Structural and Functional Analysis of Hepatitis C Virus Strain JFH1 Polymerase

Abstract

The hepatitis C virus (HCV) isolate JFH1 represents the only cloned wild-type sequence capable of efficient replication in cell culture, as well as in chimpanzees. Previous reports have pointed to the viral polymerase NS5B as a major determinant for efficient replication of this isolate. To understand the underlying mechanisms, we expressed and purified NS5B of JFH1 and of the closely related isolate J6, which replicates below the limit of detection in cell culture. The JFH1 enzyme exhibited a 5-to 10-fold-higher specific activity in vitro, consistent with the polymerase activity itself contributing to efficient replication of JFH1. The higher in vitro activity of the JFH1 enzyme was not due to increased RNA binding, elongation rate, or processivity of the polymerase but to higher initiation efficiency. By using homopolymeric and heteropolymeric templates, we found that purified JFH1 NS5B was significantly more efficient in de novo initiation of RNA synthesis than the J6 counterpart, particularly at low GTP concentrations, probably representing an important prerequisite for the rapid replication kinetics of JFH1. Furthermore, we solved the crystal structure of JFH1 NS5B, which displays a very closed conformation that is expected to facilitate de novo initiation. Structural analysis shows that this closed conformation is stabilized by a sprinkle of substitutions that together promote extra hydrophobic interactions between the subdomains "thumb" and "fingers." These analyses provide deeper insights into the initiation of HCV RNA synthesis and might help to establish more efficient cell culture models for HCV using alternative isolates.Hepatitis C virus (HCV) is an enveloped positive-strand RNA virus belonging to the genus Hepacivirus in the family Flaviviridae. The genome of HCV encompasses a single ϳ9,600-nucleotide (nt)-long RNA molecule carrying one large open reading frame flanked by nontranslated regions (NTRs), which is primarily translated into one polyprotein. The polyprotein precursor is cleaved by cellular and viral proteases into at least 10 different products (for a review, see reference 5). The nonstructural proteins NS3 to NS5B are necessary and sufficient for autonomous RNA replication. They form a membraneassociated replication complex in which NS5B is the RNAdependent RNA polymerase (RdRp), the key enzyme of viral-RNA replication.Studies of HCV have long been hampered by the lack of efficient cell culture systems. The advent of subgenomic replicons derived from genotype 1 isolates allowed for the first time a concise analysis of authentic viral-RNA replication (29).However, these isolates required adaptive mutations for efficient propagation in cells (9,21,27). Based on these adapted viral sequences, it was not possible to generate infectious virus, since most of these mutations seemed to interfere with virus assembly (38), and only one cell culture-adapted genotype 1 isolate has yet given rise to moderately efficient production of infectious virus (49). This restriction was abolished by a particu...

A Comprehensive Structure-Function Comparison of Hepatitis C Virus Strain JFH1 and J6 Polymerases Reveals a Key Residue Stimulating Replication in Cell Culture across Genotypes

“…Murayama et al took advantage of the properties of these isolates, generating chimeric replicons and demonstrating that the NS3 helicase-coding region, NS5B, and the 3ЈNTR were the major elements for efficient JFH1 replication, in which NS5B was the most important single determinant (32). In a subsequent study we found that the JFH1 polymerase indeed exhibited a 5-to 10-fold-higher specific activity in vitro than the J6 enzyme, consistent with the polymerase activity itself contributing to efficient replication of JFH1 (41). This was due to significantly more efficient de novo RNA synthesis of JFH1 than of J6 NS5B.…”

“…Differences in main-chain conformation between pairs of molecules were objectively assessed using the program ESCET (39). As in our previous work (20,41), we used significance values (ESCET "lolim" parameter) of 5.0 sigmas to identify broader conformational differences and 2.5 sigmas to take into account all significant conformational differences. When comparing the two high-resolution structures reported here, we also used a lolim value of 2.0.…”

“…Glucose (1%) was added to repress NS5B expression in all media except the induction medium. Proteins for biochemical analysis were expressed and purified exactly as described previously (41). Purified NS5B was quantified by the Bradford method and stored in small aliquots at Ϫ70°C.…”

“…For the analysis of the enzymatic activities of NS5B from JFH1 and J6 and chimeras thereof, the corresponding regions of subgenomic replicons (see above) were transferred into expression plasmids encoding the different NS5B proteins lacking the 21 C-terminal amino acids (⌬C21) and C-terminally fused to 6ϫHis in a pET21b vector that has been described previously (41).…”

Norovirus RNA-Dependent RNA Polymerase Is Phosphorylated by an Important Survival Kinase, Akt

“…The differences in enzyme kinetics between the wild-type and phosphomimetic RdRp suggest that the phosphorylation of Thr33 may modulate the function of the polymerase. In HCV, it has been shown that disruptions to the interactions between the finger and thumb domains lead to a reduction in de novo RdRp activity whereas efficient primer extension activity is maintained (9,10,24). Since NoV utilizes both de novo and proteinprimed RNA synthesis, it is possible that phosphorylation of NoV RdRp could alter the balance between these modes of RNA synthesis.…”