Coen D M, Irmiere A F, Jacobson J G, Kerns K M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Virology. 1989 Feb;168(2):221-31. doi: 10.1016/0042-6822(89)90261-4.
Herpes simplex virus mutant KG111 contains a nonsense mutation at codon 44 of the viral thymidine kinase (tk) gene and produces low amounts of a truncated tk polypeptide. We tested mutant KG111 and related viruses that specify varying amounts of similar truncated tk polypeptides for their sensitivities to antiviral nucleoside analogs at different temperatures using plaque reduction assays. The results of these assays showed that the nonsense mutation confers high resistance to bromovinyldeoxyuridine (BVdU) at any temperature and temperature-dependent resistance to acyclovir (ACV), buciclovir (BCV), ganciclovir (DHPG), and fluoroiodoarabinouracil (FIAU). Above relatively low threshold levels of tk that varied depending on the drug tested, viruses exhibited full sensitivity to ACV, BCV, DHPG, and FIAU at 34 degrees. Below these threshold levels, however, decreases in drug sensitivity were linear with decreases in tk levels, forming the basis of a pharmacological assay for tk gene expression. Studies of thymidine (TdR) anabolism in infected 143 tk-cells showed that when high TdR concentrations were added to the medium, KG111 directed thymidine monophosphate (TMP) formation at rates consonant with the amount of tk polypeptide produced by the mutant. When low concentrations to TdR were added to the medium, however, KG111 directed TMP formation at a rate similar to that directed by wild-type virus, indicating that the truncation of the tk polypeptide had little or no effect on tk activity at 34 degrees. Subsequent anabolism to thymidine diphosphate and thymidine triphosphate was reduced in KG111-infected cells, indicating a defect in TMP kinase activity that explains this mutant's resistance to BVdU. Despite the low levels of tk and TMP kinase activity expressed by KG111, this mutant established reactivatable latent infections as efficiently as wild-type virus in a mouse model.
单纯疱疹病毒突变体KG111在病毒胸苷激酶（tk）基因的第44密码子处含有一个无义突变，并产生少量截短的tk多肽。我们使用蚀斑减少试验，测试了突变体KG111和能产生不同数量相似截短tk多肽的相关病毒在不同温度下对抗病毒核苷类似物的敏感性。这些试验结果表明，该无义突变使病毒在任何温度下对溴乙烯脱氧尿苷（BVdU）具有高抗性，并对阿昔洛韦（ACV）、布昔洛韦（BCV）、更昔洛韦（DHPG）和氟碘阿糖脲嘧啶（FIAU）具有温度依赖性抗性。在取决于所测试药物的相对较低的tk阈值水平之上，病毒在34℃时对ACV、BCV、DHPG和FIAU表现出完全敏感性。然而，在这些阈值水平以下，药物敏感性的降低与tk水平的降低呈线性关系，形成了tk基因表达药理学检测的基础。对感染的143 tk细胞中胸苷（TdR）合成代谢的研究表明，当向培养基中添加高浓度TdR时，KG111指导胸苷一磷酸（TMP）形成的速率与该突变体产生的tk多肽量一致。然而，当向培养基中添加低浓度TdR时，KG111指导TMP形成的速率与野生型病毒指导的速率相似，这表明tk多肽的截短在34℃时对tk活性几乎没有影响。在KG111感染的细胞中，随后向二磷酸胸苷和三磷酸胸苷的合成代谢减少，表明TMP激酶活性存在缺陷，这解释了该突变体对BVdU的抗性。尽管KG111表达的tk和TMP激酶活性水平较低，但在小鼠模型中，该突变体与野生型病毒一样有效地建立了可再激活的潜伏感染。
