探討登革病毒前驅膜/外套膜蛋白質之功能及其與殼蛋白質之間的交互作用

Study of the functions of precursor membrane and envelope proteins of dengue virus and their interactions with capsid protein

胡賢屏(Hsien-Ping Hu)

指導教授 ： 王維恭

國立臺灣大學/醫學院/微生物學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.10279

摘要

四種血清型的登革病毒(DENV)在熱帶及亞熱帶造成最重要的經由節肢動物傳播的疾病，包括登革熱及嚴重型的登革出血熱。在過去30年來，全球每年登革出血熱之病例數及報告出現登革出血熱的國家數目增加4倍以上。登革病毒的前驅膜蛋白質(precursor membrane)及外套膜蛋白質(envelope)可形成異質雙體位於病毒顆粒表面，而另一個結構性蛋白質殼蛋白質(capsid)及RNA基因體則位於病毒顆粒內部。前驅膜/外套膜蛋白質是登革病毒細胞嗜性的主要決定因子，也是與保護性或促進疾病有密切相關的中和性抗體及促進性抗體的主要標的。探討前驅膜/外套膜蛋白質的功能以及其與殼蛋白質及病毒顆粒之形成，對於登革疾病致病機轉的瞭解與疫苗之設計與策略將提供重要訊息。
發展登革病毒的全長及嵌合型感染性克隆，可以在完整病毒顆粒的狀態下研究前驅膜蛋白質及外套膜蛋白質的功能，但是由於操作起來費時費力，限制了其廣泛的應用。本研究的第一個目標，是要發展以反轉錄病毒為基礎的報告病毒系統來探討登革病毒前驅膜/外套膜蛋白質的功能。我們已成功的建立了以反轉錄病毒為核心，由第二型登革病毒前驅膜/外套膜蛋白質所構成的偽報告病毒D2(HIVluc)。利用蔗糖梯度分析偽報告病毒，顯示大部分登革病毒的外套膜蛋白質及人類免疫缺乏病毒(HIV-1)的核心蛋白質沉澱在同一個濃度梯度。此外，在免疫沉澱實驗中，利用抗外套膜蛋白質單株抗體沉澱偽報告病毒可以偵測到人類免疫缺乏病毒的核心蛋白質，這些證據顯示登革病毒的外套膜蛋白質及人類免疫缺乏病毒的核心蛋白質是組裝在偽報告病毒中。偽報告病毒的感染力可以被趨溶酶體性(lysosomotropic)藥物抑制，顯示偽報告病毒感染進入細胞是經由受pH值影響的機制完成。另一方面我們也建立了由其他三型登革病毒的前驅膜/外套膜蛋白質所構成的偽報告病毒。把外套膜蛋白質上融合環(fusion loop)中參與外套膜與細胞膜融合步驟之重要胺基酸，即第107位置的白胺酸(leucine)改變為離胺酸(lysine)時，會造成偽報告病毒感染力嚴重的影響，顯示偽報告病毒進入細胞與外套膜蛋白質所主導的融合步驟有關。隨著越來越多的登革病毒序列在全球爆發疫情之中國家陸續被報告出來，我們所發展的四型登革病毒之偽報告病毒是靈敏度高又方便的工具，有潛力可促進對田野登革病毒株前驅膜蛋白質及外套膜蛋白質分子特性之瞭解，並且可助於未來抑制登革病毒進入細胞藥物的篩選。
