Yamagishi Junya, Burnett Erik D, Harwood Steven H, Blissard Gary W
Boyce Thompson Institute at Cornell University, Ithaca, NY 14853, USA.
Virology. 2007 Aug 15;365(1):34-47. doi: 10.1016/j.virol.2007.02.034. Epub 2007 Apr 30.
The pp31 gene of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes a phosphorylated DNA binding protein that associates with virogenic stroma in the nuclei of infected cells. Prior studies of pp31 by transient late expression assays suggested that pp31 may play an important role in transcription of AcMNPV late genes [Todd, J. W., Passarelli, A. L., and Miller, L. K. (1995). Eighteen baculovirus genes, including lef-11, p35, 39K, and p47, support late gene expression. J. Virol. 69, 968-974] although genetic studies of the closely related BmNPV pp31 gene suggested that pp31 may be dispensable [Gomi, S., Zhou, C. E., Yih, W., Majima, K., and Maeda, S. (1997). Deletion analysis of four of eighteen late gene expression factor gene homologues of the baculovirus, BmNPV. Virology 230 (1), 35-47]. In the current study, we examined the role of the pp31 gene in the context of the AcMNPV genome during infection. We used a BACmid-based system to generate a pp31 knockout in the AcMNPV genome. The pp31 knockout was subsequently rescued by reinserting the pp31 gene into the polyhedrin locus of the same virus genome. We found that pp31 was not essential for viral replication although the absence of pp31 resulted in a lower viral titer. Analysis of viral DNA replication in the absence of pp31 showed that the kinetics of viral DNA replication were unaffected. An AcMNPV oligonucleotide microarray was used to compare gene expression from all AcMNPV genes in the presence or absence of pp31. In the absence of pp31, a modest reduction in transcripts was detected for many viral genes (99 genes) while no substantial increase or decrease was observed for 43 genes. Transcripts from 6 genes (p6.9, ORF 97, ORF 60, ORF 98, ORF 102 and chitinase) were reduced by 66% or more compared to the levels detected from the control virus. Microarray results were further examined by qPCR analysis of selected genes. In combination, these data show that deletion of the pp31 gene was not lethal and did not appear to affect viral DNA replication but resulted in an apparent modest down-regulation of a subset of AcMNPV genes that included both early and late genes.
苜蓿银纹夜蛾多粒包埋核型多角体病毒（AcMNPV）的pp31基因编码一种磷酸化的DNA结合蛋白，该蛋白与受感染细胞细胞核中的病毒发生基质相关联。先前通过瞬时晚期表达分析对pp31的研究表明，pp31可能在AcMNPV晚期基因的转录中起重要作用[Todd, J. W., Passarelli, A. L., and Miller, L. K. (1995). 包括lef-11、p35、39K和p47在内的18个杆状病毒基因支持晚期基因表达。J. Virol. 69, 968 - 974]，尽管对密切相关的家蚕核型多角体病毒（BmNPV）pp31基因的遗传学研究表明pp31可能是可有可无的[Gomi, S., Zhou, C. E., Yih, W., Majima, K., and Maeda, S. (1997). 杆状病毒BmNPV的18个晚期基因表达因子基因同源物中4个的缺失分析。Virology 230 (1), 35 - 47]。在本研究中，我们在感染过程中研究了pp31基因在AcMNPV基因组背景下的作用。我们使用基于BACmid的系统在AcMNPV基因组中产生pp31基因敲除。随后通过将pp31基因重新插入同一病毒基因组的多角体蛋白基因座来拯救pp31基因敲除。我们发现pp31对于病毒复制不是必需的，尽管缺少pp31会导致病毒滴度降低。对缺少pp31时的病毒DNA复制分析表明，病毒DNA复制的动力学不受影响。使用AcMNPV寡核苷酸微阵列比较存在或不存在pp31时所有AcMNPV基因的基因表达。在缺少pp31的情况下，检测到许多病毒基因（99个基因）的转录本有适度减少，而43个基因未观察到明显增加或减少。与对照病毒检测到的水平相比，6个基因（p6.9、ORF 97、ORF 60、ORF 98、ORF 102和几丁质酶）的转录本减少了66%或更多。通过对选定基因的qPCR分析进一步检查微阵列结果。综合这些数据表明，pp31基因的缺失不是致命的，似乎也不影响病毒DNA复制，但导致AcMNPV基因的一个子集明显适度下调，这些基因包括早期和晚期基因。
