Visualization of feline calicivirus replication in real-time with recombinant viruses engineered to express fluorescent reporter proteins.
Visualization of feline calicivirus replication in real-time with recombinant viruses engineered to express fluorescent reporter proteins.
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DOI：
10.1016/j.virol.2009.12.035
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发表时间：
2010-04-25
期刊：
影响因子：
3.7
通讯作者：
Green KY
中科院分区：
文献类型：
--
作者：
Abente EJ;Sosnovtsev SV;Bok K;Green KY
杯状病毒是具有单链正义RNA基因组的无包膜二十面体病毒。利用转座子介导的插入诱变技术，将转引物序列插入猫杯状病毒（FCV）基因组感染性全长cDNA克隆的随机位点。鉴定了FCV基因组的LC基因（编码衣壳前导蛋白）中可以耐受外源插入的位点，并且回收了表达与AcGFP或DsRedFP融合的LC的两种活的重组FCV变体。分析插入对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杯状病毒单次感染和共感染的进展。工程化表达外源标记的可行重组杯状病毒的能力使得能够采用新的方法来研究病毒和宿主细胞的相互作用，以及研究病毒重组，杯状病毒进化的驱动力之一。
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.
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