of human EBV-positive tumor cells, but much lower cytotoxicity than wildtype adenovirus. Our data suggest that a VA1 RNA-deleted viral mutant is a promising replication-selective oncolytic virus specifically targeting EBV-associated cancers.

Eucaryotic initiation factor 4B controls eIF3-mediated ribosomal entry of viral reinitiation factor.

The cauliflower mosaic virus reinitiation factor TAV interacts with host translation initiation factor 3 (eIF3) and the 60S ribosomal subunit to accomplish translation of polycistronic mRNAs. Interaction between TAV and eIF3g is critical for the reinitiation process. Here, we show that eIF4B can preclude formation of the TAV/eIF3 complex via competition with TAV for eIF3g binding; indeed, the eIF4B- and TAV-binding sites on eIF3g overlap. Our data indicate that eIF4B interferes with TAV/eIF3/40S ribosome complex formation during the first initiation event. Consequently, overexpression of TAV in plant protoplasts affects only second initiation events. Transient overexpression of eIF4B in plant protoplasts specifically inhibits TAV-mediated reinitiation of a second ORF. These data suggest that TAV enters the host translation machinery at the eIF4B removal step to stabilize eIF3 on the translating ribosome, thereby allowing translation of polycistronic viral RNA.

Replication of Poliovirus RNA with Complete Internal Ribosome Entry Site Deletions

cis-acting RNA sequences and structures in the 5' and 3' nontranslated regions of poliovirus RNA interact with host translation machinery and viral replication proteins to coordinately regulate the sequential translation and replication of poliovirus RNA. The poliovirus internal ribosome entry site (IRES) in the 5' nontranslated region (NTR) has been implicated as a cis-active RNA required for both viral mRNA translation and viral RNA replication. To evaluate the role of the IRES in poliovirus RNA replication, we exploited the advantages of cell-free translation-replication reactions and preinitiation RNA replication complexes. Genetic complementation with helper mRNAs allowed us to create preinitiation RNA replication complexes containing RNA templates with defined deletions in the viral open reading frame and the IRES. A series of deletions revealed that no RNA elements of either the viral open reading frame or the IRES were required in cis for negative-strand RNA synthesis. The IRES was dispensable for both negative- and positive-strand RNA syntheses. Intriguingly, although small viral RNAs lacking the IRES replicated efficiently, the replication of genome length viral RNAs was stimulated by the presence of the IRES. These results suggest that RNA replication is not directly dependent on a template RNA first functioning as an mRNA. These results further suggest that poliovirus RNA replication is not absolutely dependent on any protein-RNA interactions involving the IRES.