RESUMEN
In order to explore the mechanism of geminivirus DNA replication, we show that the Replication initiator (Rep) protein encoded by Mungbean yellow mosaic India virus (MYMIV), a member of the family Geminiviridae, binds specifically to the iterons present in the viral DNA replication origin (CR-A) in a highly ordered manner that might be a prerequisite for the initiation of replication. MYMIV Rep also acts as a helicase during the post-initiation stage and is upregulated in presence of the RPA32 subunit of Replication Protein A. The implication of these findings on the initiation and elongation stages of MYMIV DNA replication has been discussed.
Asunto(s)
ADN Helicasas/metabolismo , Replicación del ADN/fisiología , Geminiviridae/fisiología , Origen de Réplica/fisiología , Proteína de Replicación A/metabolismo , Transactivadores/metabolismo , ADN Helicasas/genética , ADN Viral/biosíntesis , Geminiviridae/genética , Proteína de Replicación A/genética , Transactivadores/genética , Transcripción Genética , Replicación Viral/genéticaRESUMEN
Geminiviruses replicate by rolling circle mode of replication (RCR) and the viral Rep protein initiates RCR by the site-specific nicking at a conserved nonamer (TAATATT downward arrow AC) sequence. The mechanism of subsequent steps of the replication process, e.g. helicase activity to drive fork-elongation, etc. has largely remained obscure. Here we show that Rep of a geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), acts as a replicative helicase. The Rep-helicase, requiring > or =6 nt space for its efficient activity, translocates in the 3'-->5' direction, and the presence of forked junction in the substrate does not influence the activity to any great extent. Rep forms a large oligomeric complex and the helicase activity is dependent on the oligomeric conformation ( approximately 24mer). The role of Rep as a replicative helicase has been demonstrated through ex vivo studies in Saccharomyces cerevisiae and in planta analyses in Nicotiana tabacum. We also establish that such helicase activity is not confined to the MYMIV system alone, but is also true with at least two other begomoviruses, viz., Mungbean yellow mosaic virus (MYMV) and Indian cassava mosaic virus (ICMV).
Asunto(s)
Begomovirus/enzimología , Begomovirus/genética , ADN Helicasas/metabolismo , Replicación del ADN , Proteínas Virales/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , ADN Helicasas/genética , ADN Viral/biosíntesis , Eliminación de Secuencia , Proteínas Virales/genéticaRESUMEN
In addition to their encapsidation function, viral coat proteins (CP) contribute to viral life cycle in many different ways. The CPs of the geminiviruses are responsible for intra- as well as inter-plant virus transmission and might determine the yield of viral DNA inside the infected tissues by either packaging the viral DNA or interfering with the viral replicative machinery. Since the cognate Rep largely controls the rolling circle replication of geminiviral DNA, the interaction between Rep and CP might be worthwhile to examine for elucidation of CP-mediated control of the viral DNA copy number. Here a reasonably strong interaction between Rep and CP of the geminivirus Mung bean yellow mosaic India virus is reported. The domain of interaction has been mapped to a central region of Rep. The replication initiation activity of Rep, i.e., its nicking and closing function, is down regulated by CP. This report highlights how CP could be important in controlling geminiviral DNA replication.
Asunto(s)
Proteínas de la Cápside/metabolismo , ADN Viral/genética , Virus del Mosaico/fisiología , Phaseolus/genética , Proteínas de Plantas/metabolismo , Replicación Viral , Cartilla de ADN , Replicación del ADN , Genes Reporteros , Glutatión Transferasa/genética , Reacción en Cadena de la PolimerasaRESUMEN
We report here the expression and purification of a truncated form of the hepatitis E virus ORF2 protein (ORF2delta111/deltaTM), from the fat bodies of Spodoptera litura larvae infected with a recombinant baculovirus. The purified protein migrated as a doublet of approximately 56 kDa on SDS-PAGE and was found to be glycosylated by staining with concanavalin A-linked horseradish peroxidase. The protein was used in a sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to HEV. The results showed complete concordance with those obtained using a commercial kit for the detection of anti-HEV antibodies. Antigen expression in the insect larvae system presents a rapid and low-cost method that obviates the need for expensive tissue culture scale-ups or special equipment.
