RESUMO
BACKGROUND/AIMS: Patients infected with hepatitis C virus (HCV) genotype 2 or 3 usually respond better to interferon (IFN) treatment than those infected with genotype 1. In this study, we investigated whether the non-structural 5A protein (NS5A) of HCV genotypes 1 and 2 (1b-NS5A and 2a-NS5A, respectively) exerted differential counteractivities against IFN treatment. METHODS: We compared the inhibitory effects of 1b-NS5As and 2a-NS5As on IFN activity. We also investigated the replication inhibition of HCV subgenomic replicons containing 1b-NS5A or 2a-NS5A in response to IFN treatment. RESULTS: 1b-NS5As exerted more profound inhibitory effects on IFN activity than 2a-NS5As. The replication of the 2a-NS5A-containing replicons was more sensitive to IFN treatment than that of the 1b-NS5A-containing replicons. Deletion of the interferon sensitivity-determining region/protein kinase R-binding domain (PKR-BD), the V3 domain, or the C-terminus region of NS5A significantly abrogated its anti-IFN activity. Domain swapping between 1b-NS5A and 2a-NS5A in the V3 domain and/or the C-terminus region resulted in a transfer of their anti-IFN activity. CONCLUSIONS: 1b-NS5As exert higher magnitudes of IFN antagonism than do 2a-NS5As. The V3 and the C-terminus regions are responsible for the differential anti-IFN effects. This phenomenon may partly explain the genotype-linked differences in the response of HCV to IFN treatment.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Interferon-alfa/farmacologia , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Farmacorresistência Viral/genética , Genótipo , Células HeLa , Hepacivirus/genética , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/fisiologia , Estrutura Terciária de Proteína/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transfecção , Células Vero , Proteínas não Estruturais Virais/químicaRESUMO
The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome coronavirus (SARS-CoV) plays key roles in viral replication and is an attractive target for anti-SARS drug discovery. In this report, a fluorescence resonance energy transfer (FRET)-based method was developed to assess the proteolytic activity of SARS-CoV 3CL(pro). Two internally quenched fluorogenic peptides, 1NC and 2NC, corresponding to the N-terminal and the C-terminal autocleavage sites of SARS-CoV 3CL(pro), respectively, were used as substrates. SARS-CoV 3CL(pro) seemed to work more efficiently on 1NC than on 2NC in trans-cleavage assay. Mutational analysis demonstrated that the His41 residue, the N-terminal 7 amino acids, and the domain III of SARS-CoV 3CL(pro) were important for the enzymatic activity. Antibodies recognizing domain III could significantly inhibit the enzymatic activity of SARS-CoV 3CL(pro). The effects of class-specific protease inhibitors on the trans-cleavage activity revealed that this enzyme worked more like a serine protease rather than the papain protease.
Assuntos
Endopeptidases/genética , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Sequência de Bases , Proteases 3C de Coronavírus , Cisteína Endopeptidases , Primers do DNA , Endopeptidases/química , Endopeptidases/imunologia , Endopeptidases/metabolismo , Transferência Ressonante de Energia de Fluorescência , Hidrólise , Cinética , Mutação , Inibidores de Proteases/farmacologia , Proteínas Virais/química , Proteínas Virais/imunologia , Proteínas Virais/metabolismoRESUMO
To determine whether the two domains of hepatitis C virus (HCV) NS3 and the NS4A interact with each other to regulate the RNA unwinding activity, this study compares the RNA unwinding, ATPase and RNA binding activities of three forms of NS3 proteins--the NS3H protein, containing only the helicase domain, the full-length NS3 protein, and the NS3-NS4A complex. The results revealed that NS3 displayed the weakest RNA helicase activity, not because it had lower ATPase or RNA binding activity than did NS3H or NS3-NS4A, but because it had the lowest RNA unwinding processivity. A mutant protein, R1487Q, which contained a mutation in the helicase domain, displayed a reduced protease activity as compared to the wild-type NS3-NS4A. Together, these results suggest the existence of interactions between the two domains of NS3 and the NS4A, which regulates the HCV NS3 protease and RNA helicase activities.
