Structure-Function Studies of a Flavivirus Non-Structural Protein

Flaviviruses are positive single-stranded RNA arthropod-borne viruses within the family Flaviviridae which includes Dengue virus (DENV), West Nile virus (WNV), Yellow fever virus (YFV), Japanese encephalitis virus (JEV) and Zika virus (ZIKV). From DENV alone approximately half of the world lives under the threat of contracting a flavivirus infection, with about 390 million infections globally per year. The non-structural protein 1 (NS1) of these viruses has been shown to be integral to several stages of the flavivirus lifecycle, impacting both intracellular and extracellular viral functions, while also directly being implicated in contributing to severe disease and pathogenesis. From an in-depth characterization of numerous NS1 variants, we identified regions within the NS1 protein that contribute to altering virus entry. Further study using purified NS1 protein and purified flavivirus particles, demonstrated that addition of NS1 to cells, concurrently with virus particles or prior to virus particle addition, increased virus attachment to cell surfaces. This increase in attachment was found to result in an enhanced viral infectivity. We were able to map this function to a charged region within the NS1 proteins C-terminus, which facilitated binding to secreted virus particles. We further demonstrated that extracellular binding of NS1 to virus particles were occurring through E protein exposed regions on these particles. This NS1-virus interaction, alongside an NS1-cell surface interaction, was found to be required for enhanced flavivirus infection. Here we show that extracellular interactions between NS1 and virus result in an increased virus infectivity and depict NS1’s extracellular roles as a virally encoded attachment factor