Xu Hong-Tao, Si Wei-Duo, Dobos Peter
Department of Microbiology, College of Biological Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1.
Virology. 2004 Apr 25;322(1):199-210. doi: 10.1016/j.virol.2004.01.024.
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.
VP1是传染性胰腺坏死病毒（IPNV）假定的与病毒粒子相关的RNA依赖性RNA聚合酶（RdRp），它在体外可以被鸟苷酸化，随后成为体外RNA合成的引物[《病毒学》208（1995）19]。模板或其他病毒粒子多肽在该反应中的作用尚不清楚。为了阐明这个问题，带有组氨酸标签的重组VP1（rVP1）在大肠杆菌和昆虫细胞中均有表达，并用于鸟苷酸化反应。与其他病毒VPg多肽不同，纯化后的rVP1自身即可在体外以不依赖模板的方式进行自我鸟苷酸化。化学和酶切结合定点诱变将鸟苷酸化位点定位到丝氨酸163。纯化后的rVP1在体外既作为引物又作为RdRp发挥作用，在存在[α(32)P] NTP和合成产生的病毒单链正链RNA作为模板的情况下产生标记的双链RNA。仅观察到一个复制周期，并且通过RNase V(1)消化可以从双链RNA中回收标记的VPg。双链RNA变性后产生基因组长度的标记单链RNA，表明RNA合成不是由3'端回折自我引发的。将rVP1的丝氨酸163突变为丙氨酸会消除其自我鸟苷酸化和聚合活性。
