Molecular, Cellular, and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, Kansas, USA.
J Virol. 2012 May;86(10):5614-25. doi: 10.1128/JVI.06252-11. Epub 2012 Mar 14.
The Autographa californica M nucleopolyhedrovirus (AcMNPV) orf79 (ac79) gene is a conserved gene in baculoviruses and shares homology with genes in ascoviruses, iridoviruses, and several bacteria. Ac79 has a conserved motif and structural similarities to UvrC and intron-encoded endonucleases. Ac79 is produced at early times during infection and concentrates in the nucleus of infected cells at late times, suggesting a cellular compartment-specific function. To investigate its function, an ac79-knockout bacmid was generated through homologous recombination in Escherichia coli. Titration assays showed that budded virus (BV) production was reduced in the ac79-knockout virus compared to control viruses, following either virus infection or the transfection of bacmid DNA. The ac79-knockout virus-infected cells produced plaques smaller than those infected with control ac79-carrying viruses. No obvious differences were observed in viral DNA synthesis, viral protein accumulation, or the formation of occlusion bodies in ac79-knockout and control viral DNA-transfected cells, indicating progression into the late and very late phases of viral infection. However, comparative analyses of the amounts of BV genomic DNA and structural proteins in a given quantity of infectious virions suggested that the ac79-knockout virus produced more noninfectious BV in infected cells than the control virus. The structure of the ac79-knockout BV determined by transmission electron microscopy appeared to be similar to that of the control virus, although aberrant capsid protein-containing tubular structures were observed in the nuclei of ac79-knockout virus-infected cells. Tubular structures were not observed for ac79 viruses with mutations in conserved endonuclease residues. These results indicate that Ac79 is required for efficient BV production.
美洲棉铃虫核多角体病毒（AcMNPV）orf79（ac79）基因是杆状病毒中保守的基因，与虹彩病毒、杆状病毒和几种细菌的基因具有同源性。Ac79 具有保守基序和与 UvrC 以及内含子编码的内切核酸酶的结构相似性。Ac79 在感染早期产生，并在晚期集中在感染细胞的核内，表明其具有细胞区室特异性功能。为了研究其功能，通过大肠杆菌中的同源重组生成了 ac79 敲除 bacmid。滴定实验表明，与对照病毒相比，在 ac79 敲除病毒感染或 bacmid DNA 转染后，芽生病毒（BV）的产生减少。感染 ac79 敲除病毒的细胞产生的斑块比感染对照携带 ac79 病毒的细胞产生的斑块小。在 ac79 敲除和对照病毒 DNA 转染的细胞中，没有观察到病毒 DNA 合成、病毒蛋白积累或包埋体形成的明显差异，表明进入病毒感染的晚期和极晚期。然而，对给定数量的感染性病毒粒子中的 BV 基因组 DNA 和结构蛋白的量进行比较分析表明，与对照病毒相比，ac79 敲除病毒在感染细胞中产生了更多的无感染性 BV。通过透射电子显微镜确定的 ac79 敲除 BV 的结构似乎与对照病毒相似，尽管在 ac79 敲除病毒感染细胞的核中观察到异常的含有衣壳蛋白的管状结构。在含有保守内切核酸酶残基突变的 ac79 病毒中未观察到管状结构。这些结果表明 Ac79 是高效 BV 产生所必需的。
