Jones F E, Smibert C A, Smiley J R
Pathology Department, McMaster University, Hamilton, Ontario, Canada.
J Virol. 1995 Aug;69(8):4863-71. doi: 10.1128/JVI.69.8.4863-4871.1995.
Herpes simplex virus (HSV) virions contain one or more factors that trigger rapid shutoff of host protein synthesis and accelerated decay of cellular and viral mRNAs in infected cells. HSV isolates bearing mutations at the virion host shutoff (vhs) locus (gene UL41) are defective for both processes, indicating that the vhs protein is required; however, it is not clear whether the role of vhs in shutoff is direct or indirect and if other virion components are also necessary. We therefore used a transient-cotransfection assay to determine if the vhs protein displays activity in the absence of other viral gene products. We found that a vhs expression vector strongly suppressed expression of a cotransfected lacZ reporter gene and that this effect was eliminated by the vhs1 point mutation that abolishes virion-induced host shutoff during HSV infection. Further evidence for the biological relevance of the transfection assay came from the demonstration that five vhs in-frame linker insertion mutations yielded concordant results when assayed in cotransfected cells and following transfer into the viral genome: three mutations eliminated activity in both assays, while two had no effect. On the basis of these results, we conclude that the vhs protein can trigger host shutoff in the absence of other HSV proteins. The cotransfection assay was used to rapidly assess the activities of a panel of linker insertion mutants spanning the vhs polypeptide. All mutations that mapped to regions conserved among the vhs homologs of alphaherpesvirus inactivated function; in contrast, four of five mutations that mapped to regions that are absent from several vhs homologs had no effect. These results further support the biological relevance of the transfection assay and begin to delineate functional domains of the vhs polypeptide.
单纯疱疹病毒（HSV）病毒粒子含有一种或多种因子，这些因子可在感染细胞中触发宿主蛋白合成的快速关闭以及细胞和病毒mRNA的加速降解。在病毒粒子宿主关闭（vhs）基因座（基因UL41）处携带突变的HSV分离株在这两个过程中均存在缺陷，这表明vhs蛋白是必需的；然而，尚不清楚vhs在关闭过程中的作用是直接的还是间接的，以及其他病毒粒子成分是否也是必需的。因此，我们使用瞬时共转染试验来确定vhs蛋白在没有其他病毒基因产物的情况下是否具有活性。我们发现，vhs表达载体强烈抑制共转染的lacZ报告基因的表达，并且这种效应被vhs1点突变消除，该突变消除了HSV感染期间病毒粒子诱导的宿主关闭。转染试验生物学相关性的进一步证据来自以下证明：五个vhs框内接头插入突变在共转染细胞中测定以及转移到病毒基因组后产生了一致的结果：三个突变在两种试验中均消除了活性，而两个则没有影响。基于这些结果，我们得出结论，vhs蛋白在没有其他HSV蛋白的情况下可以触发宿主关闭。共转染试验用于快速评估一组跨越vhs多肽的接头插入突变体的活性。所有映射到α疱疹病毒vhs同源物中保守区域的突变均使功能失活；相反，五个映射到几个vhs同源物中不存在的区域的突变中有四个没有影响。这些结果进一步支持了转染试验的生物学相关性，并开始描绘vhs多肽的功能域。
