Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression.
Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression.
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DOI：
10.1016/j.virol.2012.10.022
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发表时间：
2013-01-20
期刊：
影响因子：
3.7
通讯作者：
Thiem SM
中科院分区：
文献类型：
--
作者：
Salem TZ;Zhang F;Thiem SM
加州签名多核多角体病毒ORF34是转录单元的一部分，包括ORF32，编码病毒成纤维细胞生长因子（FGF）和ORF33。基于在病毒基因的RNAi筛选中增强报告基因的表达，我们确定ORF34是杆状病毒表达系统中缺失以提高蛋白表达的候选基因。然而，ORF34被证明是一个必需基因。为了探究ORF34的功能，我们构建并表征了缺失（KO34）和挽救（KO34）的bacmids。转染KO34的原代细胞没有感染扩散，转染KO34的细胞上清液不能感染新鲜的Sf21细胞，而转染救援bacmids的上清液可以恢复感染。此外，在转染KO34的细胞中，电镜未观察到出芽病毒，western blots也未从转染上清中检测到病毒蛋白。这表明ORF34在感染性病毒的产生中可能是一个重要的基因。
Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.
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