泛素化是一種在真核細胞中，透過泛素蛋白的轉譯後修飾以調控細胞內的生理機能。有報導指出登革病毒可利用宿主的泛素化修飾來幫助自身的複製。過去研究中發現在不影響病毒組裝的情況下，抑制泛素—蛋白酶體系統可以減少病毒的釋出。因此推測主導病毒成熟到釋出階段的病毒蛋白─前驅膜蛋白 (prM)和套膜蛋白 (E)可能具有泛素化，並且會受到蛋白酶體抑制劑的影響。在本研究中，我們發現prM在蛋白酶體抑制劑作用下表現量會大幅提升。藉由不具有離胺酸 (lysine, K)的prM (6KR)與野生型 (WT)相比，發現prM蛋白穩定度、細胞內的分布以及furin 剪切的程度皆因泛素化而有所改變。透過一系列只含有單一個lysine的泛素蛋白，確認prM的泛素化是透過K6及K33兩種鍵結。經由序列分析比對四種血清型的登革病毒後，我們建構分別只含有一個高保留lysine的prM，並發現這些lysine皆能被泛素化蛋白修飾。雖然這些泛素化不影響prM在細胞內的穩定度及分布，但我們發現prM(K52)上的泛素化是以K6的方式鍵結，並且可以提升furin剪切的效率。總結來說，我們發現泛素化改變登革prM的蛋白性質，有助於了解病毒蛋白與宿主的交互作用，並有望成為登革感染治療的新方向。

The dengue virus (DENV) utilizes ubiquitination to regulate its viral replication. Proteasomal inhibitors have been shown to reduce the release but not the assembly of the virus. Since DENV prM protein might be responsible for the proteasome inhibitor-directed effects through ubiquitination, we pulled down the prM by immunoprecipitation (IP) and checked its ubiquitination. To characterize properties of prM without ubiquitination, we designed the prM-6KR mutant that all the lysine residuals were replaced by arginine. Although both prM and E protein participated in the maturation and release processes of DENV, prM but not E protein expression level was significantly enhanced by the proteasome inhibitor. Moreover, the stability, subcellular localization, and furin cleavage efficiency were all differed between WT and 6KR. To uncover the linkage type of the prM ubiquitination, we generated a series of Ub mutants with only one lysine residue. Thus, the K6- and K33-linked ubiquitination was identified on the DENV prM protein. By the same concept using a series of prM with only one lysine residue, we found all conserved lysine among the four serotypes of DENVs could be ubiquitinated. These single-lysine prM proteins hardly affected prM stability and subcellular localization, but the furin cleavage seemed enhanced by the K6-linked ubiquitination on the prM K52 residue. In conclusion, ubiquitination might alter DENV prM stability, subcellular localization, and vulnerability to furin; whether particular linkage of Ub on specific prM residues alter virus infectivity remains to be discovered in the future.

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