Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90107
標題: The C-terminal two residues of infectious bursal disease virus VP4 protein play a role on tubule formation and enzymatic activity
傳染性華氏囊病毒 VP4 蛋白質 C 端雙殘基於柱狀體形成和酵素活性所扮演之角色
作者: 蕭鼎峰
Ting-Feng Hsiao
關鍵字: 傳染性華氏囊炎病毒
柱狀體
酵素活性
IBDV
tubular structure
enzymatic activity
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摘要: 傳染性華氏囊炎病毒 (Infectious bursal disease virus, IBDV) 是一種雙股RNA病毒科的禽類病毒,會抑制幼雞的免疫系統,進而造成養雞業重大損失。VP4 為IBDV的絲胺酸蛋白酶,能夠截切病毒多蛋白 (polyprotein),產生pVP2、VP4和VP3蛋白進而使病毒顆粒得以組裝,病毒能夠增殖。先前的文獻指出在截切完polyprotein後,VP4會透過彼此間蛋白C端的交互作用,自我組裝成柱狀。為了更精確指出是C端哪些胺基酸參與柱狀之組裝,本研究將VP4的C端減短10個胺基酸與5個胺基酸,構築了的兩個質體分別稱為VP4cd10及VP4cd5。然而這兩個construct在表現蛋白後我們發現VP4的柱狀不復存在。這結果顯示VP4的C端四個胺基酸殘基 (752LAMA755) 可能是影響柱狀形成的必要胺基酸。為了證實這項假設,我們逐一切短C端構築了VP4cd1-VP4cd4等四個construct。電子顯微鏡的觀察顯示VP4cd1維持如同全長VP4蛋白一樣的柱狀,而 VP4cd3、VP4cd4則是單體,值得注意的是,我們可以觀察到VP4cd2蛋白同時有柱狀及單體之構造。這表示C端最後兩個胺基酸對於柱狀的組裝是必須的。由酵素活性分析顯示,VP4於單體結構下是處於活性較高的狀態,而形成柱狀體時則活性被抑制,此結果說明IBDV透過VP4形成柱狀體的機制調節細胞內的VP4蛋白酶活性。目前並無文獻指出VP4柱狀結構如何形成及其對於病毒或宿主的重要性有何影響,因此本研究成果將有助於了解VP4柱狀體在細胞內組裝機制及其活性的調節方式。
Infectious bursal disease virus (IBDV), a member of the genus Avibirnavirus of the family Birnaviridae, is the causative agent of a highly immune- suppressive disease in young chicks and causes great damage to poultry industry. The IBDV viral protein 4 (VP4) is a serine protease that processes the polyprotein of pVP2-VP4-VP3 for the assembly of viral capsids and virus propagation. Previous reports demonstrated that released VP4 proteins can self-assemble into tubules through the interaction of their C-termini after the cleavage of polyproteins. To further pinpoint the involved residues for the tubule formation, two constructs with the deletions of 5 and 10 amino acids (VP4cd5 and VP4cd10) at the C-terminal end of VP4 were created. However, both of them cannot maintain the tubular conformation. These results suggest that the essential residues for the tubule formation must be in the last four amino acids of VP4 (752LAMA755). To prove it, VP4cd1-VP4cd4 were constructed by deleting one to four amino acids from C-terminus, respectively. The VP4cd1 mutant maintained the tubular structure as wild-type VP4 protein; however the VP4cd3 and VP4cd4 formed monomer. It is worth noting that both tubular and monomeric conformations were observed in VP4cd2, indicating that the last two residues are essential for VP4 to form a stable tubular structure. In enzyme kinetic assay, all truncated monomers have higher activities, whereas the activities were subsequently inhibited when the tubules were formed. These results suggest that IBDV regulates its own VP4 protease activities through the mechanism of tubule assembly. This study demonstrates that the two critical residues in the C-terminus of VP4 are involved in tubule assembly and regulation on enzymatic activities.
URI: http://hdl.handle.net/11455/90107
文章公開時間: 2017-01-30
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