RESUMO
Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus, causing severe central nerve system diseases without specific treatments. The NS2B-NS3 protease of flaviviruses mediates several cleavages on the flavivirus polyprotein, being believed to be a target for antiviral therapy. NS2B is the cofactor of the viral serine protease, correlating with stabilization and substrate recognition of the NS3 protease. In this study, we investigate the functional determinants in the JEV NS2B for the activation of the NS3 protease. Cis- and trans-cleavage assays of the deletions at the N-terminal of NS2B demonstrated that the NS2B residues Ser(46) to Ile(60) were the essential region required for both cis and trans activity of the NS3 protease. In addition, alanine substitution at the residues Trp53, Glu55, and Arg56 in NS2B significantly reduced the cis- and trans-cleavage activities of the NS3 protease. Sequence alignment and modeled structures suggested that functional determinants at the JEV NS2B residues Ser46 to Ile60, particularly in Trp53, Glu55 and Arg56 could play an important configuration required for the activity of the flavivirus NS3 protease. Our results might be useful for development of inhibitors that block the interaction between NS2B and NS3.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/fisiologia , Serina Endopeptidases/química , Proteínas não Estruturais Virais/metabolismo , Vírus da Encefalite Japonesa (Espécie)/enzimologia , Vírus da Encefalite Japonesa (Espécie)/metabolismo , RNA Helicases/química , RNA Helicases/metabolismo , Serina Endopeptidases/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/farmacologiaRESUMO
The pathogenesis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is an important issue for the treatment and prevention of severe acute respiratory syndrome. Recently, SARS-CoV has been demonstrated to induce cell apoptosis in Vero-E6 cells. The possible role of SARS-CoV 3C-like protease (3CLpro) in virus-induced apoptosis is characterized in this study. Growth arrest and apoptosis via caspase-3 and caspase-9 activities were demonstrated in SARS-CoV 3CLpro -expressing human promonocyte cells. The fluorescence intensity of dihydrorhodamine 123 staining indicated that cellular reactive oxygen species were markedly increased in SARS-CoV 3CLpro -expressing cells. Moreover, in vivo signalling pathway assay indicated that 3CLpro increased the activation of the nuclear factor-kappa B-dependent reporter, but inhibited activator protein-1-dependent transcription. This finding is likely to be responsible for virus-induced apoptotic signalling.
Assuntos
Apoptose/imunologia , Cisteína Endopeptidases/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Proteínas Virais/imunologia , Proteases Virais 3C , Anexina A5/química , Caspase 3 , Caspase 9 , Caspases/metabolismo , Linhagem Celular , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/genética , Humanos , Microscopia de Fluorescência , NF-kappa B/imunologia , Espécies Reativas de Oxigênio/metabolismo , Rodaminas/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/virologia , Transdução de Sinais , Fator de Transcrição AP-1/imunologia , Transfecção , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
The pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) is an important issue for treatment and prevention of SARS. Recently, SARS-CoV 3CL(pro) protease has been implied to be possible relevance to SARS-CoV pathogenesis. In this study, we intended to identify potential 3CL(pro)-interacting cellular protein(s) using the phage-displayed human lung cDNA library. The vacuolar-H+ ATPase (V-ATPase) G1 subunit that contained a 3CL(pro) cleavage site-like motif was identified as a 3CL(pro)-interacting protein, as confirmed using the co-immunoprecipitation assay and the relative affinity assay. In addition, our result also demonstrated the cleavage of the V-ATPase G1 fusion protein and the immunoprecipitation of cellular V-ATPase G1 by the 3CL(pro). Moreover, loading cells with SNARF-1 pH-sensitive dye showed that the intracellular pH in 3CL(pro)-expressing cells was significantly lower as compared to mock cells.