登革病毒外套膜蛋白質基因之研究：蚊媒與宿主之序列變異及形成病毒顆粒之功能

Study of the Envelope Gene of Dengue Virus: Sequence Variation in Mosquito and Human Host and Functional Role in Particle Formation

林素如(Su-Ru Lin)

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.2009.03240

摘要

登革病毒為黃病毒科，黃病毒屬之一員，有血清型1∼4型。登革病毒感染為在熱帶及亞熱帶地區流行的蟲媒病毒疾病之首位，可引起自限性的登革熱以及顏重致死的登革出血熱�登革休克症候群。官方估計全球每年約有5千萬∼1億人口感染登革病毒，其中有25萬∼50萬是登革出血熱病例。登革病毒之基因體為一正向、單股之核醣核酸，可在轉譯成3個結構性蛋白質，包含殼蛋白質、前驅膜蛋白質、外套膜蛋白質，以及7個非結構性蛋白質，包含非結構性蛋白質1、2A、2B、3、4A、4B、5。
目前已有許多致力於了解登革病毒感染及其致病機轉之臨床與流行病學方面的研究工作正在進行中，但對於登革病毒的傳播及複製仍有許多問題尚待解決。登革病毒的外套膜蛋白質是其細胞向性與病毒毒力之主要決定蛋白質，而且此蛋白質在登革病毒生活史中扮演許多重要的角色。因此本研究之整體目標為探就外套膜蛋白質於登革病毒傳播與複製時之功能。登革病毒可以在埃及斑蚊及宿主間交替複製。有研究報告指出登革病毒在被感染個體中以類種的形式存在，但其在病媒蚊體內之序列變異程度以及其類種結構在病媒蚊與人類宿主間傳播時之變化目前則未明。最近有流行病學研究報告指出登革出血熱的病例數在登革病毒流行的晚期有逐漸增多的趨勢，顯示登革病毒在同次流行中持續演化，然而其毒力決定區及其與疾病越趨嚴重的演化關係則尚缺定論。有研究指出在細胞中一起表現外套膜蛋白質與前驅膜蛋白質可以產生類病毒顆粒，其結構與抗原性質與具有感染力的病毒顆粒十分相似。外套膜蛋白質由N端外區（具有395個胺基酸）及C端柄與嵌入區（具有100個胺基酸）組成。由蜱媒介性腦炎病毒的外套膜蛋白質之連續截斷蛋白質研究顯示其柄區域對於類病毒顆粒之形成極為重要，然而影響此功能之重要殘基為何則尚待研究。
本研究的第一個目標為研究登革病毒在病媒蚊體內之序列變異程度與在傳播時其類種結構之變化。我們分析來自同一次登革病毒流行時，取樣自自然感染的病媒蚊及八位登革病人血漿中的登革病毒血清型第三型之核酸序列。以外套膜基因為例，在登革病毒病媒蚊體內的平均歧異度為（0.21％）較在登革病人體內（0.38%）為低。分析殼蛋白基因也有類似的分析結果，病媒蚊體內的殼蛋白基因平均歧異度為0.09％，低於在登革病人體內的0.23%。我們更進一步分析五隻用實驗感染登革病毒的蚊子體內的登革病毒外套膜基因（平均歧異度為0.09％）與殼蛋白基因（平均歧異度為0.1％）證實這項推論。