研究牛痘病毒鞘膜蛋白在病毒進入細胞過程中所扮演的角色

Investigation of Vaccinia Virus Attachment Proteins Involved in Virus Entry

蕭潔謙(Hsiao Jye-Chian)

指導教授 ： 張雯

臺北醫學大學/醫學院/細胞及分子生物研究所/碩士(1998年)

摘要

牛痘病毒的宿主範圍相當廣泛，幾乎能夠感染所有的細胞，故推測牛痘病毒進行感染時， 所利用的細胞受體（cellular receptor）分布應該會十分普遍。在本篇論文中，我們根 據下述的實驗結果，證實牛痘病毒會經由細胞表面的heparan sulfate以感染細胞。（一 ）soluble heparin能夠排擠（compete）宿主細胞和病毒之間的結合作用；（二）細胞經 過chlorate處理後，會抑制細胞表面heparan sulfate進行sulfation，而造成此類細胞不 易被病毒感染；（三）純化的牛痘病毒能夠直接結合至heparin beads。除此之外，我們 發現IMV之鞘膜蛋白質A27L、D8L蛋白質均會附著至細胞表面heparan sulfate，並且證明 這兩種蛋白質為牛痘病毒入侵細胞的附著蛋白質（attachment proteins）。其中D8L蛋白 質為主要和heparan sulfate結合的鞘膜蛋白質，牛痘病毒會藉著D8L蛋白質而附著至細胞 表面；A27L蛋白質則為較次要的heparan sulfate binding protein，只有在D8L蛋白質不 存在的情況下，才會影響病毒附著至細胞表面。因此，A27L蛋白質在病毒入侵細胞過程中 ，所扮演的角色在post-binding stage比較重要，研究結果顯示其N端的a.a.21-32的部分 負責與細胞表面的heparan sulfate作用，藉此方能催化細胞融合（cell fusion）的產生 。

關鍵字

牛痘病毒 ； A27L蛋白質 ； D8L蛋白質 ； 病毒入侵

並列摘要

Vaccinia virus has a wide host range and infects mammalian cells of many diffe rent species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different rec eptors are used for vaccinia virus infection of different cell types. We previ ously shown that vaccinia virus infection to BSC40 cells was blocked by solubl e heparin, suggesting that cell surface heparan sulfate mediates vaccinia viru s binding. BSC40 cells treated with sodium chlorate to prevent sulfation of ce ll surface GAGs became more resistant to vaccinia virus infection indicating t he negative charges contributed by sulfate groups are important for virus infe ction. We investigated the structure of A27L protein that is required for its binding to heparan sulfate on cells. A mutant A27L protein, named D-A27L,which removed a cluster of 12 a.a.s rich in basic residues was constructed. In cont rast to soluble A27L protein, D-A27L protein did not bind to heparin in vitro. D-A27L protein also did not compete with soluble A27L protein for binding to heparan sulfate on cells. Most importantly, soluble A27L but not D-A27L prote in interfered with vaccinia virus infection. Virus binding assays revealed tha t the interference of A27L protein was at a postbinding step. Taken together, these data demonstrated the N-terminal region of A27L protein functions as a G AGs-binding domain and is critical for its function. Since A27L protein w as thought to be involved in cell fusion under acid treatment, we investigated if cell surface GAGs also participate in A27L-dependent fusion. Mouse L cells infected by vaccinia virus developed cell fusion after brief exposure to acid ic environment. Soluble A27L protein blocked cell fusion whereas D-A27L protei n did not suggesting the GAGs binding domain plays a role in A27L-mediated cel l fusion. We also performed the converse experiment in which sog9 cells, a GAG s-free mutant cells derived from L cells, were infected with vaccinia virus an d no fusion was observed. The results demonstrated that A27L-mediated cell fus ion is triggered by its interaction with cell surface GAGs. In summary, our d ata indicate that during vaccinia virus infection, A27L protein could interact with cell surface haparan sulfate through the N-terminal region. This interac tion directly or indirectly triggers membrane fusion, resulting in virus entry . In addition, vaccinia virus may have gene redundancy in that additional vira l proteins such as D8L also binds to haparan sulfate as well.

並列關鍵字

Vaccinia Virus ； A27L Protein ； D8L Protein ； Virus Entry