title: Regulation of human norovirus VPg nucleotidylylation by ProPol and nucleoside triphosphate binding by its amino terminal sequence in vitro.
abstract: The VPg protein of human Norovirus (hNoV) is a multi-functional protein essential for virus replication. The un-cleaved viral precursor protein, ProPol (NS5-6) was 100-fold more efficient in catalyzing VPg nucleotidylylation than the mature polymerase (Pol, NS6), suggesting a specific intracellular role for ProPol. Sequential and single-point alanine substitutions revealed that several positively charged amino acids in the N-terminal region of VPg regulate its nucleotidylylation by ProPol. We provide evidence that VPg directly binds NTPs, inhibition of binding inhibits nucleotidylylation, and NTP binding appears to involve the first 13 amino acids of the protein. Substitution of multiple positively charged amino acids within the first 12 amino acids of the N-terminal region inhibits nucleotidylylation without affecting binding. Substitution of only Lys20 abolishes nucleotidylylation, but not NTP binding. These studies indicate that positively charged amino acids in the first 20 amino acids of hNoV VPg regulate its nucleotidylylation though several potential mechanisms.
authors.affiliation: Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20057, USA.
authors.forename: Alexei
authors.initials: A
authors.lastname: Medvedev
authors.affiliation: Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20057, USA.
authors.forename: Prasanth
authors.initials: P
authors.lastname: Viswanathan
authors.affiliation: Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20057, USA.
authors.forename: Jared
authors.initials: J
authors.lastname: May
authors.affiliation: Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20057, USA. Electronic address: korbabe@georgetown.edu.
authors.forename: Brent
authors.initials: B
authors.lastname: Korba
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D006801:Humans; D029322:Norovirus / Q000235:genetics*; D009711:Nucleotides / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D009711:Nucleotides; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins
keywords: Norovirus; Nucleotide binding; Nucleotidylylation; VPg