Stimulation of Hepatitis C Virus (HCV) Nonstructural Protein 3 (NS3) Helicase Activity by the NS3 Protease Domain and by HCV RNA-Dependent RNA Polymerase (original) (raw)
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Hepatitis C Virus Non-structural Protein 3 (HCV NS3): A Multifunctional Antiviral Target
Journal of Biological Chemistry, 2010
Hepatitis C virus non-structural protein 3 contains a serine protease and an RNA helicase. Protease cleaves the genomeencoded polyprotein and inactivates cellular proteins required for innate immunity. Protease has emerged as an important target for the development of antiviral therapeutics, but drug resistance has turned out to be an obstacle in the clinic. Helicase is required for both genome replication and virus assembly. Mechanistic and structural studies of helicase have hurled this enzyme into a prominent position in the field of helicase enzymology. Nevertheless, studies of helicase as an antiviral target remain in their infancy.
Journal of Virology, 2009
We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3 domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone, JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and, furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against extrapolation of data from culture-adapted replicons to infectious virus.
Analysis of NS3-mediated processing of the hepatitis C virus non-structural region in vitro
Journal of General Virology, 1994
The protease activity of the hepatitis C virus (HCV) NS3 protein has been investigated using transient expression methods in mammalian cells, as well as in vitro transcription/translation systems. We confirmed that expression of the NS3-5 polyprotein in rabbit reticulocyte lysates results in efficient cis processing at the NS3/NS4 junction. However, processing at the other predicted sites of NS3-mediated cleavage varied markedly in efficiency, the site most susceptible being that between NS5A and NS5B. Time-course analysis of the proteolytic processing of the HCV non-structural precursor showed that the cis cleavage between NS3 and NS4 occurred extremely rapidly. However, efficient cleavage at this position was dependent on the prior removal of the NS2 protein. Furthermore, the presence of uncleaved NS2 sequences on the enzyme severely impeded NS3-mediated proteolysis at downstream sites in the polyprotein. This suggests therefore that efficient cleavage at the NS2/NS3 junction is a pivotal event in HCV replication. During the course of this study a proteolytically inactive mutant of NS3 was characterized carrying a previously unreported amino acid substitution near the proposed active site of the enzyme. Molecular modelling suggested that the amino acid present at this position may influence the conformation of the active site of the enzyme. Recently a number of reports have described a second protease activity, located in the NS2/NS3 region, which is responsible for cleavage at the NS2/NS3 junction. We have identified an isolate of HCV, obtained from a U.K. patient, which has a virtually inactive NS2/NS3 protease. The possible implications of this observation are discussed.
Domain III of NS5A contributes to both RNA replication and assembly of hepatitis C virus particles
Journal of General Virology, 2009
The hepatitis C virus (HCV) NS5A protein plays a critical role in viral RNA replication and has recently been shown to play a role in particle production in the infectious genotype 2a HCV clone (JFH-1). Here, we show that alanine substitutions of serines 2428/2430 within the C-terminal domain III of NS5A do not affect subgenomic replicon RNA replication but do reduce particle production. In contrast, substitution of serines 2390/2391 had no effect on either RNA replication or particle production. Relative to genotype 1, all genotype 2 HCV isolates contain a 19 residue insertion near the C terminus of domain III which, when deleted (n2408-2426), resulted in a delay to both RNA replication and particle production. None of these mutations affected the ratio of basal to hyperphosphorylated NS5A, suggesting that serines between residues 2390 and 2430 are not phosphorylated. We propose that although domain III is dispensable for RNA replication, it nevertheless influences this process.
