牛痘病毒在中國倉鼠卵巢細胞內寄主限制之研究
Study of Host Restriction of Vaccinia Virus in Chinese Hamster Ovary Cells.
中文摘要
牛痘病毒(Vaccinia Virus；VV)是一種動物病毒，含有約 200Kb 的雙股
DNA。其可感染的寄主範圍相當廣，實驗證明後發現 VV 中有一部份的基因 可以使它們專一地在某些特定的寄主中複製，於是這些基因就被稱為「寄 主範圍基因(host range gene；hr gene)」。若是某 hr gene 被刪除，則
這種 VV 在感染其特異性寄主細胞後非但 VV 不能複製，而且會使寄主細胞產 生「細胞自戕(apoptosis)」。這種現象稱為：「寄主限制(host
restriction)」。所以 hr gene 的功能很可能是抑制寄主細胞的 apoptosis
而使 VV 得以在其內生長。野生型(wild-type)VV 無法在中國倉鼠卵巢細胞(
Chinese Hamster Ovary；CHO cell)中生長，且會造成細胞的 apoptosis
。但 cowpox virus 中一個 77KD 的蛋白可以使 VV 恢復生長且抑制 apoptosis 的發生，這個蛋白稱為「CP77」。CP77 是一個前期(early phase)蛋白質
，包含一段約 2kb 的 open reading frame。利用重組病毒(recombinant VV
；RCVV)的方法，我們生成一系列的突變牛痘病毒(mutant VV)分別帶有刪 除掉不同程度的 CP77 基因，發現 CP77 抑制 apoptosis 的能力與被刪除掉的 長度成反比但對於恢復 VV 在 CHO cell 內的生長卻呈現不同的趨勢，顯示 hr gene 在抑制寄主 apoptosis 及恢復 VV 的生長上可能是經由兩個不同的路徑
。 另外，為了研究在病毒感染後 CP77 在細胞內的位置(localization)，
我們將 CP77 與 GFP(green fluorescence protein)融合(fusion)並產生出 帶有此 fusion protein 的 RCVV，在共軛焦點螢光顯微鏡(confocal fluorescence microscope)下觀察被這種病毒感染的 CHO cell，在病毒感 染後 2 小時內就可以發現 CP77 大量的表現，而且在細胞內的分佈並不平均(
homogeneous)，是一種具有極性(polarity)的分佈。有趣的是，如果將 CP77-GFP 的 plasmid 直接轉染(transfect)到 CHO cell 中使其 transient expression，我們發現在細胞核周圍的一些小泡(small vesicle)會產生 perinuclear staining，而這是 GFP plasmid 所不會有的現象，CP77 在這 些細胞質中的分佈是否與細胞內某一特定胞器的分佈有相關性，未來將做 進一步的探討。另外，我們以 32P 標定時 CP77-GFP 時發現其會被磷酸化。
我們同時將 CP77 表現的時間延至後期(late phase)，發現在後期表現的
CP77 不但不能讓這種 RCVV 在 CHO cell 內的生長恢復正常，亦無法抑制因感 染而引起的細胞 apoptosis，顯示 CP77 必須在前期表現才能具有其正常的 功能。這說明了 CP77 的作用機制應亦是屬於「分級控制(cascade
regulation)」，即當在前期沒有表現上游的 CP77 時，下游的基因也就無
法在正常的時間內表現，因而造成 host restriction 的持續發生。
英文摘要
Vaccinia Virus (VV) is an animal virus that contains about 200kb double strands DNA. The host range of VV is very wild.
Recent studies showed that some genes contribute for VV growth in its specific hosts. These genes are called "host range (hr) gene". If one hr gene is deleted, this mutant VV can not only replicate in its specific host but induce programmed cell death (PCD) of host cells. This is called :"host restriction". So the function of hr genes may inhibit host cells apoptosis to rescue VV growth. The wild-type VV could not growth in CHO cells, but a cowpox protein that called CP77 can suppress host apoptosis and rescue VV growth. CP77 gene contains about 2kb OPF and encodes a 77kd early phase protein. We generate a serious mutant VV
containing a seriously deleted CP77 gene for study the
functional domain of it. We found that the suppressing apoptosis activity of CP77 is inversely between the length that be
deleted. But the virus growth function is not correlate by the deletion. It shows that reducing host apoptosis and rescuing virus growth by hr gene may go through different pathway. On the other hand, for study the subcellular localization of CP77 protein after VV infection, we fused CP77 to GFP (green fluorescence protein) and generated its recombinant VV.
Observation under confocal fluorescence microscope, we found that CP77 was expressed within the 2 h.p.i. of VV, and displayed a unhomogeneous distribution. Interestingly, if the CP77-GFP plasmid was transient expressed in CHO cells, small vesicles that exhibited perinuclear staining appeared. But GFP plasmid never showed that. We areinvestigating that whether the distribution of CP77 is correlated with some specific
organelles. We also labeled CP77-GFP and CP77 proteins with 32P- orthophosphate and found that CP77 protein was phosphorylated within the CHO cells. We also put CP77 gene under the control of a strong late promoter and found that late phase CP77 could not rescue virus growth within CHO cells and suppress host apoptosis. So the normal function of CP77 exist only when it is expressed at early stage of virus infection, shows that the
mechanism of CP77 is belongs to "cascade regulation". CP77 has to expressed at early phase to activate the downstream members participating in its pathway.
