- 學位論文
探討桿狀病毒立即早期基因 p143 對寄主 DNA 複製 的調控
Regulation of host DNA replication by baculovirus immediate early gene p143
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摘要
桿狀病毒 (Baculovirus) ，是一種專門感染節肢動物的雙股DNA病毒，目前已發現有600多種昆蟲會被感染，包含農業害蟲斜紋夜蛾（Spodoptera litura），與經濟昆蟲家蠶（Bombyx mori），前者能被加州苜蓿夜蛾核多角體病毒（AcMNPV）感染，後者能被家蠶核多角體病毒（BmNPV）感染。目前已知此兩種病毒的基因組序列高度相似，然而它們的寄主範圍並不重疊。先前的研究發現，將此兩種病毒的DNA 解旋酶基因p143種間置換使AcMNPV能夠感染非寄主範圍的細胞株，顯示解旋酶P143為寄主專一性的關鍵之一，然而目前該機制的具體細節仍不清楚。前人研究指出，哺乳類細胞能以自身的解旋酶競爭病毒的DNA複製起始點位，直接抑制病毒DNA複製作為防禦，基於此，本研究假設桿狀病毒之P143蛋白能影響寄主DNA複製。我們將病毒之p143大量表現或基因敲落，探討其對於寄主DNA複製的影響，並觀察病毒P143與寄主DNA複製起始點位的接合。結果顯示寄主在允許感染（permissive infection）下基因組DNA含量與新生成DNA含量皆呈現下降趨勢，將p143基因敲落後則影響減弱，而這些現象在非允許感染（non-permissive infection）的狀況下則無，顯示與寄主範圍的關聯性。桿狀病毒的允許感染對於寄主的葡萄糖含量、核苷酸合成、與DNA複製相關基因的表現沒有全面的顯著差異，顯示對DNA複製的影響應主要來自病毒的P143。由桿狀病毒P143蛋白與複製起始點接合的實驗可證實，桿狀病毒DNA解旋酶會接合寄主的複製起始點，與寄主自身解旋酶競爭。本研究探討了桿狀病毒運用P143蛋白抑制寄主DNA複製的機制，同時排除其他能影響DNA複製的可能途徑，以p143的作用為基礎為桿狀病毒的寄主範圍提出解釋，更好地理解桿狀病毒的生物學特性。
並列摘要
Baculovirus, a type of double-stranded DNA virus, specifically infects arthropods. Currently, over 600 insect species have been found to be susceptible to this virus, including agricultural pests such as tobacco cutworm (Spodoptera litura) and the economically significant silkworm (Bombyx mori). While the former is susceptible to the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the latter is infected by the Bombyx mori nucleopolyhedrovirus (BmNPV). Although the genomic sequences of these viruses exhibit high similarity, their host ranges do not overlap. Previous research identified mutations in the DNA helicase gene p143 of these viruses, allowing AcMNPV or BmNPV to infect non-host cell lines, indicating that the helicase P143 plays a crucial role in host specificity. However, the specific details of this mechanism remain unclear. Earlier studies suggested that mammalian cells competitively use their own helicases to impede the virus's DNA replication initiation, serving as a defense mechanism. Based on this, we hypothesized that the P143 protein of baculoviruses might affect host DNA replication. We either overexpressed or knocked down the virus's p143 gene and examined the impact on host DNA replication. Additionally, we observed the interaction between the virus's P143 and the host’s DNA replication origin. The results indicated a declining trend in both the genomic DNA content and newly synthesized DNA content of the host under permissive infection. However, this effect was mitigated upon p143 gene knockdown. These phenomena were absent under non-permissive infection, indicating their correlation with host range. Permissive infection by baculovirus showed no comprehensive impacts on the host's glucose content, nucleotide synthesis, and expression of genes related to DNA replication. This suggested that the influence on DNA replication primarily stems from the virus's P143, rather than physiological modulations. The interaction between baculovirus P143 protein and the DNA replication origin sites confirmed that the baculovirus DNA helicase binds to the host's replication origin, competing with the host's own helicase. This study delves into the mechanisms by which baculovirus utilizes the P143 protein to inhibit host DNA replication, offering insights into the host range of baculoviruses and enhancing our understanding of their biological characteristics.
參考文獻
參考文獻
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