AcMNPV 桿狀病毒lef-2基因缺失之分析

Abstract

模式桿狀病毒AcMNPV之lef-2基因為一晚期表現因子，早期研究以質體轉染實驗證明其可影響晚期基因 (vp39) 之表現，且由帶有複製起始點 (replication origin) 質體之轉染實驗中，亦證實lef-2基因為DNA 複製所必需。為了解lef-2於病毒生活史中所扮演的角色，本論文利用AcMNPV基因體之bacmid，以同源性序列重組之方法，於大腸桿菌中進行剔除病毒基因體上之lef-2基因，以得到lef-2基因剔除病毒株。此外，將lef-2基因及其啟動子，利用轉位之方法，將之構築於lef-2缺失bacmid之polyhedrin 基因座，則可得一lef-2基因回復病毒株。將野生型病毒 (bAcwt-hEG)、lef-2基因缺失病毒 (bAc△lef2-hEG) 及lef-2基因回復病毒 (bAclef2-Rep-hEG) 分別轉染至昆蟲細胞後，分析其個別的表現型 (phenotypes)。結果發現bAc△lef2-hEG 產生出芽病毒之能力，及病毒之感染能力皆較野生型差，而bAclef2-Rep-hEG之表型則是與野生型相差不大，表示可成功回復缺失lef-2之表現型。由縫孔墨漬雜交分析 (slot blot hybridization) 中，可發現缺失lef-2基因之病毒似乎無法正常複製，DNA量並無隨時間有明顯增加。但由DpnⅠ處理分析 (DpnⅠ based replication assay) 中卻可發現，lef-2基因缺失之病毒仍具少量之DNA複製，且複製起始點較野生型病毒晚，有複製延遲之現象。此外，由西方墨點法之分析，可知在缺失lef-2基因之條件下，其晚期啟動子 (vp39) 及極晚期啟動子 (polh) 皆可啟動報導基因的表現，顯示病毒之構造蛋白仍可表現，表現量雖較野生型低，但並非完全不表現。另一方面，經由免疫轉漬法及免疫金標定法偵測純化之病毒，發現LEF-2 蛋白質可被攜帶於病毒顆粒中，此特性可能與lef-2影響病毒複製相關。雖然病毒之複製受到lef-2基因缺失的影響，但由於病毒之構造蛋白仍可表現，如鞘蛋白 (VP39) 等，因此經由穿透式電子顯微鏡觀察，可知缺失lef-2之病毒仍可有正常的核酸蛋白鞘形成。最後，將lef-2基因缺失病毒以cDNA晶片分析後，可得AcMNPV基因整體轉錄之變化，結果顯示lef-2基因影響的基因大多集中於晚期基因。由前人研究中，其利用質體轉染證實lef-2為晚期基因表現及DNA複製所必需。但由本論文之結果可知，當病毒基因體缺失lef-2基因後，病毒DNA仍能有少量的複製，且晚期及極晚期基因皆可表現，並非前人研究所認為之必需基因，lef-2缺失病毒株仍有能力完成病毒之生活史。

The late expression factor 2 (lef-2) gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was originally identified by plasmid transient expression assays and shown to be important for expression of some virus-encoded late genes. Previous studies also suggested that lef-2 gene plays an essential but undefined role in viral DNA replication by using origin-dependent plasmid replication assays. To determine the role of lef-2 in AcMNPV life cycle, the lef-2 knockout (ko) mutant bacmid (bAc△lef2-hEG) was generated by homologous recombination in a wild-type (wt) bacmid (bAcwt-hEG) in Escherichia coli. Additionally, a lef-2 repair bacmid (bAclef2-Rep-hEG) was generated by transposing the lef-2 coding region along with its own promoter into the polyhedrin locus of lef-2 ko bacmid. To trace the movement of virion, DNA replication and gene expression, enhanced green fluorescence protein (eGFP) gene was inserted into bacmids, bAcwt-hEG, bAc△lef2-hEG and bAclef2-Rep-hEG. Budded viruses production of and viral spreading of bAclef2-Rep-hEG were similar to bAcwt-hEG by growth curve analyses and fluorescence microscopy, but bAc△lef2-hEG was defective in both processes. Slot blot analyses showed that the viral genome of lef-2 ko bacmid did not seem to replicate. However, by using a more sensitive replication assay based on DpnI digestion that can distinguish replicated from un-replicated DNA samples, it became clear that the lef-2 ko bacimd can replicate albeit very inefficiently. Further studies indicated that the replication level of lef-2 ko bacmid reduced substantially and onset of replication was delayed by about 48 hours. Due to late and very late gene expressions were depended on vial DNA replication, promoters from late genes, such as a capsid gene (vp39) and polyhedrin gene (polh), able to drive expression of reporter gene in the lef-2 ko background. Furthermore, LEF-2 protein was found to incorporated into the virions by using immunoblotting assay and immunogold electron microscopy, suggesting that virion-associated LEF-2 protein might be required immediately after entering cells for the initiation of viral DNA replication. However, in the absent of LEF-2, the formation of viral nucleocapsid derived from lef-2 ko bacmid appeared to be normal under transmission electron microscope, suggesting that the life cycle of lef-2 ko bacmid could proceed through DNA replication to viral pakckaging unimpededly. Finally, when the overall viral gene expression patterns were surveyed by viral cDNA microarray analyses in the absent of lef-2 gene, most of the late genes were affected. Thus, in contrast to prior studies using plasmid transient expression assay, current studies using lef-2 ko bacmid demonstrated that lef-2 was not an essential gene. lef-2 was not absolutely required for vial DNA replication and late gene expression, however without lef-2 gene in viral genome, the levels of replication and gene expression declined significantly.