Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/13043
標題: 假性狂犬病毒與多胺類及精胺酸關係之研究
A study on the relationship between pseudorabies virus, polyamines, and arginine
作者: 王咸棋
Wang, Hsien-Chi
關鍵字: pseudorabies virus
假性狂犬病毒
polyamines
arginase
多胺類
精胺酸
出版社: 獸醫學系
摘要: 假性狂犬病毒亦稱為豬第一型疱疹病毒,其屬於阿爾法疱疹病毒亞科之一員,是一種具有封套與雙股DNA病毒。多胺類(spermidine、 spermine 及putrescine等)參與細胞生長,分化以及對與環境緊迫的反應等機制。由於多胺類是帶有正電荷的分子可以中和DNA上負電荷,也參與細胞的複製。前人的研究中,發現某些病毒的感染需要多胺類,例如:人類簡單疱疹病毒,馬立克病毒以及人類巨大細胞病毒等等。本論文之研究主要分為三大部分。 (一):實驗發現,添加外源性的spermine或spermidine不會影響病毒在細胞中之病毒斑的形成,以及病毒的蛋白質合成亦不受影響。這些添加的多胺類會輕微刺激假性狂犬病毒立即早期蛋白(IE)基因啟動子。結果顯示,假性狂犬病毒的感染並不會因增加外源性或消耗內源性多胺類而受到影響。 (二):精胺酸(arginine)是帶正電荷之鹼性氨基酸,可被精胺酸脢水解成ornithine以及urea,是多胺類物質的前趨物之一。先前有報告指出人類腺病毒、單純疱疹病毒的複製需要精胺酸的參與。因此在本實驗中,我們以添加精胺酸酵素(arginase)來除去精胺酸,研究假性狂犬病毒的感染是否需要精胺酸。在病毒斑的測試中,其病毒斑的形成會受到精胺酸酵素處理的抑制,而此抑制現象會因再添加外源性精胺酸而回復。長時間的精胺酸酵素作用下,病毒蛋白(gB,gE,UL47以及UL48)的總量皆受到影響。而病毒蛋白合成則不受影響。以native gel電泳分析則發現病毒蛋白的mobility會受到精胺酸酵素處理的影響,推斷蛋白的三級結構因缺乏精胺酸而有所改變,進而影響其於膠體分析時之mobility移動。另外,熱休克蛋白參與蛋白的折疊,本研究發現熱休克啟動子也會受外源性精胺酸酵素處理而受到抑制。因此我們認為精胺酸及熱休克蛋白都參與假性狂犬病毒蛋白的折疊行為。 (三):細胞的Spermidine/ spermine N1- acetyltransferase (SSAT)在多胺類分解路徑中扮演極重要的角色。當SSAT將多胺類乙醯化後使得喪失與DNA的親和力,再將這些代謝後的產物運送出細胞外,因此細胞的生長分裂,也部分受到限制。本實驗中,我們選殖人類SSAT啟動子基因來測試病毒的立即早期蛋白IE 180與早期蛋白EP0是否會影響SSAT基因的表現。在共同轉染實驗中發現,IE 180會刺激SSAT啟動子而EP0則會輕微抑制,而病毒感染的前六小時中亦發現,隨感染時間的增長, SSAT啟動子的活性漸漸上升,代表病毒的某些產物能刺激此啟動子。
Pseudorabies virus (PrV) is a member of alphahepesviruses; it is an enveloped virus with a double-stranded DNA genome. Polyamines are ubiquitous in animal cells and participate in cellular proliferation, differentiation, and response for environment stress. Acetylated polyamines lose positive charge, thus decrease the affinity for DNA or RNA. In previous studies, polyamines were reported to help in some virus infection, such as human herpes simplex virus (HSV), Marek's disease virus, and human cytomegalovirus (HCMV). In this study, we found the plaque formation of PrV was not affected by spermine, spermidine, or DFMO (an ornithine decarboxylase inhibitor) treatment. The synthesis of viral proteins was not affected by these drugs in radioactive methionine labeling. Furthermore, the effects of these agents on the transcription of PrV immediate-early gene (IE 180) promoter were examined by chloramphenicol acetyltransferase (CAT) assay, and results showed that the transcription was slightly stimulated by these drugs. Taken together, our results demonstrated that lytic infection of PrV was not influenced by addition of exogenous polyamines or by the depletion of endogenous polyamines. Arginine is a basic amino acid with net positive charges at neutral pH. Arginine, a precursor of polyamines, was hydrolyzed to urea and ornithine by arginase. Arginine is required as well for some virus replication, such as adenovirus type II, and herpesvirus. In this work, we have investigated the role of arginine in PrV infection. It was found that the plaque formation of PrV was inhibited by arginase treatment, and this inhibitory effect could be reversed by supplying exogenous arginine, suggesting that arginine is essential for PrV proliferation. The production of four PrV structural proteins (gB, gE, UL47, and UL48) were apparently decreased in a long time arginase treatment. However, the overall protein synthesis machinery was basically not influenced by arginase treatment either in mock or PRV-infected cells. Based on the analysis on native gel, we found that arginase treatment did affect the mobility of PrV structural proteins, suggesting a conformational change of viral proteins may be resulted from arginine depletion. Our results also showed that long time arginase treatment could lead to a reduce in the expression of cellular heat shock proteins 70 and the down regulation of heat shock protein 70 gene promoter was potentially one of the mechanisms accounted for this effect. We thus suggested that both arginine and heat shock proteins 70 participate in the folding of PrV proteins. Spermidine / spermine N1- acetyltransferase (SSAT) is a key enzyme in polyamine catabolism pathway. It acetylates polyamine, neutralized the positive discharges on the polyamines, and decreased their affinity for DNA or RNA. According to our study, transcription of PRV immediate-early gene (IE 180) promoter was weakly stimulated by polyamines. We had examined the effect of IE 180 and EP0 on SSAT promoter. The results indicated that IE 180 could stimulate SSAT promoter, but EPO would slightly suppressed SSAT promoter. The SSAT promoter was activated by PrV infection, suggesting that certain viral products can activate this promoter directly or indirectly.
URI: http://hdl.handle.net/11455/13043
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