Nodamura virus RNA replication in Saccharomyces cerevisiae:: Heterologous gene expression allows replication-dependent colon formation
Nodamura virus RNA replication in Saccharomyces cerevisiae:: Heterologous gene expression allows replication-dependent colon formation
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DOI：
10.1128/jvi.79.1.495-502.2005
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发表时间：
2005-01-01
影响因子：
5.4
通讯作者：
Johnson, KL
中科院分区：
文献类型：
--
作者：
Price, BD;Eckerle, LD;Johnson, KL
新村病毒(NoDamura Virus，NOV)和鸡舍病毒(Flock House Virus，FHV)属于诺达韦科。诺达病毒基因组由两个正义RNA片段组成：RNA1编码病毒依赖于RNA的RNA聚合酶，RNA2编码衣壳蛋白前体。在RNA复制过程中，编码非结构蛋白B1和B2的小亚基因组RNA3由RNA1转录而来。此前，FHV被证明在瞬时转基因的酵母细胞中既复制了它的基因组RNA，又转录了RNA3。含有RNA聚合酶11启动子的质粒也可表达FHV RNAs及其衍生物。在这里，我们展示了所有这些特征可以概括为NOV，这是唯一一种能够有效感染哺乳动物的诺达病毒。利用可诱导的基于质粒的系统研究了酿酒酵母对NOVRNA1和RNA2的RNA复制需求。诱导的NOVRNA1复制能力很强。之前描述的三个NOVRNA1突变体在酵母中的行为与它们在哺乳动物细胞中的行为相同。酵母菌落从表达Nov RNA1的细胞中选择，RNA2复制子编码酵母营养标记的复制粒子从质粒中选择。出乎意料的是，这些依赖NOV RNA复制的酵母菌落的恢复频率比类似的FHV系统低10(4)倍。分子分析显示，在复制的病毒RNA中，一些依赖于NOV RNA的克隆在NOV B2开放阅读框中存在突变。此外，我们发现Nov RNA1可以支持表达酵母HIS3选择标记的异源FHV RNA2的删除衍生物的有限复制，导致His+克隆的形成。
Nodamura virus (NoV) and Flock House virus (FHV) are members of the family Nodaviridae. The nodavirus genome is composed of two positive-sense RNA segments: RNA1 encodes the viral RNA-dependent RNA polymerase and RNA2 encodes the capsid protein precursor. A small subgenomic RNA3, which encodes nonstructural proteins B1 and B2, is transcribed from RNA1 during RNA replication. Previously, FHV was shown to replicate both of its genomic RNAs and to transcribe RNA3 in transiently transfected yeast cells. FHV RNAs and their derivatives could also be expressed from plasmids containing RNA polymerase 11 promoters. Here we show that all of these features can be recapitulated for NoV, the only nodavirus that productively infects mammals. Inducible plasmid-based systems were used to characterize the RNA replication requirements for NoV RNA1 and RNA2 in Saccharomyces cerevisiae. Induced NoV RNA1 replication was robust. Three previously described NoV RNA1 mutants behaved in yeast as they had in mammalian cells. Yeast colonies were selected from cells expressing NoV RNA1, and RNA2 replicons that encoded yeast nutritional markers, from plasmids. Unexpectedly, these NoV RNA replication-dependent yeast colonies were recovered at frequencies 10(4)-fold lower than in the analogous FHV system. Molecular analysis revealed that some of the NoV RNA replication-dependent colonies contained mutations in the NoV B2 open reading frame in the replicating viral RNA. In addition, we found that NoV RNA1 could support limited replication of a deletion derivative of the heterologous FHV RNA2 that expressed the yeast HIS3 selectable marker, resulting in formation of HIS+ colonies.