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
Investigation of poxviral host range genes that are required for vaccinia virus growth in HeLa cells
中文摘要
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。
英文摘要
Vaccinia virus infection into Hela was previously shown to trigger apoptosis of the infected cells. In addition, translation of viral intermediate genes was blocked resulting in abortive infection 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 monitor CP77 intracellular
location in the infected cells. Expression of GFP-CP77 in the infected cells indicated that early after infection CP77 expresses in the cytoplasm whereas later after infection CP77 was mainly detected in the nuclei of the infected cells.
In order to understand how CP77 is translocated between nucleus and cytoplasm various truncations of CP77 were generated and transfected into Hela cells.
Immunofluorescence analyses were performed with these transfected cells to determine which region of CP77 regulates protein movement in cells. The
experiments are still in progress and the results indicated that regions, 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 activate same signaling pathways in cells for virus rescue. Previously, by two hybrid analyses we have identified 5 cellular cDNAs that bind to CP77. One of the cDNAs, HMG20A, also interacts with K1L and C7L in two hybrid analyses. I have generated GFP-K1L and GFP-C7L constructs 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 between viral host range genes and cellular targets.
