EFFECT OF AN AMBER MUTATION IN THE HERPES-SIMPLEX VIRUS THYMIDINE KINASE GENE ON POLYPEPTIDE-SYNTHESIS AND STABILITY
EFFECT OF AN AMBER MUTATION IN THE HERPES-SIMPLEX VIRUS THYMIDINE KINASE GENE ON POLYPEPTIDE-SYNTHESIS AND STABILITY
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DOI：
10.1016/0042-6822(89)90260-2
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发表时间：
1989-02-01
期刊：
影响因子：
3.7
通讯作者：
COEN, DM
中科院分区：
文献类型：
--
作者：
IRMIERE, AF;MANOS, MM;COEN, DM
KG 111是单纯疱疹病毒（HSV）科斯株的突变体，表现出温度依赖性耐药性。例如，它在39 ℃几乎与胸苷激酶（tk）缺陷型病毒一样具有抗性，但在34 ℃对阿昔洛韦相对敏感。利用标记转移技术，我们已经将KG 111中赋予温度依赖性耐药性的突变定位到tk基因的5“部分。该区域的测序揭示了密码子44处的琥珀突变，其位于tk多肽的第一和第二甲硫氨酸密码子之间。该突变与TK 4中发现的突变相同，TK 4是一种来源于C1101（L. Haidian等人，1985，J. Virol. 56，512-519）。从KG 111-和TK 4-感染细胞的免疫沉淀tk蛋白的分析表明，KG 111和TK 4不合成全长tk多肽，而是产生截短形式的蛋白质。少量的类似截短的tk多肽也产生在野生型感染的细胞中，并被认为是在下游AUG起始产生的。与野生型相比，突变tk蛋白的相对量和大小与消除全长多肽翻译的琥珀突变一致，并导致下游AUG密码子起始利用率增加4 - 5倍。由KG 111和TK 4指定的截短的多肽在39 °下比全长多肽稳定性差，这可能导致条件性耐药表型。另一方面，通常由野生型病毒以低水平表达的截短多肽和来自缺失突变体的更高表达的截短tk多肽在39 °相对稳定。这些结果表明，截短的tk多肽的稳定性受存在的tk量的影响。
KG111 is a mutant of herpes simplex virus (HSV), strain KOS, that exhibits temperature-dependent drug resistance. For example, it is almost as resistant as a thymidine kinase (tk)-deficient virus at 39.degree., but is relatively sensitive to acyclovir at 34.degree.. Using marker transfer techniques, we have mapped the mutation conferring temperature-dependent drug resistance in KG111 to the 5'' portion of the tk gene. Sequencing of this region revealed an amber mutation at codon 44, which lies between the first and second methionine codons of the tk polypeptide. This mutation is identical to that found in TK4, an HSV mutant derived from Cl 101 (L. Haarr et al., 1985, J. Virol. 56, 512-519). Analyses of immunoprecipitated tk proteins from KG111- and TK4-infected cells showed that KG111 and TK4 do not synthesize full-length tk polypeptides, but instead produce a truncated form of the protein. Small amounts of a similar truncated tk polypeptide are also produced in wild-type-infected cells and are thought to arise from initiation at a downstream AUG. The relative amounts and size of the mutant tk proteins compared with those of the wild-type are consistent with the amber mutation eliminating translation of full-length polypeptide and causing a four- to fivefold increase in the utilization of downstream AUG codons for initiation. The truncated polypeptides specified by KG111 and TK4 are less stable than the full-length polypeptide at 39.degree., which may contribute to the conditional drug-resistant phenotype. On the other hand, the truncated polypeptides normally expressed by wild-type virus at low levels and the more highly expressed truncated tk polypeptides from a deletion mutant are relatively stable at 39.degree.. These results suggest that stability of the truncated tk polypeptide is influenced by the amount of tk present.