cis-acting RNA elements required for replication of bovine viral diarrhea virus-hepatitis C virus 5' nontranslated region chimeras.

Pestiviruses, such as bovine viral diarrhea virus (BVDV), share many similarities with hepatitis C virus (HCV) yet are more amenable to virologic and genetic analysis. For both BVDV and HCV, translation is initiated via an internal ribosome entry site (IRES). Besides IRES function, the viral 5' nontranslated regions (NTRs) may also contain cis-acting RNA elements important for viral replication. A series of chimeric RNAs were used to examine the function of the BVDV 5' NTR. Our results show that: (1) the HCV and the encephalomyocarditis virus (EMCV) IRES element can functionally replace that of BVDV; (2) two 5' terminal hairpins in BVDV genomic RNA are important for efficient replication; (3) replacement of the entire BVDV 5' NTR with those of HCV or EMCV leads to severely impaired replication; (4) such replacement chimeras are unstable and efficiently replicating pseudorevertants arise; (5) pseudorevertant mutations involve deletion of 5' sequences and/or acquisition of novel 5' sequences such that the 5' terminal 3-4 bases of BVDV genome RNA are restored. Besides providing new insight into functional elements in the BVDV 5' NTR, these chimeras may prove useful as pestivirus vaccines and for screening and evaluation of anti-HCV IRES antivirals.     

Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication

Sequences in the 5' and 3' termini of plus-strand RNA viruses harbor cis-acting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flaviviridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5' end of the genome. This sequence contains an internal ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5' NTR required for RNA replication. We show that deletions introduced into the 5' terminal 40 nucleotides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5' NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5' NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5' NTR are sufficient for RNA replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of signals in the 5' NTR of HCV RNA essential for replication of this virus.     

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.     

Assessing IRES activity in the HIF-1alpha and other cellular 5' UTRs

Dicistronic reporter plasmids, such as the dual luciferase-containing pR-F plasmid, have been widely used to assay cellular and viral 5' untranslated regions (UTRs) for IRES activity. We found that the pR-F dicistronic reporter containing the 5' UTRs from HIF-1alpha, VEGF, c-myc, XIAP, VEGFR-1, or Egr-1 UTRs all produce the downstream luciferase predominantly as a result of cryptic promoter activity that is activated by the SV40 enhancer elements in the plasmid. RNA transfection experiments using dicistronic or uncapped RNAs, which avoid the complication of cryptic promoter activity, indicate that the HIF-1alpha, VEGF, c-myc, and XIAP UTRs do have some IRES activity, although the activity was much less than that of the viral EMCV IRES. The translation of transfected monocistronic RNAs containing these cellular UTRs was greatly enhanced by the presence of a 5' cap, raising questions as to the strength or mechanism of IRES-mediated translation in these assays.     

Genetic analysis of a poliovirus/hepatitis C virus (HCV) chimera: interaction between the poliovirus cloverleaf and a sequence in the HCV 5' nontranslated region results in a replication phenotype

Internal ribosomal entry sites (IRESs) can function in foreign viral genomes or in artificial dicistronic mRNAs. We describe an interaction between the wild-type hepatitis C virus (HCV)-specific sequence and the poliovirus (PV) 5'-terminal cloverleaf in a PV/HCV chimeric virus (containing the HCV IRES), resulting in a replication phenotype. Either a point mutation at nucleotide (nt) 29 or a deletion up to nt 40 in the HCV 5' nontranslated region relieved the replication block, yielding PV/HCV variants replicating to high titers. Fortuitous yet crippling interactions between an IRES and surrounding heterologous RNA must be considered when IRES-based dicistronic expression vectors are being constructed.     

Interaction of Poly(rC) Binding Protein 2 with the 5′ Noncoding Region of Hepatitis A Virus RNA and Its Effects on Translation

Utilization of internal ribosome entry segment (IRES) structures in the 5′ noncoding region (5′NCR) of picornavirus RNAs for initiation of translation requires a number of host cell factors whose distribution may vary in different cells and whose requirement may vary for different picornaviruses. We have examined the requirement of the cellular protein poly(rC) binding protein 2 (PCBP2) for hepatitis A virus (HAV) RNA translation. PCBP2 has recently been identified as a factor required for translation and replication of poliovirus (PV) RNA. PCBP2 was shown to be present in FRhK-4 cells, which are permissive for growth of HAV, as it is in HeLa cells, which support translation of HAV RNA but which have not been reported to host replication of the virus. Competition RNA mobility shift assays showed that the 5′NCR of HAV RNA competed for binding of PCBP2 with a probe representing stem-loop IV of the PV 5′NCR. The binding site on HAV RNA was mapped to nucleotides 1 to 157, which includes a pyrimidine-rich sequence. HeLa cell extracts that had been depleted of PCBP2 by passage over a PV stem-loop IV RNA affinity column supported only low levels of HAV RNA translation. Translation activity was restored upon addition of recombinant PCBP2 to the depleted extract. Removal of the 5′-terminal 138 nucleotides of the HAV RNA, or removal of the entire IRES, eliminated the dependence of HAV RNA translation on PCBP2.     

