登革病毒外套膜蛋白質於內質網之定位與產生病毒顆粒之研究

Abstract

登革病毒(dengue virus, DENV)在分類上屬於黃病毒科中的黃病毒屬，四種血清型的登革病毒(DENV1-DENV4)在熱帶及亞熱帶地區造成最重要的節肢動物傳播疾病。 外套膜蛋白質(Envelope, E)是登革病毒細胞嗜性的主要決定因子，也是中和性抗體及促進性抗體的主要標的。根據結晶繞射的結果，其N 端包含三個區域(domain)。而C 端的一百個胺基酸形成柄狀(stem)及穿膜區域(transmembrane, TMD)，各自包含兩個螺旋結構(α-helices, T1 及T2)。黃病毒在複製的過程中會出現似病毒顆粒(virus-like particle, VLP)，在結構上及物理化學的性質上類似具有感染力的病毒顆粒。同時表現黃病毒屬的前驅膜蛋白質(Precusor membrane, PrM)及E 蛋白質，可有效率地產生似病毒顆粒，可應用於研究PrM 及E 蛋白質的功能及病毒的組裝。目前知道含有套膜的病毒會在分泌途逕(secretory pathway)中的不同胞器形成病毒顆粒。其關鍵步驟是病毒核糖蛋白質(nucleocapsid)，病毒結構蛋白質與複製過程中產生的病毒核酸之相互作用，因此病毒以何種機制滯留在不同胞器內對於病毒顆粒的形成十分重要。 登革病毒的複製發生在內質網(Endoplasmic reticulum, ER)所衍生出來的膜系上，病毒的組裝也在此形成，並藉由分泌路徑將未成熟的病毒顆粒送至細胞外。一般認為黃病毒的複製複合體是藉由嵌入在ER 膜系上的病毒非結構性蛋白質與ER 膜系相互作用。關於病毒結構性蛋白質，已有研究指出C 型肝炎病毒的E1E2 蛋白質及黃熱病病毒的PrM/E 蛋白質之穿膜區域具有內質網滯留訊號(ER retention signal)，這顯示E 蛋白質的穿膜區域具有內質網滯留訊號，但其機制仍尚未被釐清。本研究的整體目的是利用DENV 的PrM/E 蛋白質及形成的VLPs當作模式，用以研究黃病毒的E 蛋白質如何滯留在內質網的機制及組裝成病毒顆粒。 在第一個研究目標中，根據之前的研究推測含有日本腦炎病毒(Japanese encephalitis virus, JEV)的柄狀-穿膜區域之DENV2 的外套膜蛋白質滯留於內質網能力較差，且似病毒顆粒的產量較低，我們探討此兩種E 蛋白質的特性。進ㄧ步建構僅含有JEv 的柄狀或穿膜區域DENV2 E 之重組蛋白質，結果發現增強DENV2 似病毒顆粒的分泌需要同時具備JEV 的柄狀-穿膜區域，且並不影響前驅膜/外套膜蛋白質雙體間的交互作用。此外，藉由分析E 蛋白質在細胞內之定位，我們發現JEV 的柄狀-穿膜區域會促進PrM/E 蛋白質由膜系上釋出而產生較多似病毒顆粒。因此我們建構一系列含有JEV 或DENV2 的柄狀-穿膜區域之chimericCD4 重組質體，其結果顯示兩者的柄狀-穿膜皆具有內質網滯留訊號。進一步地將DENV2 及DENV4 的穿膜區域構築在CD4 蛋白質，結果顯示內質網滯留訊號位於穿膜區域。 在第二個研究目標，我們進ㄧ步探討DENV2 E 蛋白質滯留於內質網的決定性胺基酸及機制。以點突變的方法置換T1 的N 端及T2 的N 端或C 端非疏水性的胺基酸成疏水性胺基酸，結果顯示這些突變會影響chimeric CD4 蛋白質在細胞內的定位，及蛋白質糖化作用之模式。此外，以七個疏水性胺基酸增長T1 的長度也顯示相似的結果，由此推測E 蛋白質非疏水性的胺基酸與穿膜區域的長度是滯留在ER 的重要決定因子。 在第三個研究目標中，我們研究不同程度的內質網滯留現象與形成似病毒顆粒的相關性。雖然藉由點突變增加疏水性胺基酸及T1 增長的DENV2 突變E蛋白質對前驅膜/外套膜蛋白質雙體的相互作用影響不大，但分析胞外似病毒顆粒的分泌，我們發現這些突變E 蛋白質具有較弱的內質網滯留訊號，且形成似病毒顆粒的能力比較強。這可能是由於改變穿膜區域的特性使得病毒E 蛋白質容易由膜系上彎曲釋出，但詳細機制仍未明。這些實驗結果顯示改變E 蛋白質的穿膜區域可以增加似病毒顆粒的分泌，未來可應用在疫苗之生產與臨床非感染性血清學抗原的製備。 綜合本研究的結果，我們發現DENV E 蛋白質的穿膜區域具有內質網滯留訊號。T1 的N 端及T2 的N 端或C 端之非疏水性胺基酸，以及T1 的長度是決定內質網滯留的重要因子。此外，較弱的內質網滯留訊號促進產生較多的似病毒顆粒，這顯示改變穿膜區域的特性可以促進似病毒顆粒的產生。我們的結果支持病毒E 蛋白質以穿膜區域的特性來定位在不同的胞器上，這些資訊將有助於將來針對病毒組裝的抗病毒藥物的發展。

