Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens

Summary

The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide ¹ . No approved antivirals are available to date and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates ² . Also hepatitis C virus (HCV) remains a major medical problem with 160 million chronically infected patients and only expensive treatment on the market ³ . Despite distinct differences in pathogenesis and mode of transmission, the two viruses share common replication strategies ⁴ . A detailed understanding of the host functions that determine viral infection is lacking. Here, we utilized a pooled CRISPR genetic screening strategy ^{5, 6} to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified ER-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER associated degradation. Dengue virus replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as critical step requiring the OST complex. Moreover, we showed that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects ⁷ . In contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA binding proteins and enzymes involved in metabolism. We discovered an unexpected link between intracellular FAD levels and HCV replication. This study shows remarkable divergence in host dependency factors between DENV and HCV and illuminates novel host targets for antiviral therapy.