Suppression of hepatitis A virus genome translation and replication by siRNAs targeting the internal ribosomal entry site

Small interfering RNAs (siRNAs) targeting the coding region of hepatitis A virus (HAV) were shown to specifically inhibit viral genome replication. Compared to the coding region, the HAV internal ribosomal entry site (IRES) in the 5' non-coding region is highly sequence-conserved and folds into stable secondary structures. Here, we report efficient and sustained RNA interference mediated by both RNase III-prepared siRNA (esiRNA) and vector-derived short hairpin RNAs (shRNAs) that are targeted to various domains of the HAV IRES. Using reporter constructs, and the DNA-based HAV replicon system, we found that shRNAs targeting the HAV IRES domains IIIc and V sustainably suppressed genome translation and replication whereas the IRES domains IIIa and IV were resistant to RNA interference. Our study suggests that some HAV IRES domains might be used as a universal and effective target for specific inhibition of HAV infection.     

Stability of a stem-loop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA.

The initiation of translation on the positive-sense RNA genome of hepatitis C virus (HCV) is directed by an internal ribosomal entry site (IRES) that occupies most of the 341-nt 5' nontranslated RNA (5'NTR). Previous studies indicate that this IRES differs from picornaviral IRESs in that its activity is dependent upon RNA sequence downstream of the initiator AUG. Here, we demonstrate that the initiator AUG of HCV is located within a stem-loop (stem-loop IV) involving nt -12 to +12 (with reference to the AUG). This structure is conserved among HCV strains, and is present in the 5'NTR of the phylogenetically distant GB virus B. Mutant, nearly genome-length RNAs containing nucleotide substitutions predicted to enhance the stability of stem-loop IV were generally deficient in cap-independent translation both in vitro and in vivo. Additional mutations that destabilize the stem-loop restored translation to normal. Thus, the stability of the stem-loop is strongly but inversely correlated with the efficiency of internal initiation of translation. In contrast, mutations that stabilize this stem-loop had comparatively little effect on translation of 5' truncated RNAs by scanning ribosomes, suggesting that internal initiation of translation follows binding of the 40S ribosome directly at the site of stem-loop IV. Because stem-loop IV is not required for internal entry of ribosomes but is able to regulate this process, we speculate that it may be stabilized by interactions with a viral protein, providing a mechanism for feedback regulation of translation, which may be important for viral persistence.     

Domains I and II in the 5' nontranslated region of the HCV genome are required for RNA replication.

Hepatitis C virus (HCV), a hepacivirus member of the Flaviviridae family, has a positive-stranded RNA genome, which consists of a single open reading frame (ORF) and nontranslated regions (NTRs) at the 5' and 3' ends. The 5'NTR was found to contain an internal ribosomal entry site (IRES), which is required for the translation of HCV mRNA. Moreover, the 5'NTR is likely to play a key role in the replication of viral RNA. To identify the cis-acting element required for viral RNA replication, chimeric subgenomic replicons of HCV were generated. Dissection of the replication element from the translation element was accomplished by inserting the polioviral IRES between the serially deleted 5'NTR of HCV and ORF encoding neomycin phosphotransferase. The deletions of the 5'NTR of HCV were performed according to the secondary structure of HCV. Replicons containing domains I and II supported RNA replication and further deletion toward the 5' end abolished replication. The addition of domain III and the pseudoknot structure of the 5'NTR to domains I and II augmented the colony-forming efficiency of replicons by 100-fold. This indicates that domains I and II are necessary and sufficient for replication of RNA and that almost all of the 5'NTR is required for efficient RNA replication.     

Structural analysis of the interaction of the pyrimidine tract-binding protein with the internal ribosomal entry site of encephalomyocarditis virus and foot-and-mouth disease virus RNAs.