以低溫電子顯微鏡研究登革病毒顆粒顯示病毒的核心及膜之間有一個明顯的空隙，這個發現引發一個重要的問題，病毒的前驅膜蛋白質與外套膜蛋白質在病毒組裝的過程中是否與殼蛋白質交互作用而影響其組裝。在前驅膜蛋白質與外套膜蛋白質的C端中各有兩個穿膜區域(transmembrane domains)，而在兩個穿膜區域之間均有一個在許多黃病毒科成員中都高度保守性的正電性胺基酸，包括精胺酸(arginine)或是離胺酸(lysine)。在本研究的第二個目標中，我們使用點突變法以第一型及第四型登革病毒的前驅膜/外套膜蛋白質表現質體，探討這兩個胺基酸在似病毒顆粒(virus-like particles)形成以及組裝核心蛋白質的過程中扮演的角色。當前驅膜蛋白質的精胺酸或是離胺酸被取代為麩胺酸(glutamic acid)會降低似病毒的形成量。然而，將外套膜蛋白質胺基酸471的精胺酸(arginine)取代為丙胺酸(alanine) (E471RA)或是麩胺酸(glutamic acid) (E471RE)會增加殼蛋白質組裝進入似病毒顆粒。這種殼蛋白質在組裝時增加的情形，也會在殼蛋白質α1上端的兩個正電性胺基酸突變為負電性胺基酸時發生，但是同樣在殼蛋白質α2上有兩個正電性胺基酸突變負電性胺基酸，則不會增加殼蛋白質組裝進入似病毒顆粒。除此之外，殼蛋白質可以在同時表現殼蛋白質、前驅膜蛋白質及外套膜蛋白質的細胞中被抗外套膜蛋白質之單株抗體免疫沉澱下來。但是將缺乏前驅膜/外套膜蛋白質的複製子(replicon)與突變之前驅膜/外套膜蛋白質E471RA或是E471RE一同送入細胞中，殼蛋白質組裝進入似病毒顆粒反而會減少。總結來說，這些結果暗示著在病毒顆粒組裝的過程中，外套膜蛋白質會與殼蛋白質交互作用，並且外套膜蛋白質E471R會調節殼蛋白質組裝進入似病毒顆粒。我們提出一個模式來解釋這些發現，外套膜蛋白質胺基酸471的精胺酸以及周圍非疏水性(non-hydrophobic)胺基酸暴露在細胞膜的細胞溶質側(cytosolic side)而非埋在細胞膜內。並且，外套膜蛋白質胺基酸471的正電性可以阻止與新合成切割好的鹼性(basic)殼蛋白質的結合以及組裝，同時也可以等候殼蛋白質將新合成的RNA基因體組包裹好後再組裝起來。這兩個位於α1螺旋的正電性胺基酸可能參與在一開始外套膜蛋白質胺基酸471的精胺酸與負電性的細胞膜表面交互作用。未來利用建立在全長感染性克隆中前驅膜/外套膜蛋白質及殼蛋白質上相似的突變，探討其產生具感染性病毒顆粒的情形，可以進一步的証實這個模式。
總結本研究的目的是要建立一個四型登革病毒的報告偽病毒系統，有助於前驅膜/外套膜蛋白質的分子特性之研究，同時探討前驅膜/外套膜蛋白質與殼蛋白質在形成病毒顆粒時的交互作用。未來將擴展這個系統及觀察不僅可證明我們提出的模式及其應用性，而且可就我們對前驅膜/外套膜蛋白質及殼蛋白質的功能、交互作用及致病機轉的瞭解，提供新的啟示。

