Defined mutations in the 5'' nontranslated sequence of Sindbis virus RNA.

We have constructed 24 deletion mutants which contain deletions of from 1 to 15 nucleotides in the 5' nontranslated region of Sindbis virus RNA and tested the effect of these mutations on virus replication. The results showed that the first 44 nucleotides, which are capable of forming a hairpin structure, are important for virus replication, as all deletions tested in this region were either lethal or resulted in virus that grew poorly in comparison to the parental virus. Many of these deletions had different effects in mosquito cells than in chicken cells, suggesting that cellular factors, presumably proteins, bind to this region. This domain may function in at least two processes in viral replication. It seems likely that in the minus strand, this sequence element is bound by the viral replicase and promotes RNA replication. In the plus strand, this element may modulate initiation of translation of the nonstructural proteins. The results suggest that the hairpin structure itself is important. All deletions within it had deleterious effects on virus replication, and in particular, deletion of one of the G residues at nucleotide 7 or 8 or of one of the C residues at nucleotide 36 or 37 which are theoretically base-paired with these G's resulted in temperature-sensitive viruses that behaved very similarly. In contrast, large deletions between the 44-nucleotide hairpin and the translation start site at nucleotides 60 to 62 resulted in virus that grew as well as or better than the parental virus in both chicken and mosquito cells. The A residue at position 5 of the HRSP strain used was examined in more detail. Deletion of this A was lethal, whereas substitution by G resulted in a virus that grew poorly, despite the fact that G is present at position 5 in the AR339 parent of HRSP. U at position 5 resulted in a virus that grew less well than the A5 strain but better than the G5 mutant.     

Conserved tertiary structural elements in the 5' nontranslated region of cardiovirus, aphthovirus and hepatitis A virus RNAs.

Statistical analyses of RNA folding in 5' nontranslated regions (5'NTR) of encephalomyocarditis virus, Theiler's murine encephalomyelitis virus, foot-and-mouth disease virus, and hepatitis A virus indicate that two highly significant folding regions occur in the 5' and 3' portions of the 5'NTR. The conserved tertiary structural elements are predicted in the unusual folding regions (UFR) for these viral RNAs. The theoretical, common structural elements predicted in the 3' parts of the 5'NTR occur in a cis-acting element that is critical for internal ribosome binding. These structural motifs are expected to be highly significant from extensive Monte Carlo simulations. Nucleotides (nt) in the conserved single-stranded polypyrimidine tract for these RNAs are involved in a distinctively tertiary interaction that is located at about 15 nt prior to the initiator AUG. Intriguingly, the proposed common tertiary structure in this study shares a similar structural feature to that proposed in human enteroviruses and rhinoviruses. Based on these common structural features, plausible base pairing models between these viral RNAs and 18 S rRNA are suggested, which are consistent with a general mechanism for regulation of internal initiation of cap-independent translation.     

Mutations within the 5'' nontranslated region of hepatitis A virus RNA which enhance replication in BS-C-1 cells.

Passage of human hepatitis A virus (HAV) in cell culture results in attenuation of the virus as well as progressive increases in the efficiency of virus replication in cell culture. Because the presence of identical mutations within the 5' nontranslated regions (5'NTRs) of several independently isolated cell culture-adapted HAV variants suggests that the 5'NTR may play a role in determining this change in virus host range, we constructed chimeric infectious cDNA clones in which portions of the 5'NTR of cell culture-adapted HM175/p35 virus were replaced with cDNA from either wild-type virus (HM175/wt) or a second independently isolated, but closely related cell culture-adapted virus (HM175/p16). Substitution of the complete 5'NTR of HM175/p35 with the 5'NTR of HM175/wt resulted in virus with very small replication foci in continuous African green monkey kidney (BS-C-1) cells, indicating that 5'NTR mutations in HM175/p35 virus are required for optimal growth in these cells. A chimera with the 5'NTR sequence of HM175/p16 retained the large foci of HM175/p35 virus, while the growth properties of other viruses having chimeric 5'NTR sequences indicated that mutations at bases 152 and/or 203 to 207 enhance replication in BS-C-1 cells. These findings were confirmed in one-step growth experiments, which also indicated that radioimmunofocus size is a valid measure of virus replication competence in cell culture. An additional mutation at base 687 of HM175/p16 had only a minor role in enhancing growth. In contrast to their effect in BS-C-1 cells, these 5'NTR mutations did not enhance replication in continuous fetal rhesus monkey kidney (FRhK-4) cells. Thus, mutations at bases 152 and/or 203 to 207 enhance the replication of HAV in a highly host cell-specific fashion.     

Selective stimulation of hepatitis C virus and pestivirus NS5B RNA polymerase activity by GTP

NS5B of the hepatitis C virus is an RNA template-dependent RNA polymerase and therefore the key player of the viral replicase complex. Using a highly purified enzyme expressed with recombinant baculoviruses in insect cells, we demonstrate a stimulation of RNA synthesis up to 2 orders of magnitude by high concentrations of GTP but not with ATP, CTP, UTP, GDP, or GMP. Enhancement of RNA synthesis was found with various heteropolymeric RNA templates, with poly(C)-oligo(G)12 but not with poly(A)-oligo(U)12. Several amino acid substitutions in polymerase motifs B, C, and D previously shown to be crucial for RdRp activity were tested for GTP stimulation of RNA synthesis. Most of these mutations, in particular those affecting the GDD motif (motif C) strongly reduced or completely abolished activation by GTP, suggesting that the same NTP-binding site is used for stimulation and RNA synthesis. Since GTP did not affect the overall RNA binding properties or the elongation rate, high concentrations of GTP appear to accelerate a rate-limiting step at the level of initiation of RNA synthesis. Finally, enhancement of RNA synthesis by high GTP concentrations was also found with NS5B of the pestivirus classical swine fever virus, but not with the 3D polymerase of poliovirus. Thus, stimulation of RdRp activity by GTP is evolutionarily conserved between the closely related hepaciviruses and pestiviruses but not between these and the more distantly related picornaviruses.     

Core-associated non-duplex sequences distinguishing the genomic and antigenomic self-cleaving RNAs of hepatitis delta virus.

The two ribozymes found in hepatitis delta virus RNA form related but non-identical secondary structures and display similar cleavage properties in vitro. Three of the non-duplex elements hypothesized to contribute nucleotides to the catalytic core vary slightly in length between the two ribozymes and the differences are conserved in clinical isolates. Possible functional relationships of the core sequence elements were tested by systematically exchanging sequences between the two ribozymes. It was found that switching two of the elements (L3 and J4/2) from one ribozyme to the other reduced cleavage activity in both. On the other hand, exchanging the third region (J1/4) resulted in enhanced activity for one ribozyme and a smaller increase in activity for the other. Combining exchanges did not reveal any compensatory interactions involving these particular elements nor did a pattern emerge that would suggest an optimal combination of core sequences for a generalized HDV ribozyme. Non-compensatory behavior reinforces the idea that the non-duplex sequences may form sequence-specific contacts with duplex portions of the ribozyme, but, in addition, these data suggest that there may be selective pressures on the ribozyme sequences in the virus that are not reflected in the in vitro self-cleavage assays.     

Secondary structure and hybridization accessibility of hepatitis C virus 3'-terminal sequences

The 3'-terminal sequences of hepatitis C virus (HCV) positive- and negative-strand RNAs contribute cis-acting functions essential for viral replication. The secondary structure and protein-binding properties of these highly conserved regions are of interest not only for the further elucidation of HCV molecular biology, but also for the design of antisense therapeutic constructs. The RNA structure of the positive-strand 3' untranslated region has been shown previously to influence binding by various host and viral proteins and is thus thought to promote HCV RNA synthesis and genome stability. Recent studies have attributed analogous functions to the negative-strand 3' terminus. We evaluated the HCV negative-strand secondary structure by enzymatic probing with single-strand-specific RNases and thermodynamic modeling of RNA folding. The accessibility of both 3'-terminal sequences to hybridization by antisense constructs was evaluated by RNase H cleavage mapping in the presence of combinatorial oligodeoxynucleotide libraries. The mapping results facilitated identification of antisense oligodeoxynucleotides and a 10-23 deoxyribozyme active against the positive-strand 3'-X region RNA in vitro.     