Initiation of translation of a subset of eukaryotic mRNAs results from internal ribosomal entry. This process is exemplified by encephalomyocarditis virus (EMCV), which contains an internal ribosomal entry site (IRES) within its 5' nontranslated region that is approximately 450-nt long and consists of a series of stem-loops designated H-L. We have previously identified a cellular 58-kDa polypeptide that binds specifically to this IRES and that is implicated in its function as the pyrimidine tract-binding protein PTB. We have now mapped PTB binding sites directly on the IRES elements of EMCV and the related foot-and-mouth disease virus (FMDV) using structure-specific enzymatic probes and base-specific chemical probes. PTB bound to six sites on the EMCV IRES: site 1 (UCUU401) is upstream of domain H, site 2 is the basal helix of domain H (nt 407-410 and 440-443), site 3 (UCUUU423) is the apical loop of domain H, site 4 is the apical helix and adjacent internal bulged loop of domain K, site 5 (CUUUA750) is the apical loop of domain K, and site 6 (CCUUU815) is downstream of domain L. PTB bound to sites on the FMDV IRES that correspond precisely to EMCV sites 3, 5, and 6. These sites have the consensus sequence CUUU and form two groups that are located near to the 5' and 3' borders of these IRES elements. Their position, and the effects of mutation of them on IRES function are consistent with PTB's role in IRES-mediated initiation being to bind to multiple sites in the IRES, thereby stabilizing a specific active conformation.     

Poliovirus 5'-terminal cloverleaf RNA is required in cis for VPg uridylylation and the initiation of negative-strand RNA synthesis

Chimeric poliovirus RNAs, possessing the 5' nontranslated region (NTR) of hepatitis C virus in place of the 5' NTR of poliovirus, were used to examine the role of the poliovirus 5' NTR in viral replication. The chimeric viral RNAs were incubated in cell-free reaction mixtures capable of supporting the sequential translation and replication of poliovirus RNA. Using preinitiation RNA replication complexes formed in these reactions, we demonstrated that the 3' NTR of poliovirus RNA was insufficient, by itself, to recruit the viral replication proteins required for negative-strand RNA synthesis. The 5'-terminal cloverleaf of poliovirus RNA was required in cis to form functional preinitiation RNA replication complexes capable of uridylylating VPg and initiating the synthesis of negative-strand RNA. These results are consistent with a model in which the 5'-terminal cloverleaf and 3' NTRs of poliovirus RNA interact via temporally dynamic ribonucleoprotein complexes to coordinately mediate and regulate the sequential translation and replication of poliovirus RNA.     

Cell type-specific proteins which interact with the 5' nontranslated region of hepatitis A virus RNA.

The 5' nontranslated region (5'NTR) of hepatitis A virus (HAV) RNA contains structural elements which facilitate 5' cap-independent initiation of virus translation and are likely to interact with cellular proteins functioning as translation initiation factors. To define these interactions, we characterized the binding of ribosome-associated proteins from several cell types to synthetic RNAs representing segments of the 5'NTR by using a UV cross-linking/label transfer assay. Four major proteins (p30, p39, p57, and p110) were identified. p30 and p39 were present in ribosomal salt washes prepared only from HAV-permissive BS-C-1 and FRhK-4 cells, while p57 was found only in HeLa cells and rabbit reticulocyte lysates. p110 was present in all cell types. Both p30 and p39 bound to multiple sites within the 5'NTR. Efficient transfer of label to p30 occurred with minimal RNA probes representing nucleotides (nt) 96 to 155, 151 to 354, and, to a much lesser extent, 634 to 744, while label transfer to p39 occurred with probes representing nt 96 to 155 and 634 to 744. All of these probes represent regions of the 5'NTR which are rich in pyrimidines. Competitive inhibition studies indicated that both p30 and p39 bound with greater affinity to sites in the 5' half of the NTR (a probe representing nt 1 to 354) than to the more 3' site (nt 634 to 744). Binding of p39 to the probe representing nt 96 to 155 was inhibited in the presence of an equal amount of proteins derived from HeLa cells, suggesting that p39 shares binding site specificity with one or more HeLa cell proteins. A 57-kDa protein in HeLa cell protein extracts reacted with antibody to polypyrimidine tract-binding protein in immunoblots, but no immunoreactive protein was identified in a similar BS-C-1 protein fraction. These results demonstrate that ribosome-associated proteins which bind to the 5'NTR of HAV vary substantially among different mammalian cell types, possibly accounting for differences in the extent to which individual cell types support growth of the virus. Mutations in the 5'NTR which enhance the growth of HAV in certain cell types may reflect specific adaptive responses to these or other proteins.     

