Alanine Scanning of the Hepatitis C Virus Core Protein Reveals Numerous Residues Essential for Production of Infectious Virus

Murray, Catherine; Jones, Christopher T.; Tassello, Jodie; Rice, Charles M.

doi:10.1128/jvi.00793-07

Cited by 104 publications

(148 citation statements)

References 57 publications

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“…Several experiments have provided strong evidence of extensive epistasis in RNA viruses, confirming that during viral evolution certain substitutions at different sites may occur in a coordinated manner (7)(8)(9)(10)(11)(12). Experimental studies into the adaptation of genetically modified HCV genomes propagated in cell culture also have shown antagonistic epistatic interactions between deleterious mutations exhibited in the form of compensatory mutations (13)(14)(15). These results suggest the importance of long-range interactions among HCV constitutive amino acids that place additional constraints on HCV heterogeneity.…”

mentioning

confidence: 76%

Coordinated evolution of the hepatitis C virus

Campo

Dimitrova

Mitchell

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Hepatitis C virus is a genetically heterogeneous RNA virus that is a major cause of liver disease worldwide. Here, we show that, despite its extensive heterogeneity, the evolution of hepatitis C virus is primarily shaped by negative selection and that numerous coordinated substitutions in the polyprotein can be organized into a scale-free network whose degree of connections between sites follows a power-law distribution. This network shares all major properties with many complex biological and technological networks. The topological structure and hierarchical organization of this network suggest that a small number of amino acid sites exert extensive impact on hepatitis C virus evolution. Nonstructural proteins are enriched for negatively selected sites of high centrality, whereas structural proteins are enriched for positively selected sites located in the periphery of the network. The complex network of coordinated substitutions is an emergent property of genetic systems with implications for evolution, vaccine research, and drug development. In addition to such properties as polymorphism or strength of selection, the epistatic connectivity mapped in the network is important for typing individual sites, proteins, or entire genetic systems. The network topology may help devise molecular intervention strategies for disrupting viral functions or impeding compensatory changes for vaccine escape or drug resistance mutations. Also, it may be used to find new therapeutic targets, as suggested in this study for the NS4A protein, which plays an important role in the network.complex systems ͉ scale-free network ͉ covariation ͉ natural selection ͉ epistasis H epatitis C virus (HCV) is a major cause of liver disease worldwide. The global prevalence of HCV infection is estimated to be 2.2%, representing 130 million people (1). HCV causes chronic infection in 70-85% of infected adults (2). There is no vaccine against HCV and current antiviral therapy is relatively toxic, being effective in 50-60% of patients treated (3). HCV is a single-stranded RNA virus of Ϸ9.4 kb belonging to the Flaviviridae family (4). The positive-sense genome of HCV contains one large ORF that encodes a polyprotein that can undergo proteolytic cleavage into 10 mature proteins (C-E1-E2-P7-NS2-NS3-NS4A-NS4B-NS5A-NS5B). The structural proteins, the core (C) and envelope glycoproteins E1 and E2, are present in the N-terminal part of the polyprotein and presumably self-assemble to form the virion. The nonstructural (NS) proteins have various functions and form the replication complex (5).The HCV genome continually mutates during virus replication. Although a high rate of mutation significantly contributes to the enormous adaptability of RNA viruses, it also limits the size of viral genomes by causing error catastrophe (6). The small size of viral genomes imposes strong evolutionary constraints on their organization, as a result of which each genomic region may encode multiple and often conflicting functions. Such genomic organization requires a tight coor...

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mentioning

confidence: 76%

Coordinated evolution of the hepatitis C virus

Campo

Dimitrova

Mitchell

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…However, only recently has a cell culture system been made available for HCV, supporting production of infectious virus in the Huh-7 human hepatoma cell line [4][5][6][7]. The HCV virion is considered to contain a capsid built of the virally encoded core protein, whose structural integrity has been shown to be critical for production of infectious virus in this model [8]. Beside its role as a structural component of the virion, HCV core protein has been assigned a number of regulatory functions and roles in the virus-induced pathogenesis, including liver steatogenesis and carcinogenesis [2,9,10].…”

Section: Introductionmentioning

confidence: 99%

Sequential processing of hepatitis C virus core protein by host cell signal peptidase and signal peptide peptidase: a reassessment

