登革熱屬於黃病毒科，具有四種血清型。登革病毒顆粒主要由單鏈RNA基因組、脂質雙層膜和結構蛋白質組成。其中，結構蛋白質主要包括：包膜蛋白 (E)、膜蛋白 (M)和衣殼蛋白 (C)。在病毒複製過程中，新合成的登革病毒顆粒在內質網中組裝成未成熟顆粒，未成熟顆粒的結構具有前驅膜蛋白 (pr-M)，病毒釋放出細胞過程中，弗林蛋白酶除去前驅分子 (pr)，形成具有感染性的成熟登革病毒。然而，弗林蛋白酶切割被認為是低效的。未成熟的登革病毒缺乏感染細胞的能力，但登革病毒感染細胞過程中，會產生高比例的不具感染性的未成熟病毒顆粒，這些顆粒會藉由依賴性增強作用進入含有Fc接受體的細胞內，增加病毒增值的機會，亦是造成感染異型登革病毒產生重症原因。我們去年與中興大學趙老師發表文章說明，成熟的登革類病毒顆粒可以中和四型血清型，加上因為不含有感染基因組，因此具安全性，是個潛在的候選疫苗。我的研究目的是使用低溫電子顯微鏡解析未成熟類登革病毒結構，結構能告訴我們顆粒成熟過程中，表面蛋白的形態變化，及抗原決定位的分佈變化。為了得到高解析度結構，我們從兩方面著手，一是找到最佳樣品製備參數，降低樣品顆粒異質性，二是我們使用高階電子顯微鏡及影像擷取系統，並使用自動化大量收集影像。目前初步結構顯示未成熟類登革病毒的表面有突起，與成熟類登革病毒表面平滑不同，經過表面蛋白的嵌入 (fitting) 發現此突起是pr組成，此外，未成熟類登革病毒的表面蛋白E與成熟類登革病毒E的排列方式都是遵從T=1的排列，此與病毒顆粒是T＝3排列不同。日後期待將結構解析度再提高，更細微探討穿膜區域的變化，此外研究抗原決定位的變化，與成熟類登革病毒結構的抗原決定位做比對 (mapping)，這些結構資訊將有助於應用疫苗與藥物製劑上的開發。

Dengue fever belongs to Flaviviridae family and has four serotypes. Dengue virus particles are mainly composed of a single-stranded RNA genome, a lipid bilayer membrane, and structural proteins. Among them, structural proteins are mainly composed of envelope (E), membrane (M) and capsid (C) proteins. In the process of virus replication, newly synthesized DENVs are assembled as immature particles which have pr-M protein. The furin then cleaves away pr so that the mature DENV contains only M protein and is infectious. However, the furin cleavage is thought to be inefficient. It is believed that a completely immature DENV lacks the ability to infect cell, but cells infected with dengue virus release a high proportion of immature virions and the immature DENV could enter to the cells containing Fc receptors by dependence enhancement, increasing the chance of virus value-adding, and causing serious causes of infection with heterologous dengue virus. Our previous study showed that mature dengue viral like particle (mD2VLP) could neutralize four serotypes and was therefore suggested to be a safe vaccine candidate. My study is to solve the structure of dengue VLP in immature form (imDENV VLP) using cryo-EM. The structure of imDENV VLP could tell us the surface protein rearrangement and epitope exposure changes during maturation process. To obtain the high-resolution structure, first, we optimized the sample preparation protocol; second, we used the advanced cryo-EM and image taking system which is equipped with automatic data collection. Current imDENV VLP structure showed that there were protrusions on the imDENV VLP surface, the E lied parallelly on the surface and followed T=1 lattice arrangement which resembled the mD2VLP. In the future, we would like to improve the resolution, and map the epitopes and TM/stem regions with mature VLP. The structure information gathered could provide us the hints of vaccine development and drug design.

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