Initiation of protein synthesis by internal entry of ribosomes into the 5' nontranslated region of encephalomyocarditis virus RNA in vivo.

Expression vectors that yield mono-, di-, and tricistronic mRNAs upon transfection of COS-1 cells were used to assess the influence of the 5' nontranslated regions (5'NTRs) on translation of reporter genes. A segment of the 5'NTR of encephalomyocarditis virus (EMCV) allowed translation of an adjacent downstream reporter gene (CAT) regardless of its position in the mRNAs. A deletion in the EMCV 5'NTR abolishes this effect. Poliovirus infection completely inhibits translation of the first cistron of a dicistronic mRNA that is preceded by the capped globin 5'NTR, whereas the second cistron preceded by the EMCV 5'NTR is still translated. We conclude that the EMCV 5'NTR contains an internal ribosomal entry site that allows cap-independent initiation of translation. mRNA containing the adenovirus tripartite leader is also resistant to inhibition of translation by poliovirus.     

Cell type-specific enhancement of hepatitis C virus internal ribosome entry site-directed translation due to 5' nontranslated region substitutions selected during passage of virus in lymphoblastoid cells

Low-level replication of hepatitis C virus (HCV) in cultured lymphoblastoid cells inoculated with H77 serum inoculum led to the appearance of new virus variants containing identical substitutions at three sites within the viral 5' nontranslated RNA (5'NTR): G(107)-->A, C(204)-->A, and G(243)-->A (N. Nakajima, M. Hijikata, H. Yoshikura, and Y. K. Shimizu, J. Virol. 70:3325-3329, 1996). These results suggest that virus with this 5'NTR sequence may have a greater capacity for replication in such cells, possibly due to more efficient cap-independent translation, since these nucleotide substitutions reside within the viral internal ribosome entry site (IRES). To test this hypothesis, we examined the translation of dicistronic RNAs containing upstream and downstream reporter sequences (Renilla and firefly luciferases, respectively) separated by IRES sequences containing different combinations of these substitutions. The activity of the IRES was assessed by determining the relative firefly and Renilla luciferase activities expressed in transfected cells. Compared with the IRES present in the dominant H77 quasispecies, an IRES containing all three nucleotide substitutions had significantly greater translational activity in three of five human lymphoblastoid cell lines (Raji, Bjab, and Molt4 but not Jurkat or HPBMa10-2 cells). In contrast, these substitutions did not enhance IRES activity in cell lines derived from monocytes or granulocytes (HL-60, KG-1, or THP-1) or hepatocytes (Huh-7) or in cell-free translation assays carried out with rabbit reticulocyte lysates. Each of the three substitutions was required for maximally increased translational activity in the lymphoblastoid cells. The 2- to 2.5-fold increase in translation observed with the modified IRES sequence may facilitate the replication of HCV, possibly accounting for differences in quasispecies variants recovered from liver tissue and peripheral blood mononuclear cells of the same patient.     

Interaction of hepatitis C virus core protein with viral sense RNA and suppression of its translation

To clarify the binding properties of hepatitis C virus (HCV) core protein and its viral RNA for the encapsidation, morphogenesis, and replication of HCV, the specific interaction of HCV core protein with its genomic RNA synthesized in vitro was examined in an in vivo system. The positive-sense RNA from the 5' end to nucleotide (nt) 2327, which covers the 5' untranslated region (5'UTR) and a part of the coding region of HCV structural proteins, interacted with HCV core protein, while no interaction was observed in the same region of negative-sense RNA and in other regions of viral and antiviral sense RNAs. The internal ribosome entry site (IRES) exists around the 5'UTR of HCV; therefore, the interaction of the core protein with this region of HCV RNA suggests that there is some effect on its cap-independent translation. Cells expressing HCV core protein were transfected with reporter RNAs consisting of nt 1 to 709 of HCV RNA (the 5'UTR of HCV and about two-thirds of the core protein coding regions) followed by a firefly luciferase gene (HCV07Luc RNA). The translation of HCV07Luc RNA was suppressed in cells expressing the core protein, whereas no significant suppression was observed in the case of a reporter RNA possessing the IRES of encephalomyocarditis virus followed by a firefly luciferase. This suppression by the core protein occurred in a dose-dependent manner. The expression of the E1 envelope protein of HCV or beta-galactosidase did not suppress the translation of both HCV and EMCV reporter RNAs. We then examined the regions that are important for suppression of translation by the core protein and found that the region from nt 1 to 344 was enough to exert this suppression. These results suggest that the HCV core protein interacts with viral genomic RNA at a specific region to form nucleocapsids and regulates the expression of HCV by interacting with the 5'UTR.     

