Flavivirus nonstructural protein 1 (NS1) is a unique secreted non-structural glycoprotein. Although it is absent from the flavivirus virion, intracellular and extracellular forms of NS1 have essential roles in viral replication and the pathogenesis of infection. The fate of NS1 in insect cells has been more controversial, with some reports suggesting it is exclusively cell-associated. Here, we confirm NS1 secretion from cells of insect origin and characterize its physical, biochemical, and functional properties in the context of dengue virus (DENV) infection. Unlike mammalian cell-derived NS1, which displays both high mannose and complex type N-linked glycans, soluble NS1 secreted from DENV-infected insect cells contains only high mannose glycans. Insect cell-derived secreted NS1 also has different physical properties including smaller and more heterogeneous sizes, and the formation of less stable NS1 hexamers. Both mammalian and insect cell-derived NS1 bind to complement proteins C1s, C4 and C4 binding protein as well as to a novel partner, mannose-binding lectin (MBL). Binding of NS1 to MBL protects DENV virus against MBL-mediated neutralization by the lectin pathway of complement activation. As we detected secreted NS1 and DENV virus together in the saliva of infected Aedes aegypti mosquitoes, these findings suggest a mechanism of viral immune evasion at the very earliest phase of infection.

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