Mapping the site of guanylylation on VP1, the protein primer for infectious pancreatic necrosis virus RNA synthesis.
VP1, the putative virion-associated RNA-dependent RNA polymerase (RdRp) of infectious pancreatic necrosis virus (IPNV) can be guanylylated in vitro whereupon it becomes a primer for in vitro RNA synthesis [Virology 208 (1995) 19]. The role of a template or other virion polypeptides in the reaction is unknown. To shed light on this question, his-tagged recombinant VP(1) (rVP1) was expressed both in Escherichia coli and insect cells and used in the guanylylation reaction. Unlike other viral VPg polypeptides, the purified rVP1 alone could guanylylate itself in vitro in a template-independent manner. Chemical and enzymatic cleavage in combination with site-directed mutagenesis mapped the site of guanylylation to serine 163. The purified rVP1 functioned as a primer as well as an RdRp in vitro, producing labeled dsRNA in the presence of [alpha(32)P] NTP and synthetically produced viral ss + RNA as a template. Only a single cycle of replication was observed and labeled VPg could be recovered from the dsRNA by RNase V(1) digestion. Denaturation of the dsRNA yielded genome-length labeled ssRNA, indicating that RNA synthesis was not initiated by 3'-end snap-back self-priming. Mutating serine 163 to alanine of rVP1 abolished both its self-guanylylating and polymerizing activity.
Xu
Hong-Tao
HT
Department of Microbiology, College of Biological Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1.
Si
Wei-Duo
WD
Dobos
Peter
P
D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D002874:Chromosome Mapping; D018061:Infectious pancreatic necrosis virus / Q000235:genetics / Q000378:metabolism*; D012330:RNA, Double-Stranded; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D012330:RNA, Double-Stranded; D012367:RNA, Viral; D015678:Viral Structural Proteins; D012324:RNA-Dependent RNA Polymerase