Assessment of tick-borne flavivirus host factors through genome-scale screens

Powassan virus (POWV) is the only tick-borne flavivirus (TBFV) known to circulate in North America. Although there are relatively few documented cases of POWV disease, investigations into POWV are justified due to the increasing incidence of infection and significant case fatality rate associated with this virus. To better describe the molecular biology of the POWV replication cycle in mammalian cells, we performed genome-scale screens to uncover host factors required for viral replication. Putative proviral host factors were identified by infecting pools of cells containing knockout mutations in non-essential genes with POWV, followed by analysis of cells resistant to virus-induced cell death. Many endoplasmic reticulum membrane complex proteins were revealed in these screens, suggesting that TBFVs share some common host cell hijacking strategies with mosquito-borne flaviviruses. Candidate proteins that function in cell-matrix adhesion, glycosylation, or RNA binding in uninfected cells were the focus of validation studies. We used single-gene knockout cell lines to investigate possible proviral roles for specific proteins in the replication cycle of either POWV or Langat virus (LGTV), a non-pathogenic and model TBFV. Proteins identified in the POWV screens were not necessarily critical for LGTV replication. These results suggest that our screens were able to identify both pan-flaviviral, as well as POWV-specific, host gene products exploited during virus replication. Ongoing work is focused on characterizing distinct host-cell requirements of diverse flaviviruses. This genetic assessment of POWV replication factors, in combination with ensuing mechanistic studies, will provide possible avenues for the development of host-targeting countermeasures.