登革熱第二型病毒膜蛋白藉由胺基酸K51及K241與人類Ubc9蛋白產生交互作用

Abstract

登革熱為一經蚊蟲叮咬而感染人類之病毒，在全球暖化之氣候影響之下，其威脅性已不再侷限於熱帶/亞熱帶而成為全球性之流行疾病。登革熱病毒之外套膜蛋白屬於結構性蛋白，在感染宿主細胞時扮演很重要之角色，同時也是主要抗原之一。Chapter I利用重組蛋白技術表現登革熱第二型病毒strain PL046之外套膜蛋白，該重組蛋白不包含C端之疏水性結構(transmembrane domain)，而以一段S peptide取代作為純化之用。在E. coli表現之結果雖然形成inclusion body，但經過重新□合後，此重組蛋白之結構未受影響。在病毒感染的同時加入重組膜蛋白能有效抑制病毒斑(plaque)之形成，顯示該重組膜蛋白經由與病毒顆粒本身之膜蛋白競爭，減少病毒入侵宿主細胞之機會。Chapter II研究病毒膜蛋白與宿主細胞內蛋白之交互作用，藉由Functional Yeast Array篩選500個人類蛋白是否與登革熱第二型病毒膜蛋白發生交互作用。篩選的結果找出5個人類蛋白，其中一個為Ubc9。利用co-precipitation assay可再次驗證Ubc9與DV2E之蛋白質交互作用，點突變結果顯示Ubc9可能以K51與K241作為與DV2E作用之位置。以共軛焦顯微鏡觀察於BHK-21細胞內表現DV2E-GFP及Flag-Ubc9兩種蛋白質之位置，發現當共同表現DV2E-GFP及Flag-Ubc9時，DV2E-EGFP的位置由原本分布於細胞質範圍逐漸往細胞核趨近。另外，若於BHK-21細胞內大量表現Ubc9能減少病毒感染之病毒斑(plaque)之形成，上述結果顯示在登革熱病毒感染時，Ubc9可能扮演一重要之角色。

Dengue viruses (DVs) are mosquito-borne infectious pathogens. They have become an expanding public health problem in the tropics and subtropics. The dengue envelope (E) protein is one of the viral structure proteins responsible mainly for the virus attachment and entry onto host cells. It is also the major immunogen for virus neutralization. In chapter I, I have constructed a recombinant plasmid expressing a truncated E protein of DV-2 virus PL046 strain. The C-terminal hydrophobic domain of the E protein was removed and replaced with the sequence of S peptide to facilitate expression and purification. When expressed in Escherichia coli, the recombinant E proteins were found to be in the form of aggregated state. Through denaturation and dialysis processes, the receptor-interacting function of the purified recombinant E proteins was maintained, which was demonstrated by its ability to inhibit the DV-2 plaque-forming efficiency on mammalian BHK-21 host cells. In chapter II, to identify the human cellular proteins interacting with the envelope protein of dengue virus serotype 2 inside host cells, I have performed a screening with the yeast two-hybrid-based “Functional Yeast Array”. Interestingly, the Small Ubiquitin-like Modifier-1 Conjugating Enzyme 9 protein, modulating cellular processes such as those regulating signal transduction and cell growth, was one of the candidates interacting with the dengue virus envelope protein. With co-precipitation assay, it is demonstrated that the dengue envelope protein indeed could interact directly with the Ubc9 protein. Site-directed mutagenesis has demonstrated that Ubc9 might interact with the E protein via amino acid residues K51 and K241. Furthermore, immunofluorescence microscopy has shown that the DV2E-EGFP proteins tended to progress toward the nucleus membrane and co-localized with Flag-Ubc9 proteins around the nucleus membrane in the cytoplasm side, and DV2E-EGFP also shifted the distribution of Flag-Ubc9 from evenly in the nucleus toward concentrating around the nuclear membrane in the nucleic side. In addition, over-expression of Ubc9 could reduce the plaque formation of the dengue virus in mammalian cells. This is the first report that DV envelope proteins can interact with the protein of sumoylation system and Ubc9 may involve in the host defense system to prevent virus infection.