ISOLATION AND CHARACTERIZATION OF HERPES-SIMPLEX VIRUS TYPE-1 MUTANTS WHICH FAIL TO INDUCE DUTPASE ACTIVITY
ISOLATION AND CHARACTERIZATION OF HERPES-SIMPLEX VIRUS TYPE-1 MUTANTS WHICH FAIL TO INDUCE DUTPASE ACTIVITY
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DOI：
10.1016/0042-6822(86)90414-9
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发表时间：
1986-01-15
期刊：
影响因子：
3.7
通讯作者：
PRESTON, VG
中科院分区：
文献类型：
--
作者：
FISHER, FB;PRESTON, VG
通过将HindIII寡核苷酸接头序列插入克隆基因编码区内的Kpn I位点，使单纯疱疹病毒（HSV）1型dUTR基因失活。通过标记拯救将突变基因引入野生型单纯疱疹病毒，通过获得基因组图谱坐标0.69至0.70内的HindIII位点和未能诱导病毒特异性dUTR活性来鉴定重组体。从该转染实验中还分离到一种自发的dUTR缺陷型突变体，其具有与野生型病毒相同的限制性内切酶DNA模式。两种类型的dUTPase-negative突变体未能诱导病毒特异性的39，000 mol wt多肽。用插入突变体感染的细胞反而含有约40，000 mol wt的新多肽。在感染自发突变体的细胞中未检测到异常的病毒特异性多肽。我们得出结论，由野生型HSV-1诱导的39，000 mol wt多肽是病毒编码的dUTR。由于两种类型的突变体在不存在野生型辅助病毒的情况下在指数生长和血清饥饿的组织培养细胞中生长良好，因此在这些条件下病毒复制不需要dUTR。通过将胸苷激酶插入突变重组到dUTR缺陷型病毒中，构建胸苷激酶缺陷型、dUTR缺陷型双突变体。这些突变体在正常组织培养细胞和缺乏细胞胸苷激酶的细胞中也与野生型病毒一样生长。
The herpes simplex virus (HSV) type 1 dUTPase gene was inactivated by insertion of HindIII oligonucleotide linker sequences into the KpnI site within the coding region of the cloned gene. The mutated gene was introduced into wild type herpes simplex virus by marker rescue and the recombinants were identified by the acquisition of a HindIII site within genome map coordinates 0.69 to 0.70 and the failure to induce virus-specific dUTPase activity. A spontaneous dUTPase deficient mutant, which had an identical restriction endonuclease DNA pattern to wild type virus, was also isolated from this transfection experiment. Both types of dUTPase-negative mutants failed to induce a virus-specific 39,000 mol wt polypeptide. Cells infected with the insertional mutant contained instead a novel polypeptide about 40,000 mol wt. No abnormal virus specific polypeptide was detected in cells infected with the spontaneous mutant. We conclude that the 39,000 mol wt polypeptide induced by wild type HSV-1 is the virus-coded dUTPase. Since both types of mutants grew well in exponentially growing and serum-starved tissue culture cells in the absence of wild type helper virus, the dUTPase is not required for virus replication under these conditions. Thymidine kinase deficient, dUTPase deficient double mutants were constructed by recombination of a thymidine kinase insertional mutation into dUTPase deficient virus. These mutants also grew as well as wild type virus both in normal tissue culture cells and cells lacking the cellular thymidine kinase.