HERPES-SIMPLEX VIRUS TYPE-1-INDUCED RIBONUCLEOTIDE REDUCTASE-ACTIVITY IS DISPENSABLE FOR VIRUS GROWTH AND DNA-SYNTHESIS - ISOLATION AND CHARACTERIZATION OF AN ICP6 LACZ INSERTION MUTANT
HERPES-SIMPLEX VIRUS TYPE-1-INDUCED RIBONUCLEOTIDE REDUCTASE-ACTIVITY IS DISPENSABLE FOR VIRUS GROWTH AND DNA-SYNTHESIS - ISOLATION AND CHARACTERIZATION OF AN ICP6 LACZ INSERTION MUTANT
复制标题
DOI：
10.1128/jvi.62.1.196-205.1988
复制
发表时间：
1988-01-01
影响因子：
5.4
通讯作者：
WELLER, SK
中科院分区：
文献类型：
--
作者：
GOLDSTEIN, DJ;WELLER, SK
单纯疱疹病毒（HSV）编码一种由两个亚基（140和38千道尔顿）组成的核糖核苷酸还原酶，其基因在病毒基因组上的坐标为0.56至0.60。产生含有HSV 1型毒株KOS还原酶亚基基因的HpaI F片段的宿主细胞系（坐标0.535 ~ 0.620）。用含有立即早期ICPO基因的质粒转染这些细胞，可导致ICP6的表达；有趣的是，ICP4质粒未能诱导表达，这表明ICP6的调节模式不寻常。利用其中一株细胞系（D14）分离出lacZ结构基因插入ICP6基因的突变体，使lacZ基因在ICP6的n端区读取。该突变体产生了一个包含434个氨基酸（38%）的ICP6 N端融合到。β上的蛋白质。-半乳糖苷酶受内源性ICP6启动子控制。用5-溴-4-氯-3-吲哚基- β染色病毒斑块，极大地促进了病毒重组体的筛选。-D-galactosde(半乳糖苷)。感染BHK细胞的酶分析表明，突变体不能诱导病毒核糖核苷酸还原酶活性。令人惊讶的是，虽然斑块大小大大减少，但突变病毒的产量与在指数增长的Vero细胞中生长的野生型相比只减少了4到5倍。与在相同条件下生长的野生型相比，血清饥饿细胞中突变病毒的斑块大小、产量和合成病毒DNA的能力受到更严重的损害。虽然我们的证据表明，HSV 1型核糖核苷酸还原酶不是分裂细胞中病毒生长和DNA复制所必需的，但它可能是非分裂细胞生长所必需的。
Herpes simplex virus (HSV) encodes a ribonucleotide reductase consisting of two subunits (140 and 38 kilodaltons) whose genes map to coordinates 0.56 to 0.60 on the viral genome. Host cell lines containing the HpaI F fragment which includes the reductase subunit genes of HSV type 1 strain KOS (coordinates 0.535 to 0.620) were generated. Transfection of these cells with a plasmid containing the immediate-early ICPO gene resulted in the expression of ICP6; interestingly, ICP4 plasmids failed to induce expression, indicating an unusual pattern of ICP6 regulation. One such cell line (D14) was used to isolate a mutant with the structural gene of lacZ inserted into the ICP6 gene such that the lacZ gene is read in frame with the N-terminal region of ICP6. This mutant generated a protein containing 434 amino acids (38%) of the N terminus of ICP6 fused to .beta.-galactosidase under control of the endogenous ICP6 promoter. Screening for virus recombinants was greatly facilitated by staining virus plaques with 5-bromo-4-chloro-3-indoyl-.beta.-D-galactosde (X-gal). Enzyme assays of infected BHK cells indicated that the mutant is incapable of inducing viral ribonucleotide reductase activity. Surprisingly, although plaque size was greatly reduced, mutant virus yield was reduced only four-to fivefold compared with that of the wild type grown in exponentially growing Vero cells. Mutant virus plaque size, yields, and ability to synthesize viral DNA were more severely compromised in serum-starved cells as compared with the wild type grown under the same condition. Although our evidence suggests that the HSV type 1 ribonucleotide reductase is not required for virus growth and DNA replication in dividing cells, it may be required for growth in nondividing cells.