Construction of recombinant DNA molecules by the use of a single stranded DNA generated by the polymerase chain reaction: its application to chimeric hepatitis A virus/poliovirus subgenomic cDNA.

In order to study the importance of VP4 in picornavirus replication and translation, we replaced the hepatitis A virus (HAV) VP4 with the poliovirus (PV1) VP4. Using a modification of oligonucleotide site directed mutagenesis and the polymerase chain reaction (PCR), we created a subgenomic cDNA chimera of hepatitis A virus in which the precise sequences coding for HAV VP4 capsid protein were replaced by the sequences coding for the poliovirus VP4 capsid protein. The method involved the use of PCR primers corresponding to the 3' and 5' ends of the poliovirus VP4 sequence and that had HAV VP4 3' and 5' flanking sequences on their 5'ends. Single stranded DNA of 240 and 242 nt containing the 204 nt coding for the complete poliovirus VP4 were produced by using a limiting amount of one of the primers in a PCR reaction. These single stranded PCR products were used like mutagenic oligonucleotides on a single stranded phagemid containing the first 2070 bases of the HAV genome. Using this technique, we precisely replaced the HAV VP4 gene by the poliovirus VP4 gene as determined by DNA sequencing. The cDNA was transcribed into RNA and translated in vitro. The resulting protein could be precipitated by antibody to poliovirus VP4 but not to HAV VP4.     

Inherent instability of poliovirus genomes containing two internal ribosome entry site (IRES) elements supports a role for the IRES in encapsidation

Previous studies have described poliovirus genomes in which the internal ribosome entry (IRES) for encephalomyocarditis virus (EMCV) is positioned between the P1 and P2-P3 open reading frames of the poliovirus genome. Although these dicistronic poliovirus genomes were replication competent, most exhibited evidence of genetic instability, and the EMCV IRES was deleted upon serial passage. One possible reason for instability of the genome is that the dicistronic genome was at least 108% larger than the wild-type poliovirus genome, which could reduce the efficiency of encapsidation. To address this possibility, we have constructed dicistronic poliovirus replicons by substituting the EMCV IRES and the gene encoding luciferase in place of the poliovirus P1 region; the resulting dicistronic replicons are smaller than the wild-type poliovirus genome. One dicistronic genome was constructed in which the poliovirus 5' nontranslated region was fused to the gene encoding luciferase, followed by the complete EMCV IRES fused to the P2-P3 region of the poliovirus genome (PV-Luc-EMCV). A second dicistronic genome, EMCV-Luc-PV, was constructed with the first 108 nucleotides of the poliovirus genome fused to the EMCV IRES, followed by the gene encoding luciferase and the poliovirus IRES fused to the remaining P2-P3 region of the poliovirus genome. Both dicistronic replicons expressed abundant luciferase following transfection of in vitro-transcribed RNA into HeLa cells at 30, 33, or 37 degrees C. The luciferase activity detected from PV-Luc-EMCV increased rapidly during the first 4 h following transfection and then plateaued, peaking after approximately 24 h. In contrast, the luciferase activity detected from EMCV-Luc-PV increased for approximately 12 h following transfection; by 24 h posttransfection, the overall levels of luciferase activity were similar to that of PV-Luc-EMCV. To analyze encapsidation of the dicistronic replicons, we used a system in which the capsid protein (P1) is provided in trans from a recombinant vaccinia virus (VV-P1). The PV-Luc-EMCV replicon was unstable upon serial passage in the presence of VV-P1, with deletions of the EMCV IRES region detected even during the initial transfection at 37 degrees C. Following serial passage in the presence of VV-P1 at 33 or 30 degrees C, we detected deleted genomes in which the luciferase gene was fused with the P2-P3 genes of the poliovirus genome so as to maintain the translational reading frame. In contrast, the EMCV-Luc-PV replicon was genetically stable during passage with VV-P1 at 33 or 30 degrees C. The encapsidation of EMCV-Luc-PV was compared to that of monocistronic replicons encoding luciferase with either a poliovirus or EMCV IRES. Analysis of the encapsidated replicons after four serial passages with VV-P1 revealed that the dicistronic replicon was encapsidated more efficiently than the monocistronic replicon with the EMCV IRES but less efficiently than the monicistronic replicon with the poliovirus IRES. The results of this study suggest a genetic predisposition for picornavirus genomes to contain a single IRES region and are discussed with respect to a role of the IRES in encapsidation.