RESUMO
The complete nonstructural NS5 gene of Japanese encephalitis virus (JEV) was amplified and cloned into an expression vector. The NS5 protein was expressed in Escherichia coli and purified by His-tag based affinity chromatography. This recombinant NS5 protein exhibited RNA-dependent RNA polymerase (RdRp) activity in vitro in the absence of other viral or cellular factors. The RNA polymerase activity was dependent on divalent cations, and Mn(2+) was found to be 20 times more effective than Mg(2+) in coordinating the catalytic reaction of RdRp, while Ca(2+) inhibited enzyme activity. The optimal reaction conditions for the in vitro RdRp reaction were established. Characterization of the RdRp reaction products demonstrated that the JEV NS5 protein can initiate RNA synthesis through a de novo initiation mechanism in our in vitro reaction system. Comparing the efficiency of different RNA templates, we found that JEV NS5 protein was more efficient in using negative-strand RNA templates, indicating that the JEV NS5 protein is involved in regulating the ratio of positive- to negative-strand RNA. Four amino acid sequence motifs crucial for RdRp activity were also identified using site-directed mutagenesis analysis. All substitutions of the conserved residues within these motifs led to a complete inactivation or severe loss of enzyme activity.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas não Estruturais Virais/metabolismo , Aedes/citologia , Aedes/virologia , Substituição de Aminoácidos , Animais , Cálcio/metabolismo , Catálise , Cátions Bivalentes/metabolismo , Linhagem Celular , Clonagem Molecular , Vírus da Encefalite Japonesa (Espécie)/genética , Vírus da Encefalite Japonesa (Espécie)/isolamento & purificação , Escherichia coli/genética , Humanos , Concentração de Íons de Hidrogênio , Magnésio/metabolismo , Manganês/metabolismo , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , RNA Polimerase Dependente de RNA/análise , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Temperatura , Moldes Genéticos , Fatores de Tempo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificaçãoRESUMO
A reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of Chikungunya virus infection. Based on the nonstructural protein 1 (nsP1) and glycoprotein E1 (E1) genes of Chikungunya, two primer sets were designed. Total RNA were extracted from the cell culture fluid of Aedes albopictus C6/36 cells inoculated with the S27 prototype virus, isolated in Tanzania in 1953, and the Malaysian strains (MALh0198, MALh0298, and MALh0398), isolated in Malaysia in 1998. For both sets of RNA samples, the expected 354- and 294-base pair (bp) cDNA fragments were amplified effectively from the nsP1 and E1 genes, respectively. Phylogenetic analysis was conducted for the Malaysian strain and other virus strains isolated from different regions in the world endemic for Chikungunya, using partial E1 gene sequence data. The Malaysian strains isolated during the epidemics of 1998 fell into a cluster with other members of the Asian genotype.
Assuntos
Infecções por Alphavirus/diagnóstico , Infecções por Alphavirus/epidemiologia , Vírus Chikungunya/classificação , Vírus Chikungunya/isolamento & purificação , Surtos de Doenças , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Aedes , Infecções por Alphavirus/virologia , Animais , Células Cultivadas , Vírus Chikungunya/genética , Genótipo , Humanos , Malásia/epidemiologia , Dados de Sequência Molecular , Filogenia , Sensibilidade e Especificidade , Análise de Sequência de DNA , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Apoptosis is a highly regulated process of cellular self-destruction with diverse functions in multicellular organisms. It is known to be one of the mechanisms of viral pathogenesis. St. Louis encephalitis virus (SLEV), an arthropod-borne flavivirus, causes encephalitis disease of varying severity mostly in North America and in some regions of South America. This virus induces cytopathic effects in vertebrate cell lines, however, the mechanism by which this occurs is yet to be elucidated. SLEV induced cytopathic effects in K562 cells, a human mononuclear cell line, and in Neuro 2a cells, a mouse neuroblastoma cell line. SLEV-infected K562 and Neuro 2a cells underwent apoptotic cell death, whereas neither the cells inoculated with UV-inactivated virus nor the mock-infected cells developed cytopathic effects. The gene expression of regulators of apoptosis was investigated in K562 cells. A rise in the expression of the pro-apoptotic bax gene was detected specifically in the SLEV-infected K562 cells. These findings suggest that up-regulation of bax mRNA is correlated with cytopathic effects in SLEV-infected K562 cells.
