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Investigation of the Interaction between the Novel Avian Influenza A (H7N9) Virus NEP and M1 Proteins Affected by pH change
|Publication Year :||2017|
|Abstract:||研究發現禽流感病毒的NEP (nuclear export protein) 扮演許多重要生理角色，包括運輸、調控病毒基因以及幫助病毒離開宿主細胞。NEP透過與M1交互作用幫助複製完成病毒基因運送出宿主細胞核以利病毒組裝。然而目前尚未有研究探討pH改變對於NEP與M1交互作用的影響。本研究在進行膠體過濾實驗時，意外發現H7N9病毒之NEP會隨著使用不同pH值之緩衝液，而使其流洗出的位置不同，因此環境pH的改變似乎能使NEP之分子大小或構形隨之改變。此外，當NaCl濃度改變時，pH對於NEP流洗體積影響趨勢仍相同，但是隨著鹽濃度提升，pH改變對於NEP影響漸弱。由於已知NEP能夠透過與M1交互作用幫助複製完成的vRNP運送離開宿主細胞核，以利病毒組裝，因此推測若pH造成NEP的構形改變或許會影響NEP與M1交互作用力改變，造成生理功能上的影響。然而在pH 7至pH 9的實驗條件下，經過crosslinking以及CD光譜分析，發現NEP聚體之產生或其二級結構並未受到此pH改變而影響；反之H7N9病毒之基質蛋白M1卻會隨著pH升高而產生較多的多聚體。透過pulldown實驗，的確發現M1與NEP之結合現象隨著pH值提升而減少。由於M1-M1間交互作用的區域與M1-NEP交互作用的位置有所重疊，因此當pH提升後，M1傾向形成多聚體，而阻擋了M1與NEP之結合，進而使M1與NEP形成複合體之現象減少。然而此推論尚需更充足的實驗證據佐證。|
Studies showed that NEP has many important biological functions, including transportation and regulation of viral genome and virus budding from host cells. However, the effect of pH change on the interaction between M1 and NEP is unknown.
In this study, I unexpectedly found that the retention volume of NEP would change while performing gel filtration in different pH conditions. In addition, the elution volume of NEP would also change by using buffers with different NaCl concentration at the same pH. It seemed that change of pH might alter the molecular size or conformation of NEP. Besides, it was known that M1-NEP interaction would help export replicated vRNP from nucleus for assembly of viral particles. Hence, it was hypothesized that if the conformation of NEP would change at different pH, it might interrupt M1-NEP interaction and affect its biological function. However, the results of crosslinking and CD experiment showed that the polymerization and secondary structure of NEP were not different between pH 7 and pH 9. In contrast, the matrix protein M1 of H7N9 tended to undergo polymerization as pH increases. Moreover, the interaction between M1 and NEP decreased as pH increases according to the result of pulldown assay. Previous studies have shown that the regions of M1 for interacting with M1 and NEP were overlapped for some extent. Therefore, the formation of the M1-NEP complex was reduced due to the polymerization of M1 and blocking the interaction site between M1 and NEP upon pH change. More investigation should be conducted for elucidating this assumption.
|Appears in Collections:||生化科技學系|
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