Expression and mutational analysis of the baculovirus very late factor 1 (vlf-1) gene.
Expression and mutational analysis of the baculovirus very late factor 1 (vlf-1) gene.
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DOI：
10.1006/viro.1998.9152
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发表时间：
1998-05
期刊：
影响因子：
3.7
通讯作者：
S. Yang;L. Miller
中科院分区：
文献类型：
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作者：
S. Yang;L. Miller
我们检查了苜蓿银纹夜蛾核多角体病毒基因 vlf-1 的表达和功能，已知该基因编码非常晚期基因转录的调节因子。 Western blot分析显示vlf-1在感染后期表达，主要是感染后15至24小时。 VLF-1定位于细胞核，也存在于病毒颗粒的核衣壳中。通过引物延伸对 vlf-1 mRNA 进行图谱显示，转录起始于 vlf-1 开放阅读框上游 71 bp 的 TAAG 基序。在瞬时表达测定中，破坏该 TAAG 基序会消除 vlf-1 刺激极晚期多角体蛋白基因 (polh) 启动子转录的能力，表明 vlf-1 表达受 TAAG 基序控制。使用高效系统构建带有 vlf-1 修饰的重组病毒，我们证实 TAAG 基序是必需的。此外，构建 vlf-1 无效突变体的努力失败了，这表明 vlf-1 是病毒复制的必需基因。计算机辅助序列同源性搜索将 vlf-1 置于 lambda 噬菌体整合酶家族中（McLachlin 和 Miller，1994）。 vlf-1 中发现的该家族严格保守的残基在病毒基因组中均不会发生改变，这意味着 VLF-1 的推定整合酶活性与 vlf-1 的基本功能相关。然而，关键活性位点酪氨酸的突变并不影响 vlf-1 在瞬时表达测定中反式激活 polh 启动子的能力，表明 VLF-1 的极晚期转录活性不需要整合酶活性。
We have examined the expression and function of a gene, vlf-1, of Autographa californica nuclear polyhedrosis virus that is known to encode a regulator of very late gene transcription. Western blot analysis revealed that vlf-1 is expressed during the late phase of infection, primarily from 15 to 24 h postinfection. VLF-1 localized in the cell nucleus and was also present in the nucleocapsids of virus particles. Mapping of vlf-1 mRNA by primer extension showed that transcription initiates at a TAAG motif 71 bp upstream of the vlf-1 open reading frame. Disruption of this TAAG motif abolished the ability of vlf-1 to stimulate transcription from the very late polyhedrin gene (polh) promoter in transient expression assays, suggesting that vlf-1 expression is controlled by the TAAG motif. Using a highly efficient system to construct recombinant viruses with modifications in vlf-1, we confirmed that the TAAG motif was essential. Furthermore, efforts to construct null mutants of vlf-1 failed, suggesting that vlf-1 is an essential gene for virus replication. Computer-assisted sequence homology searches place vlf-1 in the lambda phage integrase family (McLachlin and Miller, 1994). None of the strictly conserved residues of this family which are found in vlf-1 could be changed in the viral genome, implying that the putative integrase activity of VLF-1 is associated with the essential function of vlf-1. However, mutation of a crucial active-site tyrosine did not affect the ability of vlf-1 to transactivate the polh promoter in transient expression assays, indicating that the very late transcriptional activity of VLF-1 does not require the integrase activity.