Impact of N69 glycosylation site of premembrane protein on viral fitness in dengue virus

Dengue virus (DENV) is a vector-borne, single-stranded positive-sense RNA virus with four distinct serotypes (DENV1-4). The four serotypes of DENV cause about 390 million human infections each year. The maturation of DENV during viral replication affects infectivity and antibody neutralization of the virus. The premembrane (prM) protein composition and the cleavage of the prM protein are determinants of DENV maturation. Across all four serotypes, the prM region is highly conserved. In particular, an N-linked glycosylation site that is located at the prM-N69 amino acid position is conserved across all but two strains of complete genomes provided by the Virus Pathogen Resource (ViPR) database. DENV prM glycosylation has not been extensively studied and there is no known function of the prM-N69 glycosylation site. To investigate the impact of N69 glycosylation of the prM protein, we generated three DENV mutants with loss-of-N69 glycosylation mutations. Subsequently, we derived four more DENV constructs from the previous three mutants with loss-of-N69-glycosylation mutations and a new gain-of-glycosylation mutation at a site proximally near the prM-N69 region. Our results demonstrate no sign of viral production in any of the loss-of-N69-glycosylation constructs. In contrast, we show widespread signs of viral production in one construct, DENV4∆ADD2 (a glycosylation moving mutant). Our result suggests 1) the N69 glycosylation is critical for viral fitness and 2) glycan presence is sufficient for viral fitness. By understanding the importance of the DENV structures like pRM, we’ll be more informed for designing effective DENV vaccines for the future.Bachelor of Scienc