Journal of Virology, 2001
The hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be involved in viral RNA replication. We have expressed and purified recombinant NS3 (protease and helicase domains) and ⌬pNS3 (helicase domain only) and examined their abilities to interact with the 3-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were chosen because initiation of RNA synthesis is likely to occur at or near the 3 untranslated region (UTR). The results presented here demonstrate that NS3 (and ⌬pNS3) interacts efficiently and specifically with the 3-terminal sequences of both positive-and negative-strand RNA but not with the corresponding complementary 5-terminal RNA sequences. The interaction of NS3 with the 3-terminal negative strand [called 3(؊) UTR 127 ] was specific in that only homologous (and not heterologous) RNA competed efficiently in the binding reaction. A predicted stem-loop structure present at the 3 terminus (nucleotides 5 to 20 from the 3 end) of the negative-strand RNA appears to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and ⌬pNS3) binding. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggested that both a double-stranded structure and the 3-proximal guanosine residues in the stem were important determinants of protein binding. In contrast to the relatively stringent requirement for 3(؊) UTR binding, specific interaction of NS3 (or ⌬pNS3) with the 3-terminal sequences of the positive-strand RNA [3(؉) UTR] appears to require the entire 3(؉) UTR of HCV. Deletion of either the 98-nucleotide 3-terminal conserved region or the 5 half sequence containing the variable region and the poly(U) and/or poly(UC) stretch significantly impaired RNA-protein interaction. The implication of NS3 binding to the 3-terminal sequences of viral positive-and negative-strand RNA in viral replication is discussed.
Internal processing of hepatitis C virus NS3 protein
Virology, 1999
Hepatitis C virus (HCV) NS3 protein contains at least three enzymatic activities: NS2-3 protease, NS3 serine protease, and NTPase/RNA helicase. It has been shown that NS2/3 cleavage is mediated by NS2-3 protease, whereas NS3 serine protease is responsible for the other four cleavage sites of the nonstructural (NS) region. In this study, we showed that the internal cleavage of NS3 protein produced two products of 49 kDa (NS3a) and 23 kDa (NS3b) when the entire NS3 region (aa 1027-1657) or the whole open reading frame (aa 1-3010) was expressed in mammalian and insect cells. By means of site-directed mutagenesis, we demonstrated that NS3a/NS3b cleavage occurs within the RNA helicase sequence motif that is highly conserved in the Flaviviridae family and that neither NS2-3 protease nor NS3 serine protease was responsible for this cleavage. The NS3 protease of flaviviruses, dengue virus type 2, for example, has been shown to mediate the internal cleavage of NS3. The NS3 proteins of HCV an...
Molecular Biology of Hepatitis C Virus: An Overview
The Hepatitis C virus (HCV) is an enveloped, positive strand RNA virus and has been classified as a separate genus Hepacivirus of the family Flaviviridae. The HCV genome is a linear molecule with a length of approximately 9.6 kb that is flanked by 5` and 3' untranslated regions (UTRs) and encodes to a nearly 3,010 amino acids polyprotein precursor. It is cleaved by combination of viral and host proteases into structural (C, P7, El, and E2) and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B), essential for viral replication and viron formation in post-translational process. The 5` and 3' UTRs are highly conserved regions in HCV genome, crucial for molecular processes such as replication and translation. In this review, we summarize the current knowledge about HCV molecular and structural HCV biology. This knowledge may help to improve treatment strategies and development of vaccine against HCV.
Hepatology, 1999
The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is considered to possess RNA-dependent RNA polymerase (RdRp) activity and to play an essential role for the viral replication. In this study, we expressed the NS5B protein of 65 kd by a recombinant baculovirus. With the highly purified NS5B protein, we established an in vitro system for RdRp activity by using poly(A) as a template and a 15-mer oligo(U) (oligo(U) 15 ) as a primer. Optimal conditions of temperature and pH for primer-dependent polymerase activity of the NS5B were 32ЊC and pH 8.0. The addition of 10 mmol of Mg 2؉ increased the activity. The importance of three motifs conserved in RdRp among other positivestrand RNA viruses was confirmed by introduction of an Ala residue to every amino acid of the motifs by sitedirected mutagenesis. All mutants lost RdRp activity, but retained the RNA binding activity, except one mutant at Thr 287 /Asn 291 . Deletion mutant analysis indicated that the N-terminal region of NS5B protein was critical for the RNA binding. Inhibition of RdRp activity by (؊)-L-2Ј,3Ј-dideoxy-3Ј-thiacytidine 5Ј-triphosphate (3TC; lamivudine triphosphate) and phosphonoacetic acid (PAA) was observed after screening of nucleoside analogs and known polymerase inhibitors. These data provide us not only important clues for understanding the mechanism of HCV replication, but also a new target of antiviral therapy. (HEPATOLOGY 1999;29: 1227-1235.)