The biological role of Asn-linked glycosylations of DENV-2 E protein

Abstract

The envelope proteins of many viruses are glycosylated, where the glycans on the virion surface have been shown to be important, in addition to their strictly structural role, in various aspects of the virus life cycle, such as receptor-binding and escape from antibody recognition. Dengue virus (DENV) is in terms of human disease frequency the most important member of the flaviviruses, a group of small, enveloped, plus-strand RNA viruses. The four DENV serotypes differ from all other flaviviruses by the presence of two glycans on the envelope (E) protein, one linked to Asn-153, a glycosylation which is well conserved among the flaviviruses, and a second linked to Asn-67, which is unique to DENY. While the presence of glycan at Asn-153 has been shown by others to influence fusion of viral with cellular membranes, infectivity, assembly and/or release and virulence, the glycan at Asn-67 has been suggested to be critical in virus attachment to the cell surface molecule, DC-SIGN, a putative cellular receptor for DENV. In this study, I have used a reverse genetics approach to prevent glycosylation at Asn-67, Asn-153, or both carbohydrate acceptor sites, in order to investigate their physiological function(s). Single or multiple point mutation were introduced at the Asn-153 and Asn-67 glycosylation sites, respectively, on the E protein ofDENV-2 strains NGC and PUO-218, using two infectious cDNA clones. DENV -2 strain, NGC, is a laboratory-adapted virus, while strain PUO-218 is a low-passage natural isolate from Toxorhynchites splendens mosquitoes. ... I also show that removal of both glycans from the DENV-2 E protein prevents the recovery of viable virus with a stable mutant genotype (Chapters 3 and 5). Nevertheless, given the importance of strain origin and type of substitution used for deletion of the carbohydrate acceptor sites that I have demonstrated in this thesis, it is likely that the engineering of a viable DENV variant lacking both E protein glycans is feasible.