Aedes aegypti calreticulin promotes dengue virus replication and transmission by glycan-dependent binding to the prM protein.

Mosquito saliva is coinoculated with dengue virus (DENV) during blood feeding, potentially influencing infection at bite sites; however, the key salivary factors and underlying mechanisms remain unclear. This study identifies Aedes aegypti calreticulin (CRT) as a dual-function enhancer of DENV replication and transmission. Far-western blotting and coimmunoprecipitation revealed that mosquito salivary proteins interact with the viral precursor membrane (prM) protein. Furthermore, enzymatic deglycosylation (via endoglycosidase H) and lectin-competition assays demonstrated that this interaction relies on N-linked glycans bearing N-acetylglucosamine residues on viral structural proteins. Liquid chromatography-mass spectrometry analysis of saliva-virion complexes identified nine Ae. aegypti candidates, and immunoblotting confirmed CRT as a prM-binding partner. Oral infection models showed that DENV markedly upregulates CRT in salivary glands, and immunofluorescence colocalized CRT with calnexin in the endoplasmic reticulum (ER). RNA interference-mediated CRT knockdown significantly reduced both viral RNA and salivary-gland prM expression, as well as infectious titers in a plaque assay, underscoring CRT's essential role in DENV replication. Relative to control saliva, CRT-depleted mosquito saliva conferred markedly reduced infectivity to C6/36 cells, establishing CRT as a saliva-derived enhancer of host cell infection. Collectively, these findings support a model in which Ae. aegypti CRT acts intracellularly as an ER chaperone facilitating DENV replication and extracellularly as a salivary factor binding prM glycans to promote early infection. By identifying a glycan-dependent interface between a vector chaperone and a viral structural protein, this study reveals a conserved mechanism of arbovirus transmission and highlights mosquito CRT-glycan interactions as potential targets for disrupting dengue transmission.