Wansley Elizabeth K, Parks Griffith D
Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1064, USA.
J Virol. 2002 Oct;76(20):10109-21. doi: 10.1128/jvi.76.20.10109-10121.2002.
The V protein of the paramyxovirus simian virus 5 (SV5) is responsible for targeted degradation of STAT1 and the block in alpha/beta interferon (IFN-alpha/beta) signaling that occurs after SV5 infection of human cells. We have analyzed the growth properties of a recombinant SV5 that was engineered to be defective in targeting STAT1 degradation. A recombinant SV5 (rSV5-P/V-CPI-) was engineered to contain six naturally occurring P/V protein mutations, three of which have been shown in previous transfection experiments to disrupt the V-mediated block in IFN-alpha/beta signaling. In contrast to wild-type (WT) SV5, human cells infected with rSV5-P/V-CPI- had STAT1 levels similar to those in mock-infected cells. Cells infected with rSV5-P/V-CPI- were found to express higher-than-WT levels of viral proteins and mRNA, suggesting that the P/V mutations had disrupted the regulation of viral RNA synthesis. Despite the inability to target STAT1 for degradation, single-step growth assays showed that the rSV5-P/V-CPI- mutant virus grew better than WT SV5 in all cell lines tested. Unexpectedly, cells infected with rSV5-P/V-CPI- but not WT SV5 showed an activation of a reporter gene that was under control of the IFN-beta promoter. The secretion of IFN from cells infected with rSV5-P/V-CPI- but not WT SV5 was confirmed by a bioassay for IFN. The rSV5-P/V-CPI- mutant grew to higher titers than did WT rSV5 at early times in multistep growth assays. However, rSV5-P/V-CPI- growth quickly reached a final plateau while WT rSV5 continued to grow and produced a final titer higher than that of rSV5-P/V-CPI- by late times postinfection. In contrast to WT rSV5, infection of a variety of cell lines with rSV5-P/V-CPI- induced cell death pathways with characteristics of apoptosis. Our results confirm a role for the SV5 V protein in blocking IFN signaling but also suggest new roles for the P/V gene products in controlling viral gene expression, the induction of IFN-alpha/beta synthesis, and virus-induced apoptosis.
副粘病毒猴病毒5（SV5）的V蛋白负责靶向降解信号转导和转录激活因子1（STAT1），并在人类细胞被SV5感染后阻断α/β干扰素（IFN-α/β）信号通路。我们分析了一种经基因工程改造、在靶向降解STAT1方面存在缺陷的重组SV5的生长特性。构建了一种重组SV5（rSV5-P/V-CPI-），使其含有六个天然存在的P/V蛋白突变，其中三个突变在先前的转染实验中已被证明可破坏V介导的对IFN-α/β信号通路的阻断。与野生型（WT）SV5不同，被rSV5-P/V-CPI-感染的人类细胞中STAT1水平与模拟感染细胞中的相似。发现被rSV5-P/V-CPI-感染的细胞表达的病毒蛋白和mRNA水平高于野生型，这表明P/V突变破坏了病毒RNA合成的调控。尽管无法靶向降解STAT1，但一步生长试验表明，在所有测试的细胞系中，rSV5-P/V-CPI-突变病毒比野生型SV5生长得更好。出乎意料的是，被rSV5-P/V-CPI-感染而非野生型SV5感染的细胞显示出受IFN-β启动子控制的报告基因的激活。通过IFN生物测定法证实，被rSV5-P/V-CPI-感染而非野生型SV5感染的细胞会分泌IFN。在多步生长试验的早期，rSV5-P/V-CPI-突变体的生长滴度高于野生型rSV5。然而，rSV5-P/V-CPI-的生长很快达到最终平台期，而野生型rSV5继续生长，在感染后期产生的最终滴度高于rSV5-P/V-CPI-。与野生型rSV5不同，用rSV5-P/V-CPI-感染多种细胞系会诱导具有凋亡特征的细胞死亡途径。我们的结果证实了SV5 V蛋白在阻断IFN信号通路中的作用，但也提示了P/V基因产物在控制病毒基因表达、诱导IFN-α/β合成以及病毒诱导的凋亡方面的新作用。
