Wilson G J, Wetzel J D, Puryear W, Bassel-Duby R, Dermody T S
Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
J Virol. 1996 Oct;70(10):6598-606. doi: 10.1128/JVI.70.10.6598-6606.1996.
During maintenance of L-cell cultures persistently infected with reovirus, mutations are selected in viruses and cells. Cells cured of persistent infection support growth of viruses isolated from persistently infected cultures (PI viruses) significantly better than that of wild-type (wt) viruses. In a previous study, the capacity of PI virus strain L/C to grow better than wt strain type 1 Lang (T1L) in cured cells was mapped genetically to the S1 gene (R. S. Kauffman, R. Ahmed, and B. N. Fields, Virology 131:79-87, 1983), which encodes viral attachment protein sigma1. To investigate mechanisms by which mutations in S1 confer growth of PI viruses in cured cells, we determined the S1 gene nucleotide sequences of L/C virus and six additional PI viruses isolated from independent persistently infected L-cell cultures. The S1 sequences of these viruses contained from one to three mutations, and with the exception of PI 2A1 mutations in each S1 gene resulted in changes in the deduced amino acid sequence of sigma1 protein. Using electrophoresis conditions that favor migration of sigma1 oligomers, we found that sigma1 proteins of L/C, PI 1A1, PI 3-1, and PI 5-1 migrated as monomers, whereas sigma1 proteins of wt reovirus and PI 2A1 migrated as oligomers. These findings suggest that mutations in sigma1 protein affecting stability of sigma1 oligomers are important for the capacity of PI viruses to infect mutant cells selected during persistent infection. Since no mutation was found in the deduced amino acid sequence of PI 2A1 sigma1 protein, we used T1L X PI 2A1 reassortant viruses to identify viral genes associated with the capacity of this PI virus to grow better than wt in cured cells. The capacity of PI 2A1 to grow better than T1L in cured cells was mapped to the S4 gene, which encodes outer-capsid protein sigma3. This finding suggests that in some cases, mutations in sigma3 protein in the absence of sigma1 mutations confer growth of PI viruses in mutant cells. To confirm the importance of the S1 gene in PI virus growth in cured cells, we used T1L X PI 3-1 reassortant viruses to genetically map the capacity of this PI virus to grow better than wt in cured cells. In contrast to our results using PI 2A1, we found that growth of PI 3-1 in cured cells was determined by the sigma1-encoding S1 gene. Given that the sigma1 and sigma3 proteins play important roles in reovirus disassembly, findings made in this study suggest that stability of the viral outer capsid is an important determinant of the capacity of reoviruses to adapt to host cells during persistent infection.
在维持持续感染呼肠孤病毒的L细胞培养物的过程中，病毒和细胞中会发生突变。从持续感染中治愈的细胞对从持续感染培养物中分离出的病毒（PI病毒）的支持生长能力明显优于野生型（wt）病毒。在先前的一项研究中，PI病毒株L/C在治愈细胞中比wt 1型朗（T1L）株生长得更好的能力通过遗传学方法定位到S1基因（R.S.考夫曼、R.艾哈迈德和B.N.菲尔兹，《病毒学》131:79 - 87，1983），该基因编码病毒附着蛋白σ1。为了研究S1突变赋予PI病毒在治愈细胞中生长能力的机制，我们测定了L/C病毒以及从独立的持续感染L细胞培养物中分离出的另外六种PI病毒的S1基因核苷酸序列。这些病毒的S1序列含有一到三个突变，并且除了PI 2A1外，每个S1基因中的突变都导致了σ1蛋白推导氨基酸序列的变化。使用有利于σ1寡聚体迁移的电泳条件，我们发现L/C、PI 1A1、PI 3 - 1和PI 5 - 1的σ1蛋白以单体形式迁移，而野生型呼肠孤病毒和PI 2A1的σ1蛋白以寡聚体形式迁移。这些发现表明，影响σ1寡聚体稳定性的σ1蛋白突变对于PI病毒感染在持续感染期间选择的突变细胞的能力很重要。由于在PI 2A1 σ1蛋白的推导氨基酸序列中未发现突变，我们使用T1L×PI 2A1重配病毒来鉴定与该PI病毒在治愈细胞中比wt生长得更好的能力相关的病毒基因。PI 2A1在治愈细胞中比T1L生长得更好的能力被定位到S4基因，该基因编码外衣壳蛋白σ3。这一发现表明，在某些情况下，在没有σ1突变的情况下，σ3蛋白的突变赋予了PI病毒在突变细胞中的生长能力。为了证实S1基因在PI病毒在治愈细胞中生长的重要性，我们使用T1L×PI 3 - 1重配病毒对该PI病毒在治愈细胞中比wt生长得更好的能力进行遗传学定位。与我们使用PI 2A1的结果相反，我们发现PI 3 - 1在治愈细胞中的生长由编码σ1的S1基因决定。鉴于σ1和σ3蛋白在呼肠孤病毒解体中起重要作用，本研究中的发现表明病毒外衣壳的稳定性是呼肠孤病毒在持续感染期间适应宿主细胞能力的一个重要决定因素。
