泛素化為細胞內重要的轉譯後修飾之一，許多蛋白可藉由泛素化來參與並調控各個生理反應，例如蛋白質水解、細胞週期、訊息傳遞、DNA修復及細胞凋亡等；而諸多與泛素化相關的訊息路徑中，以泛素-蛋白酶體系統最為熟知且被廣為研究。過去的文獻指出，泛素-蛋白酶體系統在病毒複製中扮演重要的角色，而且參與在不同病毒生活史的各個階段。登革病毒 (Dengue virus, DENV) 屬於黃質病毒科 (Flaviviridae)，基因體為單股正向RNA；在感染過程中，登革病毒利用宿主細胞以及登革病毒的蛋白酶轉譯出十種蛋白幫助其完成生活史，而病毒蛋白是否可藉由泛素化調控其自身功能並進而影響登革病毒的複製週期仍尚未釐清。在本研究中，我們發現登革病毒的prM及E蛋白可被泛素化修飾。以蛋白酶體抑制劑MG132阻斷細胞內的蛋白酶體功能後，登革病毒的複製亦會受到影響，顯示泛素-蛋白酶體系統於登革病毒的生活史中扮演重要的角色。在病毒感染的過程中，在不同時間點以MG132去阻斷細胞內的蛋白酶體功能，我們發現泛素-蛋白酶體系統明顯地參與在登革病毒複製的晚期。在登革病毒次基因體複製子 (subgenomic replicon)報導系統實驗中，我們發現泛素-蛋白酶體系統不影響登革病毒基因體的轉譯與複製；而在病毒釋放實驗中，MG132可明顯抑制登革病毒prM蛋白的表現以及細胞外的病毒效價。藉由TCA沈澱法濃縮並分析受感染細胞之上清液，我們發現MG132降低了上清液中病毒結構性蛋白的含量；而以MG132處理後的細胞，其產出的登革病毒仍可再於試管中以furin切割成熟，並恢復感染力，顯示MG132可抑制登革病毒的釋出。有趣的是，在試管內furin切割實驗中，高度泛素化的prM似乎更易受到furin的剪切，綜合上述，我們推測登革病毒藉由prM蛋白的泛素化修飾，調控病毒顆粒的成熟與釋放。

Protein ubiquitination, one of the post-translational modifications, regulates a wide variety of cellular processes and plays important roles for numerous viruses during infection. However, questions remained whether dengue viral proteins can be modified by ubiquitination and how the modification affects viral functions. By two independent screening assays, we found that two structural proteins of dengue virus (DENV), prM and E, were consistently ubiquitinated in infected cells. A kinetic study by pulse treatment of MG132, a proteasome inhibitor, during a single viral replication cycle showed that DENV was drastically inhibited by MG132 at a relatively late stage of replication. These results suggested that ubiquitin-proteasome system (UPS) may regulate DENV replication. By using DENV replicon and subgenomic construct containing a luciferase reporter, we found that neither DENV genomic RNA replication nor translation was the major target of MG132. However, DENV release seems to be attenuated since prM protein and DENV infectivity in the culture supernatant from MG132-treated cells were both reduced. In vitro DENV virion maturation assay showed that the infectivity of DENV, either derived from MG132- or solvent-treated cells, could be further enhanced by incubation with recombinant furin. Interestingly, in vitro furin cleavage assay revealed that prM derived from MG132-treatment, which is highly ubiqutinated, became more sensitive to furin cleavage than that derived from solvent control. Taken together, these findings suggest that prM protein is ubiquitinated to regulate DENV maturation processes. Disturbance of this fine-tuned machinery, such as MG132 treatment, may serve a novel anti-DENV strategy in the future.

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