MUTATIONAL ANALYSIS OF THE HERPES-SIMPLEX VIRUS VIRION HOST SHUTOFF PROTEIN - EVIDENCE THAT VHS FUNCTIONS IN THE ABSENCE OF OTHER VIRAL-PROTEINS
MUTATIONAL ANALYSIS OF THE HERPES-SIMPLEX VIRUS VIRION HOST SHUTOFF PROTEIN - EVIDENCE THAT VHS FUNCTIONS IN THE ABSENCE OF OTHER VIRAL-PROTEINS
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DOI：
10.1128/jvi.69.8.4863-4871.1995
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发表时间：
1995-08-01
影响因子：
5.4
通讯作者：
SMILEY, JR
中科院分区：
文献类型：
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作者：
JONES, FE;SMIBERT, CA;SMILEY, JR
单纯疱疹病毒（HSV）病毒粒子含有一种或多种因子，可触发宿主蛋白合成的快速关闭，并加速感染细胞中细胞和病毒mrna的衰变。在病毒粒子宿主关闭（vhs）位点（UL41基因）上携带突变的HSV分离株在这两个过程中都存在缺陷，表明vhs蛋白是必需的；然而，目前尚不清楚VHS在关闭中的作用是直接的还是间接的，以及是否还需要其他病毒粒子成分。因此，我们使用瞬时共转染试验来确定vhs蛋白是否在缺乏其他病毒基因产物的情况下显示活性。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，并且这种影响被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。进一步证明转染试验的生物学相关性的证据来自于在共转染的细胞中检测并随后转移到病毒基因组时，五个vhs帧内连接子插入突变产生了一致的结果：三个突变在两种检测中都消除了活性，而两个没有影响。基于这些结果，我们得出结论，在没有其他HSV蛋白的情况下，vhs蛋白可以触发宿主关闭。共转染试验用于快速评估一组连接子插入突变体的活性，这些突变体跨越vhs多肽。所有与α疱疹病毒灭活功能的vhs同系物中保守区域相关的突变；相比之下，五个突变中的四个映射到几个VHS同源物中缺失的区域，没有影响。这些结果进一步支持转染试验的生物学相关性，并开始描绘vhs多肽的功能域。
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.