The VP2 protein of grass carp reovirus (GCRV) expressed in a baculovirus exhibits RNA polymerase activity
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The double-shelled grass carp reovirus (GCRV) is capable of endogenous RNA transcription and processing. Genome sequence analysis has revealed that the protein VP2, encoded by gene segment 2 (S2), is the putative RNA-dependent RNA polymerase (RdRp). In previous work, we have ex-pressed the functional region of VP2 that is associated with RNA polymerase activity (denoted as rVP2390–900) in E. coli and have prepared a polyclonal antibody against VP2. To characterize the GCRV RNA polymerase, a recombinant full-length VP2 (rVP2) was first constructed and expressed in a baculovirus system, as a fusion protein with an attached His-tag. Immunofluorescence (IF) assays, together with immunoblot (IB) analyses from both expressed cell extracts and purified Histagged rVP2, showed that rVP2 was successfully expressed in Sf9 cells. Further characterization of the replicase activity showed that purified rVP2 and GCRV particles exhibited poly(C)-dependent poly(G) polymerase activity. The RNA enzymatic activity required the divalent cation Mg2+, and was optimal at 28 °C. The results provide a foundation for further studies on the RNA polymerases of aquareoviruses during viral transcription and replication.
Keywordsgrass carp reovirus (GCRV) VP2 protein baculovirus recombinant RNA polymerase
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- Attoui H, Fang Q, Mohd Jaafar F, Cantaloube J F, Biagini P, de Micco P, and de Lamballerie X. 2002. Common evolutionary origin of aquareoviruses and orthoreoviruses revealed by genome characterization of Golden shiner reovirus, Grass carp reovirus, Striped bass reovirus and golden ide reovirus (genus Aquareovirus, family Reoviridae). J Gen Virol, 83: 1941–1951.PubMedGoogle Scholar
- Fang Q, Zhu Z Y. 2002. Expression of Functional Domain of Grass Carp Reovirus(GCRV)RNA Dependent RNA Polymerase (RdRp)Gene in E. coli. Virol Sin, 1: 66–68.Google Scholar
- Fang Q, Ke L H, Cai Y Q. 1989. Growth characterization and high titre culture of GCHV. Virol Sin, 3: 314–319.Google Scholar
- Mertens P, Attoui H, Duncan R, Dermody T, et al. 2011. The Double Stranded RNA viruses. In Virus Taxonomy. Ninth Report of the International Committee on Taxonomy of Viruses. Edited by Andrew MQ King, Adams Michael J, Carstens Eric B, Lefkowitz Elliot J. Oxford: Elsevier: 497–650.Google Scholar
- Mertens P P C, Attoui H, Duncan R, Dermody T S et al. 2011. The Double Stranded RNA viruses. In Virus Taxonomy. Ninth Report of the International Committee on Taxonomy of Viruses. Edited by King, Andrew M Q Adams Michael J, Carstens Eric B, Lefkowitz Elliot J. Oxford: Elsevier, 497–650.Google Scholar