關鍵字

登革病毒 ； 偽報告病毒 ； 外套膜蛋白質 ； 前驅膜蛋白質 ； 核心蛋白質 ； 組裝 ； 似病毒顆粒

並列摘要

The four serotypes of dengue viruses (DENV) cause the most important arboviral diseases in the tropical and subtropical regions, including dengue fever (DF) and dengue hemorrhagic fever (DHF), a severe form of disease. During the past 30 years, the numbers of DHF cases and of countries reported to have DHF have increased by more than 4 folds. The precursor membrane (PrM) and envelope (E) proteins, which form a heterodimer, are present on the surface of virion, whereas capsid (C) protein, another structural protein, and RNA genome are present inside the virion. PrM/E proteins are the major determinants of cellular tropism and the major targets of both neutralizing and enhancing antibodies, which are known to play a critical role in protection or enhancement of disease. Studying the functions of PrM/E proteins as well as the relationships to C protein in the formation of DENV particles would provide important information not only for our understanding of the pathogenesis of dengue diseases, but also for vaccine strategies against DENV.
The development of full-length and chimeric infectious clones of DENV has made it possible to study the functions of PrM/E proteins in the context of viral particles, however, the laborious and time-consuming steps involved have restricted its wide use. The first specific aim of this study is to generate retrovirus-based reporter viral system containing PrM/E proteins of DENV to study their functions. We have successfully established a retrovirus-based reporter virus pseudotyped with the PrM/E proteins of DENV2, D2(HIVluc). Co-sedimentation of the majority of E protein and HIV-1 core proteins by sucrose gradient analysis and detection of HIV-1 core proteins by immunoprecipitation of particles with anti-E monoclonal antibody (Mab) suggested that the PrM/E proteins were incorporated into the pseudotype viral particles. The infectivity in target cells can be quickly assessed by the luciferase activity, which was found to be inhibited by the lysosomotropic agents, suggesting a pH-dependent mechanism of entry. Pseudotype reporter viruses containing the PrM/E proteins of other three serotypes, D1(HIVluc), D3(HIVluc) and D4(HIVluc), were also established. Amino acid substitutions of the leucine at position 107, a critical residue at the fusion loop of E protein, with lysine resulted in severe impairment in infectivity, suggesting that entry of the pseudotype reporter virus is mediated through the fusogenic properties of E protein. With more and more DENV sequences available from outbreaks in different countries of the world, this sensitive and convenient tool has the potential to facilitate molecular characterization of the PrM/E proteins of dengue field isolates and to screen for entry inhibitors in the future.
Cryo-electron microscopic study of DENV particles revealed a distinct gap between core and membrane, and raised the question whether PrM and E proteins interact with C protein during assembly and affect its incorporation. Between the two transmembrane (TM) domains of both PrM and E proteins, there is a positively charged residue, arginine or lysine, highly conserved by different flaviviruses. In the second specific aim of this study, we investigated the roles of these two residues in the formation of virus-like particles (VLPs) and incorporation of C protein by site-directed mutagenesis in the DENV1 and DENV4 PrM/E expressing constructs. Substitutions of the lysine or arginine of PrM protein with glutamic acid reduced the formation of VLPs. Substitutions of the arginine of E protein with alanine (E471RA) or glutamic acid (E471RE) but not lysine increased the incorporation of C protein into VLPs. The incorporation was also increased by substitutions of two positively charged residues at the upper edge of α1 helix of C protein, but not by those atα2 helix, with two negatively charged residues. Moreover, C protein was co-immunoprecipitated by anti-E Mab in cells expressing C and PrM/E proteins. Co-transfection of mutants E471RA or E471RE with a previously described replicon containing deletion of PrM/E genes decreased rather than increased the incorporation of C protein. Taken together, these findings suggest that during the process of assembly, E protein interacts with C protein and residue E471R is involved in modulating the incorporation of C protein into VLPs. We proposed a model, in which residue E471R as well as the surrounding non-hydrophobic residue is exposed on the cytosolic side rather than buried inside the membrane and the presence of the positively charged residue E471R could prevent the binding and incorporation of the newly cleaved basic C protein and await the C protein already packaged with nascent RNA genome to incorporate. The two positively charged residues atα1 helix are probably involved in the initial interaction with E471R on the negative surface of membrane. Further studies introducing similar mutations into PrM/E and C proteins in the context of full-length infectious clones are needed to verify this model in the production of infectious virions.
The overall objective of this study is to establish a reporter viral system of 4 serotypes of DENV to facilitate molecular characterization of PrM/E proteins as well as to understand how PrM/E proteins interact with C protein to form virus particles. Future studies extending the system and the observations in this study would not only validate the applications and the model established here but also provide new insights into our understanding of the functions and interaction of PrM/ E and C proteins as well as the pathogenesis of dengue disease.

並列關鍵字

dengue virus ； pseudotype reporter virus ； envelope ； precursor membrane ； capsid ； incorporation ； virus-like particles

參考文獻

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