TAILIEUCHUNG - Báo cáo khoa học: De novo RNA synthesis by a recombinant classical swine fever virus RNA-dependent RNA polymerase

Classical swine fever virus nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase, a key enzyme of the viral replication complex. To better under-stand the initiation of viral RNA synthesis and to establish anin vitroreplication system, a recombinant NS5B protein, lacking the C-terminal 24-amino acid hydrophobic domain, was expressed inEscherichia coli. The truncated fusion protein (NS5BD24) was purified on a Ni-chelating HisTrap affinity column and demonstrated to initiate either plus-or minus-strand viral RNA synthesisde novoin a primer-independent manner but not by terminal nucleotidyle transferase activity | Eur. J. Biochem. 270 4952-4961 2003 FEBS 2003 doi De novo RNA synthesis by a recombinant classical swine fever virus RNA-dependent RNA polymerase Guang-Hui Yi Chu-Yu Zhang Sheng Cao Hai-Xiang Wu and Yi Wang Institute of Virology College of Life Sciences Wuhan University Wuhan Hubei China Classical swine fever virus nonstructural protein 5B NS5B encodes an RNA-dependent RNA polymerase a key enzyme of the viral replication complex. To better understand the initiation of viral RNA synthesis and to establish an in vitro replication system a recombinant NS5B protein lacking the C-terminal 24-amino acid hydrophobic domain was expressed in Escherichia coli. The truncated fusion protein NS5BA24 was purified on a Ni-chelating HisTrap affinity column and demonstrated to initiate either plus-or minus-strand viral RNA synthesis de novo in a primerindependent manner but not by terminal nucleotidyle transferase activity. De novo RNA synthesis represented the preferred mechanism for initiation of classical swine fever virus RNA synthesis by RNA-dependent RNA polymerase in vitro. Both Mg2 and Mn2 supported de novo initiation however RNA synthesis was more efficient in the presence of Mn2 than in the presence of Mg2 . De novo initiation of RNA synthesis was stimulated by preincubation with mM GTP and a 3 -terminal cytidylate on the viral RNA template was preferred for de novo initiation. Furthermore the purified protein was also shown by North-Western blot analysis to specifically interact with the 3 -end of both p us-and minus-strand viral RNA templates. Keywords classical swine fever virus RNA-dependent RNA polymerase nonstructural protein 5B de novo RNA synthesis RNA-binding activity. Classical swine fever virus CSFV is a small enveloped positive-strand RNA virus classified in the genus of Pestivirus which also comprises bovine viral diarrhea virus BVDV and border disease virus BDV . Together with the genera Flavivirus and Hepacivirus they .

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