Glycosylation of the Premembrane Protein Is Required for Dengue Virus Replication in Both C6/36 and Vero Cells

Dengue virus (DENV) is a mosquito-borne, positive sense, single-stranded RNA virus that includes four serotypes (DENV1-4) that are further divided into distinct genotypes within a serotype. DENV causes approximately 100 million human infections each year. DENV virions encode a capisd surrounded by a lipid bilayer that encodes two glycoproteins, E and prM. During virion maturation, the premembrane protein (prM) is cleaved to form a mature virion, which includes partially mature and fully mature particles. The maturity state of the DENV virion affects both the infectivity of DENVs and the ability of antibodies to neutralize the virus. The prM glycoprotein is glycosylated and highly conserved across all DENVs. In particular, the N-linked glycosylation site at position 69 is present in over 99% of DENV genomes. In this manuscript, we investigated the importance of the N69 glycosylation site by generating both DENV2 and DENV4 loss-of-function viruses. These N69 glycosylation knockout viruses were generated by introducing a single amino acid mutation at site 69. Neither mutant virus had detectable titers despite multiple passages in vitro. To further investigate the importance of the glycosylation site, we generated another variation in which the N-linked glycosylation site was repositioned to a site proximal to residue 69. One of these proximal-glycosylation mutants showed viral replication at levels that approached wild-type (WT) DENV strains. Overall, this study demonstrated the importance of retaining an N-linked glycan on the prM protein at position 69 for DENV infectivity of mosquito cells and that inserting a glycosylation site in close proximity to position 69 may restore infectivity in some DENV4 strains.