The Second-Site Mutation in the Herpes Simplex Virus Recombinants Lacking the γ(1)34.5 Genes Precludes Shutoff of Protein Synthesis by Blocking the Phosphorylation of eIF-2α

In cells infected with the herpes simplex virus 1 (HSV-1) recombinant R3616 lacking both copies of the γ(1)34.5 gene, the double-stranded protein kinase R (PKR) is activated, eIF-2α is phosphorylated, and protein synthesis is shut off. Although PKR is also activated in cells infected with the wild-type virus, the product of the γ(1)34.5 gene, infected-cell protein 34.5 (ICP34.5), binds protein phosphatase 1α and redirects it to dephosphorylate eIF-2α, thus enabling sustained protein synthesis. Serial passage in human cells of a mutant lacking the γ(1)34.5 gene yields second-site, compensatory mutants lacking various domains of the α47 gene situated next to the U(S)11 gene (I. Mohr and Y. Gluzman, EMBO J. 15:4759–4766, 1996). We report the construction of two recombinant viruses: R5103, lacking the γ(1)34.5, U(S)8, -9, -10, and -11, and α47 (U(S)12) genes; and R5104, derived from R5103 and carrying a chimeric DNA fragment containing the U(S)10 gene and the promoter of the α47 gene fused to the coding domain of the U(S)11 gene. R5104 exhibited a protein synthesis profile similar to that of wild-type virus, whereas protein synthesis was shut off in cells infected with R5103 virus. Studies on the wild-type parent and mutant viruses showed the following: (i) PKR was activated in cells infected with parent or mutant virus but not in mock-infected cells, consistent with earlier studies; (ii) lysates of R3616, R5103, and R5104 virus-infected cells lacked the phosphatase activity specific for eIF-2α characteristic of wild-type virus-infected cells; and (iii) lysates of R3616 and R5103, which lacked the second-site compensatory mutation, contained an activity which phosphorylated eIF-2α in vitro, whereas lysates of mock-infected cells or cells infected with HSV-1(F) or R5104 did not phosphorylate eIF-2α. We conclude that in contrast to wild-type virus-infected cells, which preclude the shutoff of protein synthesis by causing rapid dephosphorylation of eIF-2α, in cells infected with γ(1)34.5(−) virus carrying the compensatory mutation, eIF-2α is not phosphorylated. The activity made apparent by the second-site mutation may represent a more ancient mechanism evolved to preclude the shutoff of protein synthesis.

在缺失两份γ(1)34.5基因拷贝的重组单纯疱疹病毒1型（herpes simplex virus 1, HSV-1）R3616感染的细胞中，双链蛋白激酶R（double-stranded protein kinase R, PKR）被激活，真核翻译起始因子2α（eIF-2α）发生磷酸化，蛋白质合成被阻断。尽管野生型病毒感染的细胞中PKR同样被激活，但γ(1)34.5基因的编码产物——感染细胞蛋白34.5（infected-cell protein 34.5, ICP34.5）——可结合蛋白磷酸酶1α并将其重定向至eIF-2α位点使其去磷酸化，从而维持蛋白质的持续合成。将缺失γ(1)34.5基因的突变株在人源细胞中连续传代培养，可获得第二位点补偿突变株，这类突变株缺失了位于U(S)11基因旁侧的α47基因的不同结构域（I. Mohr与Y. Gluzman，《欧洲分子生物学组织期刊》(EMBO J.)，1996年，15卷：4759–4766页）。

本研究构建了两株重组病毒：R5103（缺失γ(1)34.5、U(S)8、U(S)9、U(S)10、U(S)11及α47[U(S)12]基因），以及由R5103衍生而来的R5104，后者携带嵌合DNA片段，该片段包含U(S)10基因以及与U(S)11基因编码区融合的α47基因启动子。R5104感染细胞的蛋白质合成谱与野生型病毒相似，而R5103感染细胞的蛋白质合成则被阻断。

对野生型亲本株及各突变株的研究得到以下结果：(1) 亲本株或突变株感染的细胞中PKR均被激活，而假感染（mock-infected）细胞中则无此现象，与此前的研究结果一致；(2) R3616、R5103及R5104病毒感染细胞的裂解液中，缺乏野生型病毒感染细胞所特有的eIF-2α特异性磷酸酶活性；(3) 未携带第二位点补偿突变的R3616与R5103感染细胞的裂解液中，存在可在体外磷酸化eIF-2α的活性；而假感染细胞、HSV-1(F)感染细胞或R5104感染细胞的裂解液则无此活性。

综上，与野生型病毒感染细胞通过快速使eIF-2α去磷酸化从而避免蛋白质合成被阻断不同，携带补偿突变的γ(1)34.5基因缺失（γ(1)34.5(−)）病毒感染的细胞中，eIF-2α并未发生磷酸化。第二位点突变所显现的活性，可能代表了一种更为古老的、演化而来的避免蛋白质合成被阻断的机制。