我們的實驗結果顯示登革病毒在病媒蚊體內之序列變異程度普遍低於在病人體內。同時我們也以在病媒蚊與病人體內的登革病毒外套膜基因變異度分析其類種結構，結果顯示二者的主要變種序列相同，於是我們推測這個主要變種在病媒蚊與病人之間傳播。總而言之，我們的實驗結果支持「病媒蚊對於登革病毒演化之保守性有所貢獻」的假說，也就是登革病毒在病媒蚊體內保持序列較為相近的病毒族群及以主要變種傳播。
本研究的第二個目標為探討登革病毒毒力決定區及其與疾病越趨嚴重的演化關係。我們分析了來自2001至2002年台灣南部連續的兩次登革病毒血清型第二型流行時所採樣的31位病人（14個登革熱病例及17個登革出血熱病例）血漿中的登革病毒血清型第二型序列。結果顯示有五個分別在外套膜基因、非結構性蛋白基因1、4A、5的核酸變異。此結果與1997年在古巴流行的登革病毒血清型第二型之分析報告並無重複，所以我們推測與嚴重流行相關之病毒毒力決定區與造成流行之基因型有關。與其他研究兩次間隔數年的登革病毒流行之譜系交替的報告相較之下，我們的分析結果顯示2002年的高雄登革病毒來自於2001年的小部分病毒變種，而這中間只經過不到6個月的時間。我們的發現可能也代表著一種登革病毒在流行期之間的演化機制。
本研究的第三個目標為在一個可以同時表現登革病毒血清型第四型外套膜蛋白質與膜蛋白質前驅蛋白質的質體系統中，以點突變的方法尋找外套膜蛋白質柄區域中對於類病毒顆粒形成之重要殘基。我們發現位於外套膜蛋白質柄區域的第一螺旋區域（H1）之胺基酸殘基位置398、401、405、408，第二螺旋區域（H2）之胺基酸殘基位置429、436、439、446以及其間胺基酸殘基位置422若以脯胺酸取代原本胺基酸會造成類病毒顆粒之產量減少。共免疫沉澱實驗結果顯示位於H1之點突變影響外套膜蛋白質與膜蛋白質前驅蛋白質之交互作用，位於H2之點突變則未然。脂質膜結合能力實驗結果則顯示帶有造成降低類病毒顆粒形成的H2之點突變亦損害其與脂質膜之結合能力，而帶有造成降低類病毒顆粒形成的H1之點突變則未然。綜合以上實驗結果，我們發現外套膜蛋白質柄區域之H1殘基藉參與其與膜蛋白質前驅蛋白質間之交互作用而影響類病毒顆粒之形成，而H2則因具有與脂質膜結合之能力而影響類病毒顆粒之形成。
總而言之，在本研究中顯示登革病毒在病媒蚊體內以類種的形式存在，且藉著在病媒蚊體內中相似度較高的病毒族群維持其演化上的保守性，此外我們也發現了登革病毒以主要變種傳播。同時在本研究中亦藉著大量且持續的外套膜蛋白基因以及基因體全長之序列分析釐清登革病毒從一個只有少數嚴重病例的流行到一個越來越多嚴重病例的流行是如何演化。本研究中亦更進一步探討外套膜蛋白質之柄區域如何參與類病毒顆粒之形成與其在登革病毒生活史中病毒組裝的步驟之可能功能。推而廣之，本研究提供登革病毒傳播與複製之更深入的了解以及預防與控治登革病毒之新策略。

