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牛痘病毒在中國倉鼠卵巢細胞內寄主限制之研究

牛痘病毒在中國倉鼠卵巢細胞內寄主限制之研究.

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牛痘病毒在中國倉鼠卵巢細胞內寄主限制之研究

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  1. 牛痘病毒在中國倉鼠卵巢細胞內寄主限制之研究牛痘病毒在中國倉鼠卵巢細胞內寄主限制之研究 • 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(hostrestriction)」。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hrgene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(confocalfluorescence 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transientexpression，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(cascaderegulation)」，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。

  2. Study of Host Restriction of Vaccinia Virus in Chinese Hamster Ovary Cells. • Vaccinia Virus (VV) is an animal virus that contains about200kb double strands DNA. The host range of VV is very wild.Recent studies showed that some genes contribute for VV growthin its specific hosts. These genes are called "host range (hr)gene". If one hr gene is deleted, this mutant VV can not onlyreplicate in its specific host but induce programmed cell death(PCD) of host cells. This is called :"host restriction". So thefunction of hr genes may inhibit host cells apoptosis to rescueVV growth. The wild-type VV could not growth in CHO cells, but acowpox protein that called CP77 can suppress host apoptosis andrescue VV growth. CP77 gene contains about 2kb OPF and encodes a77kd early phase protein. We generate a serious mutant VVcontaining a seriously deleted CP77 gene for study thefunctional domain of it. We found that the suppressing apoptosisactivity of CP77 is inversely between the length that bedeleted. But the virus growth function is not correlate by thedeletion. It shows that reducing host apoptosis and rescuingvirus growth by hr gene may go through different pathway. Onthe other hand, for study the subcellular localization of CP77protein after VV infection, we fused CP77 to GFP (greenfluorescence protein) and generated its recombinant VV.Observation under confocal fluorescence microscope, we foundthat CP77 was expressed within the 2 h.p.i. of VV, and displayeda unhomogeneous distribution. Interestingly, if the CP77-GFPplasmid was transient expressed in CHO cells, small vesiclesthat exhibited perinuclear staining appeared. But GFP plasmidnever showed that. We areinvestigating that whether thedistribution of CP77 is correlated with some specificorganelles. We also labeled CP77-GFP and CP77 proteins with 32P-orthophosphate and found that CP77 protein was phosphorylatedwithin the CHO cells. We also put CP77 gene under the controlof a strong late promoter and found that late phase CP77 couldnot rescue virus growth within CHO cells and suppress hostapoptosis. So the normal function of CP77 exist only when it isexpressed at early stage of virus infection, shows that themechanism of CP77 is belongs to "cascade regulation". CP77 hasto expressed at early phase to activate the downstream membersparticipating in its pathway.

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