West‐Nile virus replicon particle entry requires the C‐terminal half of the carbohydrate‐recognition domain of the Dendritic cell‐specific ICAM‐3 grabbing non‐integrin related protein

Abstract

West‐Nile virus (WNV) is an arbovirus usually transmitted to humans via a mosquito vector. Infections commonly result in febrile symptoms while rare severe neuroinvasive cases may result in encephalitis or meningitis. Studies have shown that WNV infection efficiency is enhanced by expression of DC‐SIGNR on target cells, which normally do not express DC‐SIGNR. To investigate WNV tropism, we established 293T kidney epithelial cell lines that stably express vector, DC‐SIGNR and mutants of DC‐SIGNR that lack the entire carbohydrate‐recognition domain (CRD) or lack the C‐terminal half of the CRD. We demonstrate successful surface expression of DC‐SIGNR and its mutants from stably‐transfected 293T cells, but not vector‐transfected 293T cells. Further, we show that monoclonal antibody 120604 which binds specifically to the DC‐SIGNR CRD binds to DC‐SIGNR expressing 293T cells, but not to vector nor any of the DC‐SIGNR mutants expressing cells. Virus replicon particles (VRPs), replication‐incompetent viral particles containing necessary structural proteins for infection and a viral plasmid including a GFP reporter are used to safely and conveniently study viral entry. Entry assays using WNV (NY99) VRPs as well as a variant of WNV (NY99) which contains the beta‐lactamase enzyme show significant entry into DC‐SIGNR expressing cell lines, but not in controls that do not express DC‐SIGNR. Additionally, we show that WNV VRPs do not enter DC‐SIGNR expressing cells that lack the CRD or the C‐terminal half of the CRD suggesting that the C‐terminal half of the CRD is required for successful entry of WNV via DC‐SIGNR. In the future, WNV particle entry into DC‐SIGNR mutation (150(STOP), S139A, S108A) expressing cells will be tested.Support or Funding InformationWinona State University student research and travel grants, Winona State University Foundation Special Projects