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Virologica Sinica

, Volume 29, Issue 2, pp 86–93 | Cite as

The VP2 protein of grass carp reovirus (GCRV) expressed in a baculovirus exhibits RNA polymerase activity

  • Liming Yan
  • Huan Liu
  • Xiaoming Li
  • Qin FangEmail author
Research Article

Abstract

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.

Keywords

grass carp reovirus (GCRV) VP2 protein baculovirus recombinant RNA polymerase 

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Copyright information

© Wuhan Institute of Virology, CAS and Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.State Key Laboratory of Virology, Wuhan Institute of VirologyChinese Academy of SciencesWuhanChina
  2. 2.University of the Chinese Academy of SciencesBeijingChina
  3. 3.School of Basic Medical SciencesWuhan UniversityWuhanChina

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