研究牛痘病毒的寄主範圍基因對於牛痘病毒 在 HeLa 細胞生長之影響
前人的實驗結果指出，當牛痘病毒（ vaccinia virus ; VV ）感染細胞時會使 HeLa 細胞進 行細胞凋亡。並且在此類細胞中，病毒的生命週期也會被終止在中期的基因轉譯，所以 我們稱這種現象為「寄主限制」。然而，當 cowpox 的 CP77 基因被表現時，則可以抑 制細胞進行細胞凋亡並且可以幫助病毒在 HeLa 細胞中生長。
這篇研究主要的目的是去了解 CP77 基因在 HeLa 細胞中所扮演的角色。我們將 CP77 接到帶有綠色螢光的 GFP ，以觀察當病毒感染細胞時 CP77 所在的位置。實驗結果顯 示，在病毒感染細胞的早期， GFP-CP77 是在細胞的細胞質中，然而到了感染的晚期則 會移動到細胞核內。
為了了解 CP77 是如何在細胞質與細胞核之間穿梭，我做了各種不同突變的 CP77 然後 將之送到 HeLa 細胞裡，再利用螢光顯微鏡來觀察 CP77 所在細胞的位置，以推斷 CP77 哪個區域是具有調節 CP77 在細胞的移動的功能。就目前的結果顯示，在 CP77 的胺基 酸序列 79-275 和 353~441 這兩個區域，似乎對於 CP77 移動到細胞質是十分重要的。
另一部份，有兩個寄主範圍基因 K1L 和 C7L ，已經被證實在某一些細胞中，其功能可 以替代 CP77 ，所以我們有興趣想要了解這三個寄主範圍基因是否會活化相同的訊號傳 遞路徑，來幫助病毒的生長。以前我們實驗室已經利用酵母菌雙雜交篩選系統，找到五 個寄主基因能和 CP77 有結合作用，根據病毒生長的特性，我們認為這五個基因有可能 和 K1L 、 C7L 有結合作用，所以我也利用酵母菌雙雜交篩選系統，證實其中的一個寄 主基因 HMG20A 能和 K1L 和 C7L 有結合作用，現在我已經將 K1L 和 C7L 接到 GFP ， 想要找出這三個寄主範圍基因與 HMG20A 的結合位置，這些結果應該可以幫助我們了 解寄主範圍基因和寄主基因作用的分子機制。
Investigation of poxviral host range genes th at are required for vaccinia virus growth in H
eLa cells
Vaccinia virus infection into Hela was previously shown to trigger apoptosis of the infected ce lls. In addition, translation of viral intermediate genes was blocked resulting in abortive infecti on that is commonly referred to as “host restriction”. Expression of a cowpox viral CP77 gene suppressed apoptosis and rescued virus growth in Hela cells. The main focus of my study is to understand the role of CP77 in host restriction. We have fused GFP with CP77 in order to mon itor CP77 intracellular location in the infected cells. Expression of GFP-CP77 in the infected c ells indicated that early after infection CP77 expresses in the cytoplasm whereas later after inf ection CP77 was mainly detected in the nuclei of the infected cells.
In order to understand how CP77 is translocated between nucleus and cytoplasm various trunc ations of CP77 were generated and transfected into Hela cells. Immunofluorescence analyses were performed with these transfected cells to determine which region of CP77 regulates prote in movement in cells. The experiments are still in progress and the results indicated that region s, aa 79-275 and aa 353~441 appeared important for cytoplasmic localization of CP77.
Two other viral host range genes K1L and C7L have been shown to substitute the functions of CP77 in other cell types and it will be also interesting to find out whether all three genes activ ate same signaling pathways in cells for virus rescue. Previously, by two hybrid analyses we h ave identified 5 cellular cDNAs that bind to CP77. One of the cDNAs, HMG20A, also interact s with K1L and C7L in two hybrid analyses. I have generated GFP-K1L and GFP-C7L constru cts and will determine HMG20A-binding domain on CP77 in relation to these viral host range genes. These results should help us to understand molecular mechanism of interactions betwee n viral host range genes and cellular targets.
