title: Murine norovirus-1 3Dpol exhibits RNA-dependent RNA polymerase activity and nucleotidylylates on Tyr of the VPg.
abstract: We investigated the roles and biochemical properties of recombinant murine norovirus-1 (MNV-1) 3D(pol) in RNA synthesis and virus genome-linked protein (VPg) nucleotidylylation. We therefore expressed VPg and 3D(pol) of MNV-1 in Escherichia coli. MNV-1 3D(pol) exhibited RNA-dependent RNA polymerase (RdRp) activity in vitro with poly(A) RNA as a template and MnCl(2) as a cofactor. MNV-1 3D(pol) demonstrated optimum RNA-synthesis activity at pH 7.4 and 37 degrees C in the absence of a primer. Further, VPg was guanylylated by MNV-1 3D(pol) in the presence of MnCl(2) in a template-independent manner. The guanylylation reaction conducted with VPg substitution mutants (Y26F, Y40F, Y45F and Y117F) and a deletion mutant (Delta117-124) indicated that Tyr(117) was the probable target site of guanylylation. Homopolymeric RNAs did not enhance VPg guanylylation, whereas in vitro-transcribed (-) subgenomic (SG) and (+)SG RNA enhanced VPg guanylylation by 9.2 and 3.2 times, respectively. Within (-)SG RNA, the (-)ORF3 region played a critical role in enhancing VPg guanylylation, suggesting that the MNV-1 ORF3 region of negative-strand RNA contains a cis-acting element that stimulates 3D(pol)-mediated VPg guanylylation.
authors.affiliation: Department of Life Science, Sogang University, Seoul 121-742, Republic of Korea.
authors.forename: Kang Rok
authors.initials: KR
authors.lastname: Han
authors.forename: Yubin
authors.initials: Y
authors.lastname: Choi
authors.forename: Byung Sup
authors.initials: BS
authors.lastname: Min
authors.forename: Hyesook
authors.initials: H
authors.lastname: Jeong
authors.forename: Doosung
authors.initials: D
authors.lastname: Cheon
authors.forename: Jonghyun
authors.initials: J
authors.lastname: Kim
authors.forename: Youngmee
authors.initials: Y
authors.lastname: Jee
authors.forename: Sungho
authors.initials: S
authors.lastname: Shin
authors.forename: Jai Myung
authors.initials: JM
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D029322:Norovirus / Q000145:classification*; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase