Nucleotidylylation of the VPg protein of a human norovirus by its proteinase-polymerase precursor protein.
Caliciviruses have a positive strand RNA genome covalently-linked at the 5'-end to a small protein, VPg. This study examined the biochemical modification of VPg by the ProPol form of the polymerase of human norovirus strain MD145 (GII.4). Recombinant norovirus VPg was shown to be nucleotidylylated in the presence of Mn2+ by MD145 ProPol. Phosphodiesterase I treatment of the nucleotidylylated VPg released the incorporated UMP, which was consistent with linkage of RNA to VPg via a phosphodiester bond. Mutagenesis analysis of VPg identified Tyrosine 27 as the target amino acid for this linkage, and suggested that VPg conformation was important for the reaction. Nucleotidylylation was inefficient in the presence of Mg2+; however the addition of full- and subgenomic-length MD145 RNA transcripts led to a marked enhancement of the nucleotidylylation efficiency in the presence of this divalent cation. Furthermore, evidence was found for the presence of an RNA element near the 3'-end of the polyadenylated genome that enhanced the efficiency of nucleotidylylation in the presence of Mg2+.
Belliot
Gaël
G
National Institutes of Health/DHHS, NIAID/LID, Building 50, Room 6316, 9000 Rockville Pike, Bethesda, MD 20892-8007, USA. gael.belliot@u-bourgogne.fr
Sosnovtsev
Stanislav V
SV
Chang
Kyeong-Ok
KO
McPhie
Peter
P
Green
Kim Y
KY
D000595:Amino Acid Sequence; D003067:Coenzymes / Q000494:pharmacology; D020536:Enzyme Activators / Q000494:pharmacology; D015684:Gene Products, pol / Q000302:isolation & purification / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D029322:Norovirus / Q000201:enzymology / Q000378:metabolism*; D043264:Phosphodiesterase I / Q000378:metabolism; D012333:RNA, Messenger / Q000494:pharmacology; D012367:RNA, Viral / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D014542:Uridine Monophosphate / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003067:Coenzymes; D020536:Enzyme Activators; D015684:Gene Products, pol; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D014443:Tyrosine; D014542:Uridine Monophosphate; D043264:Phosphodiesterase I; D008274:Magnesium