Small interfering RNA targeted to hepatitis C virus 5' nontranslated region exerts potent antiviral effect

Hepatitis C virus (HCV) is a major cause of cirrhosis and hepatocellular carcinoma. Interferon alone or together with ribavirin is the only therapy for HCV infection; however, a significant number of HCV-infected individuals do not respond to this treatment. Therefore, the development of new therapeutic options against HCV is a matter of urgency. In the present study, we have examined vectors carrying short hairpin RNA (shRNA) targeting the 5' nontranslated conserved region of the HCV genome for inhibition of virus replication. Initially, three sequences were selected, and all three shRNAs (psh-53, psh-274, and psh-375) suppressed HCV internal ribosome entry site (IRES)-mediated translation to different degrees in Huh-7 cells. Next, we introduced siRNA into Huh-7.5 cells persistently infected with HCV genotype 2a (JFH1). The most efficient inhibition of JFH1 replication was observed with psh-274, targeted to the portion from subdomain IIId to IIIe of the IRES. Subsequently, Huh-7.5 cells stably expressing psh-274 further displayed a significant reduction in HCV JFH1 replication. The effect of psh-274 on cell-culture-grown HCV genotype 1a (H77) was also evaluated, and inhibition of virus replication and infectivity titers was observed. In the absence of a cell-culture-grown HCV genotype 1b, the effects of psh-274 on subgenomic and full-length replicons were examined, and efficient inhibition of genome replication was observed. Therefore, we have identified a conserved sequence targeted to the HCV genome that can inhibit replication of different genotypes, suggesting the potential of siRNA as an additional therapeutic modality against HCV infection.     

Secondary structure of the 3' terminus of hepatitis C virus minus-strand RNA

The 3'-terminal ends of both the positive and negative strands of the hepatitis C virus (HCV) RNA, the latter being the replicative intermediate, are most likely the initiation sites for replication by the viral RNA-dependent RNA polymerase, NS5B. The structural features of the very conserved 3' plus [(+)] strand untranslated region [3' (+) UTR] are well established (K. J. Blight and C. M. Rice, J. Virol. 71:7345-7352, 1997). However, little information is available concerning the 3' end of the minus [(-)] strand RNA. In the present work, we used chemical and enzymatic probing to investigate the conformation of that region, which is complementary to the 5' (+) UTR and the first 74 nucleotides of the HCV polyprotein coding sequence. By combining our experimental data with computer predictions, we have derived a secondary-structure model of this region. In our model, the last 220 nucleotides, where initiation of the (+) strand RNA synthesis presumably takes place, fold into five stable stem-loops, forming domain I. Domain I is linked to an overall less stable structure, named domain II, containing the sequences complementary to the pseudoknot of the internal ribosomal entry site in the 5' (+) UTR. Our results show that, even though the (-) strand 3'-terminal region has the antisense sequence of the 5' (+) UTR, it does not fold into its mirror image. Interestingly, comparison of the replication initiation sites on both strands reveals common structural features that may play key functions in the replication process.     

Long-range RNA-RNA interaction between the 5' nontranslated region and the core-coding sequences of hepatitis C virus modulates the IRES-dependent translation

Hepatitis C virus (HCV) is a positive-sense RNA virus approximately 9600 bases long. An internal ribosomal entry site (IRES) spans the 5' nontranslated region, which is the most conserved and highly structured region of the HCV genome. In this study, we demonstrate that nucleotides 428-442 of the HCV core-coding sequence anneal to nucleotides 24-38 of the 5'NTR, and that this RNA-RNA interaction modulates IRES-dependent translation in rabbit reticulocyte lysate and in HepG2 cells. The inclusion of the core-coding sequence (nucleotides 428-442) significantly suppressed the translational efficiency directed by HCV IRES in dicistronic reporter systems, and this suppression was relieved by site-directed mutations that blocked the long-range interaction between nucleotides 24-38 and 428-442. These findings suggest that the long-range interaction between the HCV 5'NTR and the core-coding nucleotide sequence down-regulate cap-independent translation via HCV IRES. The modulation of protein synthesis by long-range RNA-RNA interaction may play a role in the regulation of viral gene expression.