Dengue viruses (DENV) belong to the genus Flavivirus of the family Flaviviridae. The four serotypes of DENV (DENV1 to DENV4) are the leading cause of arboviral diseases in the tropical and subtropical areas. The envelope (E) protein of DENV is the major determinant of cell tropism and virulence, as well as the major target of neutralizing and enhancing antibodies. The N-terminal ectodomain of E protein contains three well characterized domains. The C-terminal 100 amino acid residues of E protein consist of the stem and the anchor (transmembrane domain, TMD), which consists of two α-helices (T1 and T2). A common feature of flavivirus replication is the production of virus-like particles (VLPs), which are similar to infectious virions in the structural, biochemical and antigenic properties, and have been used to investigate the function of precursor membrane (PrM)/E proteins and assembly of virus particles. Morphogenesis of many enveloped viruses is known to take place at various sites along the secretory pathway. An important step is the encounter and interaction between the viral nucleocapsid complex and viral E protein as well as matrix protein in some cases at a particular organelle. As the default pathway for cellular membrane proteins goes from ER to the plasma membrane, how the viral E protein, a membrane protein, retains in a particular intracellular organelle is critical for virus assembly. The replication of DENV occurs at the membranous structures derived from ER. Assembly also takes place in these membranous structures, where immature virions are thought to bud into the lumen of ER and transported thourgh the secretory pathway. It is generally believed that flaviviral replication complexes associate with ER membrane through their interaction with small hydrophobic nonstructural proteins, which anchored on the ER membrane. For the structural proteins, chimeric experiments between cellular proteins normally expressed on surface such as CD4 or CD8 and different domains of E1/E2 proteins of hepatitis C virus or PrM/E proteins of yellow fever virus, both flavivirus, suggested that the TMD of E protein contains an ER retention signal. However, the critical residues and elements in the TMD and the mechanism involved remains unclear. The overall objective of this study is to employ the formation of VLPs by DENV PrM/E proteins as a model system to elucidate how flaviviral E protein retains and assembles in the ER. The long-term goal of this study is to understand how E proteins of enveloped viruses contribute to the morphogenesis at different intracellular organelle, and to provide new insight into anti-viral strategies of targeting virus assembly. In the first aim of this study, we characterized two E proteins, DENV2 E protein and chimeric DENV2 E protein containing the stem-TMD of Japanese encephalitis virus (JEV), which have been reported to retain in the ER and produce VLPs with different efficiency. By analyzing chimeric PrM/E constructs, both stem and TMD of JEV E protein were found to be required for efficient production of VLPs. Then, we generated chimeric constructs of CD4 and demonstrated that both stem-TMD of DENV and JEV contained an ER retention signal with different intensity, which may account for different efficiency of VLPs production. Moreover, we generated a series of chimeric constructs between CD4 and stem-TMD or TMD alone of DENV2/DENV4 as well as reciprocal constructs, and found that TMD alone was sufficient to retain E protein in ER. In the second aim, we examined TMD of DENV2 E protein to investigate the critical residues/ elements and mechanism involved in ER retention. Substitution of non-hydrophobic residues at the N-terminus of first helix (T1), N-or C- terminus of second helix (T2) with hydrophobic residues in the chimeric CD4 and E proteins resulted in their release from ER. Moreover, insertion of 7 hydrophobic residues in T1 showed similar phenotypes, suggesting that certain non-hydrophobic residues and the short length of TMD contribute to ER retention. Analysis of enveloped viruses assembled at the plasma membrane compared to those assembled in the Golgi and ER revealed a trend of decreasing length and increasing non-hydrophobic residues of the TMD of E proteins. In the third aim, we studied the relationship between ER retention phenotypesand the production of VLPs. Although the TMD mutants, which had an increase in the hydrophobicity or length and less ER retention phenotype, did not affect the PrM-E heterodimerization and glycosylation, they increased the production of VLPs. The increased VLP production is probably due to changes in the curving and bending of membrane, though the mechanism remains to be investigated. These findings suggest that modifications of TMD could facilitate VLP production and have implications for utilizing flaviviral VLPs as serodiagnostic antigens and vaccine candidates. In summary, we demonstrated in this study that the TMD domain of DENV E protein contains an ER retention signal. Non-hydrophobic residues at the N-terminus of T1 and N- or C-terminus of T2 as well as the length of T1 contribute to the ER retention phenotype. Moreover, TMD mutants with less ER retention phenotype showed increased production of VLPs. Our findings support a TMD-dependent sorting for viral E protein. Information derived from this study may have implication for future antiviral strategies targeting the assembly of flaviviruses.