A self-excisable infectious bacterial artificial chromosome clone of varicella-zoster virus allows analysis of the essential tegument protein encoded by ORF9
A self-excisable infectious bacterial artificial chromosome clone of varicella-zoster virus allows analysis of the essential tegument protein encoded by ORF9
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DOI：
10.1128/jvi.01148-07
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发表时间：
2007-12-01
影响因子：
5.4
通讯作者：
Osterrieder, Nikolaus
中科院分区：
文献类型：
--
作者：
Tischer, B. Karsten;Kaufer, Benedikt B.;Osterrieder, Nikolaus
为了促进水痘带状疱疹病毒（VZV）（引起水痘和带状疱疹）的突变病毒的产生，我们生成了 P-Oka 菌株的全长感染性细菌人工染色体（BAC）克隆。首先，将 mini-F 序列插入预先存在的 VZV 粘粒中，并去除 SuperCos 复制子。随后，从重叠的粘粒克隆中产生含有mini-F的重组病毒，并从重组病毒中回收的全长VZV DNA在大肠杆菌中建立为感染性BAC。迷你F复制子内VZV基因组序列的反向复制导致病毒在真核细胞中重建时载体序列无标记切除。使用这种新工具，研究了 ORF9 编码的主要被膜蛋白在 VZV 复制中的作用。通过两步过路红色诱变在起始密码子中引入的无标记点突变消除了 ORF9 表达，并导致了在回复病毒中未观察到的显着生长缺陷。 ORF9对于VZV复制的本质性质最终通过杆状病毒介导的基因转移的反式互补恢复ORF9缺陷型突变病毒的生长而得到证实。
In order to facilitate the generation of mutant viruses of varicella-zoster virus (VZV), the agent causing varicella (chicken pox) and herpes zoster (shingles), we generated a full-length infectious bacterial artificial chromosome (BAC) clone of the P-Oka strain. First, mini-F sequences were inserted into a preexisting VZV cosmid, and the SuperCos replicon was removed. Subsequently, mini-F-containing recombinant virus was generated from overlapping cosmid clones, and full-length VZV DNA recovered from the recombinant virus was established in Escherichia coli as an infectious BAC. An inverted duplication of VZV genomic sequences within the mini-F replicon resulted in markerless excision of vector sequences upon virus reconstitution in eukaryotic cells. Using the novel tool, the role in VZV replication of the major tegument protein encoded by ORF9 was investigated. A markerless point mutation introduced in the start codon by two-step en passant Red mutagenesis abrogated ORF9 expression and resulted in a dramatic growth defect that was not observed in a revertant virus. The essential nature of ORF9 for VZV replication was ultimately confirmed by restoration of the growth of the ORF9-deficient mutant virus using trans-complementation via baculovirus-mediated gene transfer.