Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, MD 20892, USA.
Virology. 2010 Apr 25;400(1):18-31. doi: 10.1016/j.virol.2009.12.035. Epub 2010 Feb 4.
Caliciviruses are non-enveloped, icosahedral viruses with a single-stranded, positive sense RNA genome. Transposon-mediated insertional mutagenesis was used to insert a transprimer sequence into random sites of an infectious full-length cDNA clone of the feline calicivirus (FCV) genome. A site in the LC gene (encoding the capsid leader protein) of the FCV genome was identified that could tolerate foreign insertions, and two viable recombinant FCV variants expressing LC fused either to AcGFP, or DsRedFP were recovered. The effects of the insertions on LC processing, RNA replication, and stability of the viral genome were analyzed, and the progression of a calicivirus single infection and co-infection were captured by real-time imaging fluorescent microscopy. The ability to engineer viable recombinant caliciviruses expressing foreign markers enables new approaches to investigate virus and host cell interactions, as well as studies of viral recombination, one of the driving forces of calicivirus evolution.
杯状病毒是无包膜的二十面体病毒，具有单链、正义 RNA 基因组。转座子介导的插入诱变被用于将转引物序列插入猫杯状病毒 (FCV) 基因组的传染性全长 cDNA 克隆的随机位点。在 FCV 基因组的 LC 基因（编码衣壳蛋白前导蛋白）中鉴定出一个可以耐受外源插入的位点，并回收了两个能够表达融合了 AcGFP 或 DsRedFP 的 LC 的存活重组 FCV 变体。分析了插入对 LC 加工、RNA 复制和病毒基因组稳定性的影响，并通过实时成像荧光显微镜捕获了杯状病毒单感染和共感染的进展。能够构建表达外源标记的存活重组杯状病毒的能力为研究病毒和宿主细胞相互作用以及病毒重组提供了新方法，病毒重组是杯状病毒进化的驱动力之一。
