Mapping the site of guanylylation on VP1, the protein primer for infectious pancreatic necrosis virus RNA synthesis

Abstract

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 [α 32P] 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.