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Antigenic heterogeneity of the hepatitis C virus NS5A protein.

ABSTRACT: The effect of sequence variability between different types of hepatitis C virus (HCV) on the antigenic properties of the NS5 protein was studied by using recombinant proteins. A strong antigenic region was identified within the HCV NS5A protein at amino acids 2212 to 2313. Forty-five unique sequences encompassing this region were selected from GenBank and were compared to each other. The results of this analysis showed that the primary structure of this strong antigenic region is highly variable. Percent homology between different genotype sequences varied from 40.4 to 72.5%. Thirteen representative sequences from all six HCV genotypes were selected to design synthetic genes coding for this antigenic region. These genes were assembled by PCR from synthetic oligonucleotides and expressed in Escherichia coli as hybrid proteins with glutathione S-transferase. All 13 fusion proteins were purified from bacterial lysates and used to test a panel of anti-HCV positive sera (n = 91) obtained from patients infected with HCV genotypes 1 through 6. All but two proteins immunoreacted with 62 to 93% of HCV anti-NS5-positive serum samples. Although a variable degree of genotype-specific antigenic reactivity was detected, only one protein demonstrated a noticeable preference to immunoreact with antibodies against the homologous HCV genotype. On the other hand, closely related proteins derived from the same subtype or genotype immunoreacted with significantly different efficiency with HCV antibodies. Thus, sequence variability has a profound effect on the antigenic properties of the NS5A immunodominant regions. This observation should be taken into consideration in the development of diagnostic tests for the efficient detection of anti-HCV activity in serum specimens.

SUBMITTER: Dou XG

PROVIDER: S-EPMC120141 | biostudies-literature | 2002 Jan

REPOSITORIES: biostudies-literature

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Antigenic heterogeneity of the hepatitis C virus NS5A protein.

Dou Xiao-Guang XG Talekar Ganesh G Chang Joy J Dai Xing X Li Lixia L Bonafonte Maria-Teresa MT Holloway Brian B Fields Howard A HA Khudyakov Yury E YE

Journal of clinical microbiology 20020101 1

The effect of sequence variability between different types of hepatitis C virus (HCV) on the antigenic properties of the NS5 protein was studied by using recombinant proteins. A strong antigenic region was identified within the HCV NS5A protein at amino acids 2212 to 2313. Forty-five unique sequences encompassing this region were selected from GenBank and were compared to each other. The results of this analysis showed that the primary structure of this strong antigenic region is highly variable ...[more]

PMID: 11773093

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Project description:Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A; however, the mechanistic details of the role of this posttranslational modification in the virus life cycle remain obscure. We previously reported (D. Ross-Thriepland, J. Mankouri, and M. Harris, J Virol 89:3123-3135, 2015, doi:10.1128/JVI.02995-14) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication. A phosphoablatant (S225A) mutation resulted in a 10-fold reduction in replication and a perinuclear restricted distribution of NS5A, whereas the corresponding phosphomimetic mutation (S225D) had no phenotype. To determine the molecular mechanisms underpinning this phenotype we conducted a label-free proteomics approach to identify cellular NS5A interaction partners. This analysis revealed that the S225A mutation disrupted the interactions of NS5A with a number of cellular proteins, in particular the nucleosome assembly protein 1-like protein 1 (NAP1L1), bridging integrator 1 (Bin1, also known as amphiphysin II), and vesicle-associated membrane protein-associated protein A (VAP-A). These interactions were validated by immunoprecipitation/Western blotting, immunofluorescence, and proximity ligation assay. Importantly, small interfering RNA (siRNA)-mediated knockdown of NAP1L1, Bin1 or VAP-A impaired viral genome replication and recapitulated the perinuclear redistribution of NS5A seen in the S225A mutant. These results demonstrate that S225 phosphorylation regulates the interactions of NS5A with a defined subset of cellular proteins. Furthermore, these interactions regulate both HCV genome replication and the subcellular localization of replication complexes.IMPORTANCE Hepatitis C virus is an important human pathogen. The viral nonstructural 5A protein (NS5A) is the target for new antiviral drugs. NS5A has multiple functions during the virus life cycle, but the biochemical details of these roles remain obscure. NS5A is known to be phosphorylated by cellular protein kinases, and in this study, we set out to determine whether this modification is required for the binding of NS5A to other cellular proteins. We identified 3 such proteins and show that they interacted only with NS5A that was phosphorylated on a specific residue. Furthermore, these proteins were required for efficient virus replication and the ability of NS5A to spread throughout the cytoplasm of the cell. Our results help to define the function of NS5A and may contribute to an understanding of the mode of action of the highly potent antiviral drugs that are targeted to NS5A.

Project description:RNA viruses, such as hepatitis C virus (HCV), influenza virus, and SARS-CoV-2, are notorious for their ability to evolve rapidly under selection in novel environments. It is known that the high mutation rate of RNA viruses can generate huge genetic diversity to facilitate viral adaptation. However, less attention has been paid to the underlying fitness landscape that represents the selection forces on viral genomes, especially under different selection conditions. Here, we systematically quantified the distribution of fitness effects of about 1,600 single amino acid substitutions in the drug-targeted region of NS5A protein of HCV. We found that the majority of nonsynonymous substitutions incur large fitness costs, suggesting that NS5A protein is highly optimized. The replication fitness of viruses is correlated with the pattern of sequence conservation in nature, and viral evolution is constrained by the need to maintain protein stability. We characterized the adaptive potential of HCV by subjecting the mutant viruses to selection by the antiviral drug daclatasvir at multiple concentrations. Both the relative fitness values and the number of beneficial mutations were found to increase with the increasing concentrations of daclatasvir. The changes in the spectrum of beneficial mutations in NS5A protein can be explained by a pharmacodynamics model describing viral fitness as a function of drug concentration. Overall, our results show that the distribution of fitness effects of mutations is modulated by both the constraints on the biophysical properties of proteins (i.e., selection pressure for protein stability) and the level of environmental stress (i.e., selection pressure for drug resistance).IMPORTANCE Many viruses adapt rapidly to novel selection pressures, such as antiviral drugs. Understanding how pathogens evolve under drug selection is critical for the success of antiviral therapy against human pathogens. By combining deep sequencing with selection experiments in cell culture, we have quantified the distribution of fitness effects of mutations in hepatitis C virus (HCV) NS5A protein. Our results indicate that the majority of single amino acid substitutions in NS5A protein incur large fitness costs. Simulation of protein stability suggests viral evolution is constrained by the need to maintain protein stability. By subjecting the mutant viruses to selection under an antiviral drug, we find that the adaptive potential of viral proteins in a novel environment is modulated by the level of environmental stress, which can be explained by a pharmacodynamics model. Our comprehensive characterization of the fitness landscapes of NS5A can potentially guide the design of effective strategies to limit viral evolution.

Dataset Information

Antigenic heterogeneity of the hepatitis C virus NS5A protein.

Publications

Antigenic heterogeneity of the hepatitis C virus NS5A protein.

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