| Summary: | 碩士 === 國立成功大學 === 微生物及免疫學研究所 === 101 === Dengue virus (DENV) is a single-stranded positive-sense RNA virus of the family Flaviviridae. The DENV genome encodes a single polyprotein precursor, which is processed into three structural proteins (C, prM and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS4A, NS4B, and NS5). SUMO (Small ubiquitin-like modifier) has been identified as a reversible post-translational protein modifier. SUMOylation regulates a wide variety of cellular processes and plays important roles for numerous viruses during infection. In this study, we found that overexpression of Ubc9, the sole E2-conjugating enzyme required for protein SUMOylation, enhanced DENV replication whereas its knockdown reduced virus replication, suggesting that SUMOylation pathway is involved in DENV life cycle. We next carried out in vivo SUMOylation assay to search for the potential viral SUMO target(s). We found that DENV NS5 protein, the RNA-dependent RNA polymerase responsible for viral genome replication, is the only viral nonstructural protein which can be SUMOylated. Further, in vivo de-SUMOylation assay and in vitro SUMOylation assay were performed and confirmed that NS5 protein is a bona fide SUMO target. To delineate the region of NS5 protein in which the SUMO acceptor lysine(s) is located, expression plasmids for NS5 protein deletion mutants were generated and subjected to in vivo SUMOylation assay. We found that the SUMO acceptor sites are located in the N-terminal methyltransferase (MTase) domain of NS5 protein. Determination of the SUMOylated residues was elusive probably because of the presence of alternative SUMOylation sites. However, we found a SUMO-interacting motif (SIM) located in the MTase domain of NS5 protein and demonstrated that this SIM motif is required for NS5 protein SUMOylation. Collectively, these findings suggest that SUMOylation of NS5 protein is an important feature of DENV infection and may regulate viral replication.
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