Ting, D. H. R. | bioRxiv Science

bioRxiv2025

N153-linked glycans on envelope protein protect orthoflaviviruses from antibody-mediated clearance

The envelope (E) protein of dengue virus (DENV) is glycosylated at two highly conserved asparagine (N) sites (N67 and N153). The role and importance of these N-linked glycans in DENV pathogenesis has been elusive. Here, we report the critical role of N153-linked glycans on E protein in preventing antibody-mediated viral clearance. A DENV2 mutant lacking N153-linked glycans (N153Q mutant) was engineered and found to be mildly impaired in vitro but drastically attenuated in a symptomatic mouse model of severe dengue, as evidenced by accelerated viral clearance. In B cell-deficient mouse models, N153Q mutant displayed parental virulence and viremia profile. Homologous and heterologous passive transfers of purified IgM from infected B cell-proficient mice into B cell-deficient mice demonstrated the role of N153Q-specific IgM in N153Q attenuation and accelerated clearance, while WT DENV was unaffected by IgM from both WT- and N153Q-infected mice. Furthermore, in vitro neutralization assay supported that the accelerated clearance of N153Q mutant in mice was mediated by non-neutralizing IgM. Furthermore, using plasma samples from convalescent dengue patients and monoclonal antibodies, in vitro neutralization assays showed that N153Q virus was more susceptible than WT to IgG-mediated neutralization. Glycoproteomics combined with molecular dynamics (MD) simulations revealed that glycan composition on E protein influenced IgG binding. Our findings were extended to all DENV serotypes and ZIKV, hence supporting that the N153 glycans-mediated immune evasion strategy is conserved across orthoflaviviruses.

microbiology↗

bioRxiv2024

De-glycosylated non-structural protein 1 enhances dengue virus clearance by limiting PD-L1/PD-1 mediated T cell apoptosis

The non-structural protein 1 (NS1) of dengue virus (DENV) contains two highly conserved N-glycosylation sites at positions 130 and 207 (N130 and N207). Intracellular NS1 monomers and homo-dimers participate in viral RNA replication within membrane-bound replication complexes. Soluble multimeric NS1 (sNS1) is secreted into the extracellular milieu and represents an important virulence factor for DENV through its ability to interfere with the host complement activation cascade and to induce vascular leakage. The role of the two N-glycans in NS1 biological activities, however, has not been carefully examined. Here, stable DENV2 mutants that lack glycan at either N sites of NS1 were engineered. We showed that the lack of glycans at either N site of NS1 did not impair viral replication nor viral output in both mosquito and mammalian cell lines. In contrast, while N130 de-glycosylated DENV displayed parental in vivo fitness in IFNAR-/- mice, the N207 de-glycosylated mutant was significantly attenuated as evidenced by 100% survival rate, which correlated with accelerated viral clearance in circulation. sNS1-depletion, sNS1 exogenous administration and co-infection experiments supported that N207 de-glycosylated NS1 exerted a dominant attenuating effect during in vivo infection. Bulk RNAseq, inflammatory cytokine profile, immune phenotyping of neutrophils and T cells, immune cell depletion and immune checkpoint blockade approaches led us to propose that N207 de-glycosylated NS1 limited CD8+ T cell apoptosis mediated by the PD-L1/PD-1 axis, thereby improving viral clearance efficacy. This work uncovers a novel immune evasion strategy where N207 glycans on NS1 prevent the protein from exerting immune modulation activity that would be detrimental to DENV.

microbiology↗