Pène

Hernandez

Vauloup‐Fellous

et al. 2009

Journal of Viral Hepatitis

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SUMMARY. Hepatitis C virus (HCV) core protein is believed to play critical roles in the virus morphogenesis and pathogenesis. In HCV polyprotein, core protein terminates with a signal peptide followed by E1 envelope protein. It has remained unclear whether cleavage by host cell signal peptidase (SP) at the core-E1 junction to generate the complete form of core protein, which is anchored in the endoplasmic reticulum membrane, is absolutely required for cleavage within the signal peptide by host cell signal peptide peptidase (SPP) to liberate the mature form of core protein, which is then free for trafficking to lipid droplets. In this study, the possible sources of disagreement in published reports have been examined, and we conclude that a product generated upon inhibition of SP-catalysed cleavage at the core-E1 junction in heterologous expression systems was incorrectly identified as mature core protein. Moreover, inhibition of this cleavage in the most relevant model of human hepatoma cells replicating a full-length HCV genome was shown to abolish interaction of core protein with lipid droplets and production of infectious progeny virus. These results firmly establish that SPP-catalysed liberation of mature core protein is absolutely dependent on prior cleavage by SP at the correct core-E1 site to generate the complete form of core protein, consistent with this obligatory order of processing playing a role in HCV infectious cycle.

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“…There is evidence that mutations in the BD2 region (contains the major binding site for NS5A) Fig.1. Hepatitis C core region 1-177 depicting the domains, functions, mutations identified in the study and known epitope regions can suppress binding to lipid droplets and reduce viral particle production, infectivity and release (Murray et al, 2007;Gawlik et al, 2014). The core: R70Q and L91M mutations have been associated with poor treatment response in genotype 1b infection to IFN (Akuta et al, 2010, Alhamlan et al, 2014, resistance to telaprevir combination therapy (Akuta et al, 2010), rate of progression to HCC (Araujo et al, 2014, Nakamoto et al, 2010, Khan et al, 2010, increased steatosis and hepatic oxidative stress (Tachi et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Naturally occurring resistance mutations within the core and NS5B regions in hepatitis C genotypes, particularly genotype 5a, in South Africa

et al. 2016

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Approximately 1 million South Africans are infected with Hepatitis C virus (HCV). The standard of care (SOC) in South Africa is combination therapy (pegylated interferon and ribavirin). HCV genotypes and/or mutations in the core/ non-structural regions have been associated with response to therapy and/or disease progression. This study examines mutations in the core (29-280 amino acids, including ~90 E1 amino acids) and NS5B (241-306 amino acids) regions on pre-treatment isolates from patients attending Johannesburg hospitals or asymptomatic South African blood donors. Diversity within known CD4+ and CD8+ T-cell epitopes was also explored. Samples grouped into subtypes 1a (N=10) 1b (N=12), 3a(N=5), 4a (N=3) and 5a(N=61). Two mutations, associated with interferon resistance-R70Q and T110N-were present in 29 genotype 5a core sequences. No resistance mutation to NS5B nucleotide inhibitors, sofosbuvir was found. Six putative CD8+ and one CD4+ T-cell epitope sequence in the core region showed binding scores of <300 IC 50 nM to HLA alleles frequently observed in the South African population. No known CD8+ and CD4+ T-cell epitopes were mapped in the NS5B region. The analysis begs the question whether those infected with genotype 5a will benefit better on interferon-free combination therapies. This study provides new insight into one of the lesser studied HCV genotypes and compares the diversity seen in a large pre-treatment cohort with other subtypes. KeywordsHepatitis C, genotype 5, interferon, mutations, therapy Highlights  We examined mutational changes of hepatitis C in the core/E1 and NS5B genes. Phylogenetic analyses were performed using Bayesian inferences. Diversity within known CD4+ and CD8+ T-cell epitopes was also explored. Mutations, associated with interferon resistance, were present in genotype 5a samples at baseline. 2

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Alanine Scanning of the Hepatitis C Virus Core Protein Reveals Numerous Residues Essential for Production of Infectious Virus

Cited by 104 publications

References 57 publications

Coordinated evolution of the hepatitis C virus

Coordinated evolution of the hepatitis C virus

Sequential processing of hepatitis C virus core protein by host cell signal peptidase and signal peptide peptidase: a reassessment

Naturally occurring resistance mutations within the core and NS5B regions in hepatitis C genotypes, particularly genotype 5a, in South Africa

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