Oligomerization and Cooperative RNA Synthesis Activity of Hepatitis C Virus RNA-Dependent RNA Polymerase

Abstract

The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, K d , of Ϸ22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 Å. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data.Hepatitis C virus (HCV) belongs to the Flaviviridae family and is responsible for a significant proportion of acute and chronic hepatitis in humans worldwide (7,8). Similar to other flaviviruses, HCV is a small positive-stranded RNA virus with a genome size of Ϸ9.6 kb encoding a single polyprotein (30). This viral polyprotein is processed by both host and virally encoded proteases to generate mature structural and nonstructural proteins essential for virus replication (see references 9, 34, and 39 for review). One of the nonstructural proteins, designated NS5B, is the virally encoded RNA-dependent RNA polymerase (RdRp), which contains the GDD signature motif of RNA polymerases (3).It has been shown that NS5B is a membrane-associated protein, which contains a C-terminal domain comprising Ϸ21 hydrophobic amino acids that is responsible for membrane anchorage (41). NS5B may form a complex with cellular proteins (37) or other HCV nonstructural proteins, including NS3, the viral protease and helicase; NS4A, a cofactor of NS3 protease activity; and NS5A, a phosphoprotein containing a putative interferon sensitivity region (15). Although the HCV replication mechanism is not clearly understood, the essential role of NS5B polymerase in the HCV replication and infection process has been demonstrated in chimpanzees (22). Accordingly, it has been viewed as an attractive target for antiviral intervention.Recombinant HCV NS5B polymerase has been produced and purified from both bacterial and insect cells by several groups (3,11,16,19,25,27,31,41). The availability of highly purified protein has facilitated the biochemical characterization of HCV NS5B polymerase. Similar to other viral RdRps, purified HCV NS5B is able to synthesize RNA using various RNAs as templates in vitro (3,11,16,19,25,27,31,41). In this regard, two RNA synthesis reaction modes have been described for this enzyme: RNA elongation using a preannealed primer and RNA in...

Hepatitis c virus and host cell lipids: An intimate connection

“…71 Accordingly, NS5B's catalytic activity is believed to be regulated by conformational changes and by its ability to form oligomers. 72 The latter hypothesis is, however, discussed controversially. 73 Enzymatic activity of genotype 1b-derived NS5B might be stimulated by conformational changes that are triggered by sphingomyelin binding, 74 which also appears to contribute to NS5B localization in lipid rafts.…”

Control of Template Positioning during de Novo Initiation of RNA Synthesis by the Bovine Viral Diarrhea Virus NS5B Polymerase

“…It is therefore unlikely that the HCV enzyme contains a functionally equivalent G-specific binding site adjacent to the active site. Here the requirement for high concentrations of GTP may have different reasons, which remains to be addressed (37)(38)(39)(40)(41). For the BVDV enzyme, high concentrations of GTP, and, ultimately its binding to the G-site, could help orient the priming nucleotide, and binding of GTP may also help position the 3Ј-end of the template to facilitate the formation of the first phosphodiester bond.…”

Oligomerization of the influenza virus polymerase complex in vivo

“…At present we cannot verify the existence of the polymerase oligomeric forms in virus-infected cells but, if this were the case, these polymerase associations might be important for RNA transcription and/or replication in the infection cycle, as has been previously reported for positive-stranded RNA viruses, like poliovirus or hepatitis C virus (Lyle et al, 2002;Wang et al, 2002) and negative-stranded RNA viruses, like Sendai virus (Cevik et al, 2003;Smallwood et al, 2002). In the influenza RNA replication process, the first nascent virus RNA contains the 59-terminal conserved sequence, which is an efficient and specific binding site for the polymerase (González & Ortín, 1999;Lee et al, 2002;Tiley et al, 1994).…”