關鍵字

登革病毒 ； 序列變異 ； 病毒演化 ； 外套膜蛋白質 ； 類病毒顆粒

並列摘要

Dengue virus (DENV) belongs to the genus flavivirus in the family flavivirus. There are four serotypes of DENV (DENV1 to 4), which are the leading cause of the arboviral diseases in the tropical and subtropical areas, including a self-limiting disease, dengue fever (DF) and a severe and life-threatening disease, dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS). It has been estimated that approximately 50-100 million of DENV infection and 250-500 thousands cases of DHF occur annually throughout the world. DENV contains a positive-sense single stranded RNA genome, which encodes three structural proteins, including capsid (C), precursor membrane (PrM) and envelope (E) at the N-terminus, and seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, at the C-terminus.
While substantial efforts have been made to study the clinical and epidemiological aspects of dengue disease and pathogenesis, several fundamental questions of the transmission and replication of DENV remain unclear. The E gene of DENV is the major determinant of tropism and virulence, and is known to play important roles in the life cycles of DENV. The overall objective of this study is to focus on the E gene and investigate its roles in some poorly understood areas in the DENV transmission and replication. DENV replicates alternately in the mosquito vectors and human hosts. It has been reported that DENV is present as quasispecies in plasma of infected individuals. However, the extent of sequence variation of DENV in the mosquito vector and the quasispecies structure changes during transmission between human and mosquitoes remain unknown. Several epidemiological studies have reported recently that the proportion of severe cases, DHF, to total cases increases toward the end of an outbreak, suggesting DENV evolves during the same outbreak. However, the viral determinants and evolution linked to outbreak with increased severity remain unclear. Co-expression of PrM and E proteins is sufficient to produce virus-like particles (VLPs), which structurally and antigenically similar to infectious particles. The E protein contains 395 amino acids at the N-terminal ectodomain and 100 amino acids at its C-terminus, which consists of the stem and anchor regions. Deletional study of the E protein of the tick-borne encephalitis virus (TBEV) suggested the importance of the stem region in the formation of VLPs, however, the critical residues involved remain unknown.
In the first specific aim, we studied the extent of sequences variation of DENV in the mosquitoes and the changes of quasispecies during transmission by examining DENV3 sequences in naturally infected mosquitoes in comparison with those in eight patients from the same outbreak. For the E gene, the mean diversity in naturally infected mosquitoes was 0.21%, lower than that in patients (0.38%). Similarly, the mean diversity of C gene in naturally infected mosquitoes was 0.09%, lower than 0.23% in patients. This was further verified with five experimentally infected mosquitoes (mean diversity, 0.09 and 0.10% for the E and C genes, respectively). Our findings suggested that the extent of sequence variation in the mosquitoes was generally lower than that in the patients. Examination of the quasispecies structures of the DENV3 E sequences in the mosquitoes and patients revealed that the sequences of the major variants were the same, suggesting that the major variant was transmitted. These findings support our hypothesis that mosquitoes contribute to the evolutionary conservation of dengue virus by maintaining a more homogenous viral population and a dominant variant during transmission.
In the second specific aim, we investigated viral determinants and evolution linked to outbreak with increased severity by examining DENV2 sequences from plasma of 31 patients (14 DF, 17 DHF) continuously during two consecutive DENV2 outbreaks in southern Taiwan in 2001-2002, in which both the total cases and proportion of DHF cases increased. Analysis of E gene and full-genome sequences between viruses of the two outbreaks revealed 5 nucleotide changes in E, NS1, NS4A, and NS5 genes. None was identical to those reported in the DENV2 outbreak in Cuba in 1997, suggesting viral determinants linked to severe outbreak were genotype dependent. Compared with previous reports of lineage turnover years apart, our findings that the 2002 viruses descended from a minor variant of the 2001 viruses in less than 6 months were novel, and may represent a mechanism of evolution of DENV from one outbreak to another.
In the third specific aim, we investigated critical residues in the stem region of E protein involved in the formation of VLPs by employing site-directed muatgenesis in the context of DENV4 PrM/E expression construct. We found that proline substitutions introduced to residues 398, 401, 405, and 408 within the first helix (H1) of the stem, residues 429, 436, 439, and 446 within the second helix (H2) of the stem, and residue 422 between H1 and H2 impaired the production of VLPs. Co-immunoprecipitation experiment revealed that mutants in the H1 affected the PrM-E interaction, whereas mutants in the H2 did not. Membrane binding assay of chimeric β-gal constructs containing the stem or mutants suggested while the H1mutants did not affect the ability of the stem to bind membrane, the H2 mutants that impaired VLP production affected such membrane-binding ability. Taken together, our findings suggest that the H1 residues of the stem region are involved in the production of VLPs by participating in the interaction with PrM protein, whereas the H2 residues are involved in the production of VLPs by contributing to the membrane association of the stem region.
In summary, we showed that DENV exists as quasispecies in the mosquito vectors, and they contribute to the evolutionary conservation of DENV by maintaining a more homogenous viral population and a dominant variant during transmission. By extensive and continuous sequencing analysis of the E gene as well as full-genome, we demonstrated how DENV evolved from an outbreak with fewer severe cases to an outbreak with more severe cases. Moreover, we showed that how the stem region of DENV E protein is involved in the VLP production and presumably the assembly of DENV replication cycle. Information derived from this study would provide new insights into our understanding of the transmission and replication of DENV, as well as strategies for prevention and control of dengue diseases.

並列關鍵字

dengue virus ； sequence variation ； virus evolution ； envelope protein ； virus like particle

參考文獻

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