Genetic analysis of varicella-zoster virus ORF0 to ORF4 by use of a novel luciferase bacterial artificial chromosome system
Genetic analysis of varicella-zoster virus ORF0 to ORF4 by use of a novel luciferase bacterial artificial chromosome system
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DOI：
10.1128/jvi.02666-06
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发表时间：
2007-09-01
影响因子：
5.4
通讯作者：
Zhu, Hua
中科院分区：
文献类型：
--
作者：
Zhang, Zhen;Rowe, Jenny;Zhu, Hua
为了有效地产生水痘带状疱疹病毒（VZV）突变体，我们将细菌人工染色体（BAC）载体插入到pOka基因组中。我们发现重组VZV（VZV(BAC)）菌株是由BAC DNA高效产生的，其表现与野生型病毒没有区别。此外，VZV的细胞相关性使得表征VZV突变体生长动力学变得困难，特别是当试图监测病毒在体内的复制时。为了克服这个问题，我们创造了一个携带荧光素酶基因的VZV菌株（VZV(LUC)）。该病毒像野生型病毒一样生长，所产生的荧光素酶活性可以在体外和体内量化。利用pcr诱变技术，从VZV（LUC）基因组中分别删除开放阅读框（ORF） 0 ~ 4。转染MeWo细胞后，除ORF4缺失外，其余均出现缺失突变病毒。MeWo细胞和SCID-hu小鼠的生长曲线分析表明，ORFI、ORF2和ORF3在体外和体内都是VZV复制所必需的。有趣的是，ORF0缺失病毒在体内和体外都表现出严重的生长迟缓。ORF0和ORF4突变体的生长缺陷可以通过将这些基因的野生型拷贝引入其原生基因组位点来完全修复。这项工作验证了新型荧光素酶VZV BAC系统的使用，该系统有效地产生重组VZV变体，并简化了随后在体外和体内的病毒生长动力学分析。
To efficiently generate varicella-zoster virus (VZV) mutants, we inserted a bacterial artificial chromosome (BAC) vector in the pOka genome. We showed that the recombinant VZV (VZV(BAC)) strain was produced efficiently from the BAC DNA and behaved indistinguishably from wild-type virus. Moreover, VZV's cell-associated nature makes characterizing VZV mutant growth kinetics difficult, especially when attempts are made to monitor viral replication in vivo. To overcome this problem, we then created a VZV strain carrying the luciferase gene (VZV(LUC)). This virus grew like the wild-type virus, and the resulting luciferase activity could be quantified both in vitro and in vivo. Using PCR-based mutagenesis, open reading frames (ORF) 0 to 4 were individually deleted from VZV(LUC) genomes. The deletion mutant viruses appeared after transfection into MeWo cells, except for ORF4, which was essential. Growth curve analysis using MeWo cells and SCID-hu mice indicated that ORFI, ORF2, and ORF3 were dispensable for VZV replication both in vitro and in vivo. Interestingly, the ORF0 deletion virus showed severely retarded growth both in vitro and in vivo. The growth defects of the ORF0 and ORF4 mutants could be fully rescued by introducing wild-type copies of these genes back into their native genome loci. This work has validated and justified the use of the novel luciferase VZV BAC system to efficiently generate recombinant VZV variants and ease subsequent viral growth kinetic analysis both in vitro and in vivo.