請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/30628
標題: Hz-1病毒持續感染時與昆蟲寄主細胞之分子互動研究
Molecular Interactions Between Hz-1 Virus and Insect Cells During Persistent Viral Infection
作者: 張銘傳
Chang, Ming-Chuan
關鍵字: Hz-1 virus
Hz-1病毒
insect cells
stably transfected cells
persistent infection
pag1 gene
promoter
baculovirus
PAT1
昆蟲細胞
穩定轉染細胞
持續感染
pag1基因
啟動子
昆蟲桿狀病毒
分子研究
出版社: 昆蟲學系
摘要: 玉米穗蟲病毒(Heliothis zea virus 1, Hz-1 virus),可以在多種鱗翅目昆蟲的細胞株發生持續或急性感染,然而其生物特性傾向於持續感染的建立,所以是研究病毒持續感染機制的良好模式。Hz-1病毒在持續感染時只有一種RNA產生,稱為持續感染相關轉錄子(persistence-associated transcript 1, PAT1),而表現此RNA的基因稱為持續感染相關基因(persistence-associated gene 1, pag1),所以PAT1對於Hz-1病毒的持續感染和細胞與病毒間的分子調控上具有重要的意義。本論文探討Hz-1病毒pag1基因的產物PAT1與其持續感染的關係,並且探究Hz-1病毒以及寄主細胞對pag1基因啟動子的調節作用。本研究將pag1穩定轉染於秋行軍蟲(Spodoptera frugiperda)的細胞株Sf9中,建立可以穩定表現PAT1的細胞株SfPAGs。由Hz-1病毒感染SfPAGs與Sf9所形成持續感染的細胞群落數之比較結果,發現SfPAGs 所形成的細胞群落數為Sf9所形成者的4倍,並且較另一對照組,neo基因穩定轉染細胞株(SfPKN3H與SfPKN4H),所形成的細胞群落數多8倍。經過各株SfPAGs的PAT1產量與其所產生的持續感染細胞群落數之比較,顯示PAT1與Hz-1病毒在寄主Sf9細胞中的持續感染有正相關性。此外,以含有5''端與中間刪除的pag1啟動子(P-pag1)表現lacZ報導基因的質體pTSV(P-pag1)與pSZ(P-pag1),轉染入Sf9中偵測啟動子的表現。結果發現pag1的啟動子上有來自Sf9細胞兩個不同的壓抑子調控,其分別作用位置是在P-pag1的-315 bp至-212 bp與-158 bp至-90 bp之間的區域,而Hz-1病毒 pag1基因的產物PAT1會干擾結合在P-pag1上-315 bp至-212 bp位置的壓抑子之抑制作用,正回饋控制pag1的表現。Hz-1病毒本身可能藉此作用增強P-pag1啟動pag1基因的能力,使其表現更多的PAT1,以致Hz-1病毒更有利地進行持續感染。此或可解釋Hz-1病毒容易進行持續感染之原因。本研究另以Hz-1病毒感染家蠶(Bombyx mori)細胞株BmN,證實Hz-1病毒可以在BmN細胞中全面地進行持續感染,此結果將有助於病毒表現系統的應用。另外,以苜蓿夜蛾核多角體病毒(Autographa californica nucleopolyhedrovirus, AcMNPV)感染BmN細胞,雖然無法建立持續性感染,但是由RT-PCR可以偵測到AcMNPV各期代表基因的表現,包括ie1、egt、vp39和polh,以及AcMNPV基因體轉染入BmN後可測到病毒複製的結果,此證實AcMNPV可以在BmN細胞株中的少數細胞進行少量地複製,這一結果與Kamita and Maeda (1993)所得結果,認為BmN細胞為AcMNPV的非寄主性細胞(non-permissive cells)之論點不同。
Heliothis zea virus 1 (Hz-1 virus) is an insect virus capable of undergoing persistent and productive infections in several lepidopteran cell lines and was regarded as an ideal model for studying persistent viral infection in insect hosts. During persistent Hz-1 viral infection, the persistence-associated transcript 1 (PAT1), is the only viral specific transcript detected and is encoded by the persistence-associated gene 1 (pag1) of Hz-1 virus. One of the main objectives of thesis is to study the relationship between PAT1 and persistent Hz-1 viral infection and the regulation of pag1 promoter by both Hz-1 virus and host cells. The pag1 was first stably transfected into Spodoptera frugiperda (Sf9) cells, and PAT1 was found to be expressed continuously from these cell lines. Upon the infection of Hz-1 virus, the persistently infected colonies formed in these PAT1-expressing cell lines were four time more than those in the Sf9 cells and eight time more than those in the control cells containing a stably-transfected neomycin resistant gene. Comparing the concentrations of PAT1 expressed and the number of persistently infected colonies in pag1-stably transfected cells, a proportional relationship between the concentration of PAT1 and the extension of persistent Hz-1 viral infection was found. To further analyze the promoter of pag1, plasmids containing pag1 promoter and lacZ gene were constructed. Various promoter deletions were constructed from both 5'' end and internal sequences and their activity was analyzed by transfection of these plasmid constructs into Sf9 cells. The results showed that there are two repressor binding sites, -315 bp to -212 bp and -158 bp to -90 bp, on pag1 promoter. In addition, PAT1 enhanced pag1 promoter activity by interfering with the repressor binding onto the promoter region from -315 bp to -212 bp. This can explain that positively feedback control of PAT1 promoted expression of itself, and may be a way by which pag1 can increase its own expression and may thus enhance or maintain persistence of Hz-1 virus. In separate experiments Hz-1 virus was found to be capable of entering persistence in the Bombyx mori BmN cells. Whereas, Autograpga californica multiple nucleopolyhedrovirus (AcMNPV) was found to be able to express ie1, egt, vp39, and polh genes in the BmN cells, however, not capable of entering persistence. Since the progeny of AcMNPV could be detected from BmN cells, suggesting that BmN is semi-permissive to the infection of this virus. This finding is controversial to the viewpoint proposed by Kamita and Maeda (1993), in which BmN cells were considered to be non-permissive for AcMNPV.
URI: http://hdl.handle.net/11455/30628
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