DELETION OF THE VARICELLA-ZOSTER VIRUS LARGE SUBUNIT OF RIBONUCLEOTIDE REDUCTASE IMPAIRS GROWTH OF VIRUS IN-VITRO
DELETION OF THE VARICELLA-ZOSTER VIRUS LARGE SUBUNIT OF RIBONUCLEOTIDE REDUCTASE IMPAIRS GROWTH OF VIRUS IN-VITRO
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DOI：
10.1128/jvi.68.5.3317-3323.1994
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发表时间：
1994-05-01
影响因子：
5.4
通讯作者：
COHEN, JI
中科院分区：
文献类型：
--
作者：
HEINEMAN, TC;COHEN, JI
感染水痘带状疱疹病毒(VZV)的细胞表达一种与未感染细胞不同的病毒核苷酸还原酶。VZV开放阅读框18和19(ORF18和ORF19)分别与单纯疱疹病毒I型基因同源，编码核糖核苷酸还原酶的小亚基和大亚基。我们通过将四个重叠的粘粒DNA导入培养细胞产生了重组VZV。为了构建缺失核糖核苷酸还原酶的病毒，我们从一个粘粒中删除了VZV ORF19的97%。将此粘粒与其他亲本粘粒一起转染，经Southern印迹分析，得到了一个缺失2.3-KBP的VZV突变体。在VZV缺失ORF19的感染细胞中未检测到病毒特异性核糖核苷酸还原酶活性。与感染亲本VZV的黑色素瘤细胞相比，感染ORF19缺失VZV的黑色素瘤细胞产生的斑块气味更重。突变病毒的生长速度也略慢于亲本病毒。化学抑制VZV核糖核苷酸还原酶可增强阿昔洛韦的抗VZV活性。同样，对于VZV核糖核苷酸还原酶缺失突变体，抑制空斑形成50%所需的阿昔洛韦浓度比亲本病毒低三倍。我们的结论是，VZV核糖核苷酸还原酶大亚基在体外对病毒感染不是必需的；然而，该基因的缺失会损害VZV在细胞培养中的生长，并使该病毒更容易受到阿昔洛韦的抑制。
Cells infected with varicella-zoster virus (VZV) express a viral ribonucleotide reductase which is distinct from that present in uninfected cells. VZV open reading frames 18 and 19 (ORF18 and ORF19) are homologous to the herpes simplex virus type I genes encoding the smalt and large subunits of ribonucleotide reductase, respectively. We generated recombinant VZV by transfecting cultured cells,vith four overlapping cosmid DNAs. To construct a virus lacking ribonucleotide reductase, we deleted 97% of VZV ORF19 from one of the cosmids. Transfection of this cosmid with the other parental cosmids yielded a VZV mutant with a 2.3-kbp deletion confirmed by Southern blot analysis. Virus-specific ribonucleotide reductase activity was not detected in cells infected,vith VZV lacking ORF19. Infection of melanoma cells with ORF19-deleted VZV resulted in plaques smeller than those produced by infection with the parental VZV. The mutant virus also exhibited a growth rate slightly slower than that of the parental virus. Chemical inhibition of the VZV ribonucleotide reductase has been shown to potentiate the anti-VZV activity of acyclovir. Similarly, the concentration of acyclovir required to inhibit plaque formation by 50% was threefold lower for the VZV ribonucleotide reductase deletion mutants than for parental virus. We conclude that the VZV ribonucleotide reductase large subunit is not essential for virus infection in vitro; however, deletion of the gene impairs the growth of VZV in cell culture and renders the virus more susceptible to inhibition by acyclovir.