Glycosylation of the dengue 2 virus E protein at N67 is critical for virus growth in vitro but not for growth in intrathoracically inoculated Aedes aegypti mosquitoes

Abstract

To determine the importance of dengue 2 virus (DEN2V) envelope (E) protein glycosylation, virus mutants in one or both of the N-linked glycosylation motifs were prepared. We found that while the E2 mutant virus (N153Q) replicated in mammalian and mosquito cells, the E1 (N67Q) and E1/2 (N67Q and N153Q) mutant viruses were unable to grow in mammalian cells. Infection of C6/36 mosquito cells with either the E1 or E1/2 mutants resulted in the introduction of a compensatory mutation, K64N, restoring glycosylation in the area. All mutants replicated similarly in inoculated Aedes aegypti mosquitoes, with no change in their mutations. These results suggest that N-linked glycosylation of the E protein is not necessary for DEN2V replication in mosquitoes, however N-linked glycosylation at amino acid N67 (or nearby N64) is critical for the survival of the virus in either mammalian or insect cell culture.

Post-translational regulation and modifications of flavivirus structural proteins

“…Perhaps this is merely a reflection of differential selective pressures imposed by the mosquito midgut infection barrier (ability for initial infection to occur, probably reliant on virion infectivity) and midgut escape barrier (ability for virus to disseminate from the midgut, possibly reliant on degree of virion secretion) (Olson & Blair, 2012). This is supported by the observation that there was no apparent change in the replication of a DENV-2 N67Q mutant compared with the WT virus when the viruses were inoculated intrathoracically into Aedes mosquitoes (Bryant et al, 2007). Glycosylation of the E protein, however, was shown to be non-essential for virulence, as strains of SLEV (Monath et al, 1980) and WNV (Shirato et al, 2004a) lacking E glycosylation are not severely attenuated in mice.…”

“…Asnlinked carbohydrate modifications are always present and are relatively conserved in prM and NS1 with one to three modifications in the pr portion of prM (believed to be important in protecting the fusion peptide in E during virion egress) (Goto et al, 2005;Hanna et al, 2005; Kim et al, 2008;Li et al, 2008;Mandl et al, 1988; von Lindern et al, 2006), and modification at two to three residues in NS1 (important for multimerization of soluble NS1) (Muller & Young, 2012;Muylaert et al, 1996; Somnuke et al, 2011). Glycosylation of the E protein is more variable, usually occurring at residues 153/154 and at residue 67 in the four serotypes of DENV (Bryant et al, 2007;Lee et al, 2010). However, some strains of WNV (Beasley et al, 2005;Botha et al, 2008; Shirato et al, 2004b;Wengler et al, 1985), including KUNV (Adams et al, 1995), as well as strains of St Louis encephalitis virus (SLEV) (Vorndam et al, 1993), Alfuy virus (May et al, 2006 and YFV-17D (Post et al, 1992) lack any active carbohydrate modification in E. Therefore, such glycosylation is not absolutely required for flavivirus replication.…”

“…Investigations utilizing ectopic expression of WNV and TBEV prM and E proteins have ascertained that glycosylation of the E protein leads to a more rapid and efficient secretion of prM/E particles (Goto et al, 2005;Hanna et al, 2005). This enhanced secretion is observed with live DENV (Bryant et al, 2007;Lee et al, 2010) and WNV (Beasley et al, 2005;Li et al, 2006;Scherret et al, 2001) and has recently been demonstrated by our group using non-infectious C-deleted WNV replicons (Roby et al, 2013). However, an investigation utilizing live TBEV failed to demonstrate a difference in the degree of E secretion between glycosylation-mutant and WT viruses in both mammalian and tick cells (Yoshii et al, 2013).…”

Sequence signatures in envelope protein may determine whether flaviviruses produce hemorrhagic or encephalitic syndromes

“…It is reasonable to conclude that Asn-67 confers an adaptive advantage to DENV and that changes in this amino acid are eliminated quickly. Indeed, mutants of DENV2 where Asn-67 in envelope protein was replaced by Gln did not grow in cultured mammalian cells [21,33]. These mutants produced but did not release virus particles and, therefore, did not propagate in mammalian cells.…”

“…1a). Asn-67 is completely conserved in all the four dengue serotypes (DENV1-4) and is glycosylated in DENV envelope protein, but not in other flaviviral envelope proteins [21].…”