The major immediate-early gene ie3 of mouse cytomegalovirus is essential for viral growth
The major immediate-early gene ie3 of mouse cytomegalovirus is essential for viral growth
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DOI：
10.1128/jvi.74.23.11129-11136.2000
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发表时间：
2000-12-01
影响因子：
5.4
通讯作者：
Messerle, M
中科院分区：
文献类型：
--
作者：
Angulo, A;Ghazal, P;Messerle, M
小鼠巨细胞病毒（MCMV）的主要立即早期基因ie3和相应的人巨细胞病毒ie2基因对病毒复制的意义尚不清楚。为了研究MCMV IE3调节蛋白的功能，我们产生了两种不同的MCMV重组体，其在IE3开放阅读框（ORF）中含有大的缺失。通过细菌人工染色体诱变技术构建突变体基因组，并在表达MCMV主要立即早期基因的小鼠成纤维细胞系上重建MCMV ie3缺失突变体。ie3缺失突变体不能在正常小鼠成纤维细胞上复制，即使使用高感染复数。当IE3蛋白由互补细胞系反式提供时，复制缺陷被挽救。恢复IE3 ORF的回复突变体病毒能够在正常小鼠成纤维细胞中以野生型动力学复制，提供了ie3突变体的缺陷生长表型是由于ie3基因破坏的证据。为了描述ie3控制的病毒复制限制点，我们分析了选择性早期（beta）和晚期（gamma）基因的表达模式。虽然我们可以在感染了ie3突变体的细胞中检测到立即早期基因ie1的转录本，但我们未能检测到代表性的β和γ基因的转录本。这些数据表明，MCMV反式激活因子IE3在组织培养中的病毒复制过程中起着不可或缺的作用，暗示了人CMV ie2基因产物的类似作用。据我们所知，ie3缺失突变体代表了分离的第一个含有必需基因破坏的MCMV重组体。
The significance of the major immediate-early gene ie3 of mouse cytomegalovirus (MCMV) and that of the corresponding ie2 gene of human cytomegalovirus to viral replication are not known. To investigate the function of the MCMV IE3 regulatory protein, we generated two different MCMV recombinants that contained a large deletion in the IE3 open reading frame (ORF). The mutant genomes were constructed by the bacterial artificial chromosome mutagenesis technique, and MCMV ie3 deletion mutants were reconstituted on a mouse fibroblast cell line that expresses the MCMV major immediate-early genes. The ie3 deletion mutants failed to replicate on normal mouse fibroblasts even when a high multiplicity of infection was used. The replication defect was rescued when the IE3 protein was provided in trans by a complementing cell line. A revertant virus in which the IE3 ORF was restored was able to replicate with wild-type kinetics in normal mouse fibroblasts, providing evidence that the defective growth phenotype of the ie3 mutants was due to disruption of the ie3 gene. To characterize the point of restriction in viral replication that is controlled by ie3, we analyzed the pattern of expression of selective early (beta) and late (gamma) genes. While we could detect transcripts for the immediate-early gene ie1 in cells infected with the ie3 mutants, we failed to detect transcripts for representative beta and gamma genes. These data demonstrate that the MCMV transactivator IE3 plays an indispensable role during viral replication in tissue culture, implicating a similar role for the human CMV ie2 gene product. To our knowledge, the ie3 deletion mutants represent the first MCMV recombinants isolated that contain a disruption of an essential gene.