Genome-Wide Screening Uncovers the Significance of N-Sulfation of Heparan Sulfate as a Host Cell Factor for Chikungunya Virus Infection

The molecular mechanisms underlying chikungunya virus (CHIKV) infection are poorly characterized. In this study, we analyzed the host factors involved in CHIKV infection using genome-wide screening. Human haploid HAP1 cells, into which an exon-trapping vector was introduced, were challenged with a vesicular stomatitis virus pseudotype bearing the CHIKV E3 to E1 envelope proteins. Analysis of genes enriched in the cells resistant to the pseudotyped virus infection unveiled a critical role of N-sulfation of heparan sulfate (HS) for the infectivity of the clinically isolated CHIKV Thai#16856 strain to HAP1 cells. Knockout of NDST1 that catalyzes N-sulfation of HS greatly decreased the binding and infectivity of CHIKV Thai#16856 strain but not infectivity of Japanese encephalitis virus (JEV) and yellow fever virus (YFV). While glycosaminoglycans were commonly required for the efficient infectivity of CHIKV, JEV, and YFV, as shown by using B3GAT3 knockout cells, the tropism for N-sulfate was specific to CHIKV. Expression of chondroitin sulfate (CS) in NDST1-knockout HAP1 cells did not restore the binding of CHIKV Thai#16856 strain and the infectivity of its pseudotype but restored the infectivity of authentic CHIKV Thai#16856, suggesting that CS functions at later steps after CHIKV binding. Among the genes enriched in this screening, we found that TM9SF2 is critical for N-sulfation of HS and therefore for CHIKV infection because it is involved in the proper localization and stability of NDST1. Determination of the significance of and the relevant proteins to N-sulfation of HS may contribute to understanding mechanisms of CHIKV propagation, cell tropism, and pathogenesis.IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Chikungunya virus (CHIKV) utilizes host glycosaminoglycans to bind efficiently to its target cells. However, the substructure in glycosaminoglycans required for CHIKV infection have not been characterized. Here, we unveil that N-sulfate in heparan sulfate is essential for the efficient infection of a clinical CHIKV strain to HAP1 cells and that chondroitin sulfate does not help the CHIKV binding but does play roles at the later steps in HAP1 cells. We show, by comparing previous reports using Chinese hamster ovary cells, along with another observation that enhanced infectivity of CHIKV bearing Arg82 in envelope E2 does not depend on glycosaminoglycans in HAP1 cells, that the infection manner of CHIKV varies among host cells. We also show that TM9SF2 is required for CHIKV infection to HAP1 cells because it is involved in the N-sulfation of heparan sulfate through ensuring NDST1 activity.