Effects of Mutations of the Initiation Nucleotides on Hepatitis C Virus RNA Replication in the Cell

Abstract

Replication of nearly all RNA viruses depends on a virus-encoded RNA-dependent RNA polymerase (RdRp). Our earlier work found that purified recombinant hepatitis C virus (HCV) RdRp (NS5B) was able to initiate RNA synthesis de novo by using purine (A and G) but not pyrimidine (C and U) nucleotides (G. Luo et al., J. Virol. 74:851-863, 2000). For most human RNA viruses, the initiation nucleotides of both positive-and negative-strand RNAs were found to be either an adenylate (A) or guanylate (G). To determine the nucleotide used for initiation and control of HCV RNA replication, a genetic mutagenesis analysis of the nucleotides at the very 5 and 3 ends of HCV RNAs was performed by using a cell-based HCV replicon replication system. Either a G or an A at the 5 end of HCV genomic RNA was able to efficiently induce cell colony formation, whereas a nucleotide C at the 5 end dramatically reduced the efficiency of cell colony formation. Likewise, the 3-end nucleotide U-to-C mutation did not significantly affect the efficiency of cell colony formation. In contrast, a U-to-G mutation at the 3 end caused a remarkable decrease in cell colony formation, and a U-to-A mutation resulted in a complete abolition of cell colony formation. Sequence analysis of the HCV replicon RNAs recovered from G418-resistant Huh7 cells revealed several interesting findings. First, the 5-end nucleotide G of the replicon RNA was changed to an A upon multiple rounds of replication. Second, the nucleotide A at the 5 end was stably maintained among all replicon RNAs isolated from Huh7 cells transfected with an RNA with a 5-end A. Third, initiation of HCV RNA replication with a CTP resulted in a >10-fold reduction in the levels of HCV RNAs, suggesting that initiation of RNA replication with CTP was very inefficient. Fourth, the 3-end nucleotide U-to-C and -G mutations were all reverted back to a wild-type nucleotide U. In addition, extra U and UU residues were identified at the 3 ends of revertants recovered from Huh7 cells transfected with an RNA with a nucleotide G at the 3 end. We also determined the 5-end nucleotide of positive-strand RNA of some clinical HCV isolates. Either G or A was identified at the 5 end of HCV RNA genome depending on the specific HCV isolate. Collectively, these findings demonstrate that replication of positive-strand HCV RNA was preferentially initiated with purine nucleotides (ATP and GTP), whereas the negative-strand HCV RNA replication is invariably initiated with an ATP. Hepatitis C virus (HCV) is an enveloped RNA virus of the genus Hepacivirus of the Flaviviridae family (16, 46). It causes chronic infections of approximately 4 million people in the UnitedStates and 170 million people worldwide (10, 55). The majority (ϳ85%) of individuals with HCV infection develop chronic hepatitis, which leads to cirrhosis (10 to 20%) and hepatocellular carcinoma (1 to 5%). HCV infection results in 10,000 to 12,000 deaths each year in the United States alone, a figure expected to triple within the next 10 to 20 years if no effective in...

Mechanistic characterization of the 5′-triphosphate-dependent activation of PKR: Lack of 5′-end nucleobase specificity, evidence for a distinct triphosphate binding site, and a critical role for the dsRBD

“…Secondary structure prediction via free energy minimization (mFold) indicates that this RNA is essentially completely unstructured. (D) Experimentally determined secondary structure of ss-dsRNA (9,11) (Zheng and Bevilacqua 2004). typically require G in the first position for transcription initiation (Milligan and Uhlenbeck 1989), bacterial and viral RNAs are initiated by G or A, and even occasionally by C (Bieger and Nierlich 1989;Luo et al 2000;Cai et al 2004). Base specificity of PKR activation has implications for both specificity regarding the structural nature of the triphosphatebinding site in PKR and for the functional permissiveness of PKR activation by non-self ppp-RNAs in innate immunity.…”

MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR

“…2) (25). In our 5′ UTR-NS2 culture systems, 5′-terminal G was in all cases changed to A despite early efficient replication.…”

“…The prototype JFH1 genome naturally has A at the 5′ terminus (9). However, this change could also be an in vitro effect, because in the Con1 replicon system 5′-terminal G was changed to A upon multiple rounds of replication (25). Thus, future studies analyzing the 5′ terminus of JFH1-based 5′ UTR-NS2 HCV recombinants from in vivo transfection-derived infections would be of interest to further study this phenomenon.…”

An in vitro model of hepatitis C virion production

“…15 and 18. The G3A switch of the initial nucleotide of HCV is associated with replication in vivo and in vitro (15). A transposition from an A-T to a T-A base pairing has also been reported (18) and represents a base pair in the putative terminal stem loop of the 3Ј end of HCV.…”

“…Both the 5Ј and 3Ј ends of HCV RNA from the culture medium were successfully determined. Interestingly, a change in the most 5Ј nucleotide from G to A was noted; this change has been frequently observed in HCV RNA replicons and circulating HCV RNA in infected humans (15). In the 3Ј end, two nucleotide changes in the stem loop region were noted: U3A and A3U.…”