Murine norovirus-1 3Dpol exhibits RNA-dependent RNA polymerase activity and nucleotidylylates on Tyr of the VPg.
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.
Han
Kang Rok
KR
Department of Life Science, Sogang University, Seoul 121-742, Republic of Korea.
Choi
Yubin
Y
Min
Byung Sup
BS
Jeong
Hyesook
H
Cheon
Doosung
D
Kim
Jonghyun
J
Jee
Youngmee
Y
Shin
Sungho
S
Yang
Jai Myung
JM
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*
D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase