Molecular Analysis of Vaccinia Virus Entry

1. 牛痘病毒進入細胞之分子研究 • 摘要 牛痘病毒（vaccinia virus）是一個包有外鞘的DNA病毒，他的 • 宿主範圍很廣，可以感染許多不同種類的細胞，由電子顯微鏡的資料顯示 • ，牛痘病毒進入細胞的主要方式為membrane fusion，而其感染寄主細胞 • 所需的細胞受體至今尚未被發現。我們實驗室對於研究VV感染細胞的途徑 • 很有興趣。我們的研究方法是利用單株抗體（monoclonal antibody,mAb • ）作為尋找牛痘病毒細胞受體(receptor)的工具。這樣的mAb具有幾項特 • 性：1.能夠專一性的阻止VV感染細胞。2.認得位於細胞表面的分子。本實 • 驗室先前篩選到一個單株抗體B2，具有上述的能力，後而利用B2在lambda • expression cloning的基因表現系統中，找尋可能的細胞受體基因。最後 • 篩選到一個長約4.2Kb的cDNA clone, A1。A1的序列可轉譯一個長1300個 • 胺基酸的蛋白質，其序列經電腦分析與所有已知的蛋白質並無相似性。 • 本研究論文的目的在於研究A1 cDNA的基因產物(gene product)。並且測 • 試其功能上是否可以幫助VV進入細胞。第一步我們將A1cDNA 的5'-端與細 • 菌的Glutathione-S-transferase接合，成為融合蛋白質（fusion • protein）A1-GST。利用A1-GST蛋白質作為抗原，我們生產更多可以阻擋 • VV感染細胞的單株抗體。我們總共篩選到了四個mAbs其中1-5E-5H和5-4 • H-3B阻止病毒感染的效率達到95%以上，而另外兩個4-6G-5B，4-1D-5G的 • 阻止能力較差，分別只有29%及50%。 為了map out這些mAbs認得A1的 • 區域，我們生成一系列的A1-GST3'-deletion clones。利用mAbs認得這整 • 個panel fusion proteins的pattern來做epitope mapping。Epitopes • mapping 的結果顯示，除了1-5E-5H認得的部位與B2相同外，其他三個單 • 株抗體所辨認的epitopes均不相同，而且也都靠近A1的N-端。此外，這些 • mAbs亦可stain cell surface，可見它們認得的部位在細胞外面。綜合上 • 述結果，我們認為A1蛋白質可能屬於第一類型的transmembrane protein • 。為了求證A1基因產物是否就是牛痘病毒的受體，我們完成一個可在哺乳 • 類細胞中大量表現A1cDNA的基因產物的質體（plasmid），至於病毒結合 • 到細胞上的數量是否會因此而增加，目前仍在測試之中。 此外，為了 • 瞭解在牛痘病毒粒子上不同的外鞘蛋白質與病毒感染的關係，我們將病毒 • 的外鞘蛋白質的基因選殖到原核生物蛋白質表現載體上，利用細菌來大量 • 生產這些外鞘蛋白質，並觀察它們對於病毒的感染力是否具有影響性。另 • 一方面，利用缺乏A34R、A36R、B5R或F13L等外鞘蛋白質的變異病毒，進 • 行感染細胞的測試。結果顯示，缺乏上述之外鞘蛋白質並不會影響病毒進 • 入細胞的途徑。

2. Molecular analysis of Vaccinia Virus Entry • Vaccinia virus (VV) is an enveloped • DNA virus with wide host range andenters cells via plasma • membrane fusion. Previous studies have identified amonoclonal • antibody(mAb) B2 that recognized cell surface molecules and • neutralized 95% VV infection by blocking virus binding. In • addition, lambda expression cloning with this mAb resulted in • isolation of a 4.2 kb cDNA cloneA1. This A1 cDNA contains an • open reading frame (ORF) of 1,300 amino acidsand shares no • homology with any known sequences in database. The major • objectives of this study are to analyze the gene product of A1 • cDNA and to determine if it facilitated VV entry. A1 cDNA was • fused with bacteria glutathione-S-transferase (GST) to produce • A1-GST fusion protein,which intern was used as an antigen to • generate more monoclonal antibodies that could block VV • infection. Four mAbs 1-5E-5H mAb, 5-4H-3B mAb,4-6G-5B mAb, and • 4-1D-5G mAb were isolated. 1-5E-5H and 5-4H-3B could • approximately contribute to 95% of efficiency for blocking the • virus infection,while 4-6G-5B and 4-1D-5G mAb could only reach • 29% and 50% respecitvely. In order to map out the epitopes • within A1 protein for these mAbs, aseries of A1-GST 3'-deletion • clones were generated. The result indicated thatthese mAbs • recognize four different epitopes localized at the N-terminal • regionof A1. In addition, these mAbs also stain cell surface • indicating that the recognition sites were situated outside of • cells. Therefore, this A1 protein couldbe a type I transmenbrane • protein. To understand whether the A1 gene product serves as the • cellular receptor of VV, a plasmid expressing the full-length A1 • cDNA in mammalian cells was prepared and used to determine the • role of A1 cDNA clone in virus entry. Futur study will determine • ifoveroxperssion of the A1 gene product will result in increase • of virus binding.This study also intend to investigate the role • of viral envelope proteins in virus attachment during virus • entry. Each envelope proteins were overexpressed in E. coli and • their ability to interfere with mAb B2 interactionwith cells was • measured. On the other hand, mutant viruses defective of A34R, • A36R, B5R, or F13L of envelope proteins were also used to • determine the effect of envelope proteins in virus entry. • Results from these expermentsrevealed that lack of above • envelope protein does not affect the mechanism ofvirus entry.