Frolova E, Frolov I, Schlesinger S
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.
J Virol. 1997 Jan;71(1):248-58. doi: 10.1128/JVI.71.1.248-258.1997.
Alphaviruses synthesize large amounts of both genomic and subgenomic RNA in infected cells, but usually only the genomic RNA is packaged. This implies the existence of an encapsidation or packaging signal which would be responsible for selectivity. Previously, we had identified a region of the Sindbis virus genome that interacts specifically with the viral capsid protein. This 132-nucleotide (nt) fragment lies within the coding region of the nsP1 gene (nt 945 to 1076). We proposed that the 132-mer is important for capsid recognition and initiates the formation of the viral nucleocapsid. To study the encapsidation of Sindbis virus RNAs in infected cells, we designed a new assay that uses the self-replicating Sindbis virus genomes (replicons) which lack the viral structural protein genes and contain heterologous sequences under the control of the subgenomic RNA promoter. These replicons can be packaged into viral particles by using defective helper RNAs that contain the structural protein genes (P. Bredenbeek, I. Frolov, C. M. Rice, and S. Schlesinger, J. Virol. 67:6439-6446, 1993). Insertion of the 132-mer into the subgenomic RNA significantly increased the packaging of this RNA into viral particles. We have used this assay and defective helpers that contain the structural protein genes of Ross River virus (RRV) to investigate the location of the encapsidation signal in the RRV genome. Our results show that there are several fragments that could act as packaging signals. They are all located in a different region of the genome than the signal for the Sindbis virus genome. For RRV, the strongest packaging signal lies between nt 2761 and 3062 in the nsP2 gene. This is the same region that was proposed to contain the packaging signal for Semliki Forest virus genomic RNA.
甲病毒在受感染细胞中合成大量基因组RNA和亚基因组RNA，但通常只有基因组RNA被包装。这意味着存在一种负责选择性的衣壳化或包装信号。此前，我们已经鉴定出辛德毕斯病毒基因组中一个与病毒衣壳蛋白特异性相互作用的区域。这个132个核苷酸（nt）的片段位于nsP1基因的编码区内（核苷酸945至1076）。我们提出，这个132聚体对于衣壳识别很重要，并启动病毒核衣壳的形成。为了研究辛德毕斯病毒RNA在受感染细胞中的衣壳化，我们设计了一种新的检测方法，该方法使用自我复制的辛德毕斯病毒基因组（复制子），这些基因组缺乏病毒结构蛋白基因，并在亚基因组RNA启动子的控制下包含异源序列。通过使用含有结构蛋白基因的缺陷辅助RNA，这些复制子可以被包装成病毒颗粒（P.Bredenbeek、I.Frolov、C.M.Rice和S.Schlesinger，《病毒学杂志》67:6439 - 6446，1993）。将132聚体插入亚基因组RNA显著增加了该RNA被包装到病毒颗粒中的效率。我们使用这种检测方法和含有罗斯河病毒（RRV）结构蛋白基因的缺陷辅助病毒来研究RRV基因组中包装信号的位置。我们 的结果表明，有几个片段可以作为包装信号。它们都位于基因组中与辛德毕斯病毒基因组信号不同的区域。对于RRV，最强的包装信号位于nsP2基因的核苷酸2761至3062之间。这与被认为包含塞姆利基森林病毒基因组RNA包装信号的区域相同。
