Cohen J I, Seidel K E
Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7376-80. doi: 10.1073/pnas.90.15.7376.
Four overlapping cosmid clones were constructed that contain the complete genome of the attenuated Oka strain of VZV. Transfection of human melanoma cells with the four cosmids resulted in production of infectious VZV. A double-stranded oligonucleotide, encoding a stop codon in all three open reading frames, was inserted into one of the cosmids at the 5' end of the viral thymidylate synthetase gene. Transfection of melanoma cells with the mutant cosmid, along with the other three cosmids, resulted in VZV that does not express the viral thymidylate synthetase protein. The mutant virus grew at a rate similar to that of the parental Oka strain virus. Production of recombinant VZV using cosmid DNAs will be useful for studying the function of viral genes in VZV replication and establishment of latency. Furthermore, manipulation of the Oka strain of VZV might allow one to produce a vaccine virus that does not establish latency in the central nervous system or a virus that encodes foreign antigens for use as a polyvalent live virus vaccine.
构建了四个重叠的黏粒克隆，它们包含水痘带状疱疹病毒（VZV）减毒Oka株的完整基因组。用这四个黏粒转染人黑色素瘤细胞可产生有感染性的VZV。将一个在所有三个开放阅读框中都编码终止密码子的双链寡核苷酸插入到其中一个黏粒的病毒胸苷酸合成酶基因的5'端。用该突变黏粒以及其他三个黏粒转染黑色素瘤细胞，产生的VZV不表达病毒胸苷酸合成酶蛋白。突变病毒的生长速度与亲本Oka株病毒相似。使用黏粒DNA生产重组VZV将有助于研究病毒基因在VZV复制和潜伏建立中的功能。此外，对VZV Oka株的操作可能使人们能够生产出一种不在中枢神经系统中建立潜伏的疫苗病毒，或一种编码外源抗原用作多价活病毒疫苗的病毒。
