Characterization of Flavivirus Host-Cell Interactions

[摘要] Dengue virus (DENV) is the most prevalent arbovirus globally, infecting an estimated 400 million people each year. DENV belongs to the Flaviviridae family of viruses, and is closely related to Zika virus (ZIKV), West Nile virus (WNV), and yellow fever virus (YFV). To date, there are no approved antivirals for the treatment of dengue, in part due to the poor understanding of DENV replication and interactions with its host cell.We performed a genome-wide CRISPR-Cas9 knockout screen in human hepatoma Huh-7 cells to identify host dependency factors for DENV and reveal potential therapeutic targets. We found that two major protein complexes are necessary for DENV replication, the oligosaccharyltransferase (OST) complex and the endoplasmic reticulum membrane protein complex (EMC).We show that two mammalian OST complexes that incorporate either STT3A or STT3B, the catalytic subunits responsible for N-linked glycosylation, are necessary for DENV replication. However, the enzymatic activity of these subunits is not essential for replication. We found that expression of MAGT1 or its homolog TUSC3 is dependent on expression of STT3B, and that cells lacking MAGT1/TUSC3 are protected from DENV infection. Mutagenesis studies of MAGT1 revealed that a conserved CxxC motif shared among oxidoreductases is required for efficient DENV infection, suggesting that MAGT1 acts as an oxidoreductase to support DENV infection. We also provide evidence that the STT3B/MAGT1 OST complex interacts with NS4B and may be important for its biogenesis and folding.We also show that the EMC, which contains ten subunits EMC1 through EMC10, is also necessary for DENV replication. The EMC has been proposed to function as a chaperone and insertase for transmembrane proteins. We found that cells lacking the EMC support significantly reduced levels of flavivirus NS4A and NS4B, both of which are multi-pass transmembrane proteins that contain domains with marginal hydrophobicity. Importantly, we demonstrate an interaction between the EMC and NS4B, but not NS1 or NS2B, during viral replication. We show that deletion or mutagenesis of two marginally hydrophobic domains of NS4B render its expression no longer dependent on the EMC. Taken together, our data suggest that the EMC promotes the expression of NS4B via an interaction with marginally hydrophobic domains, a feature that may apply to other cellular EMC substrates as well.In a tangentially related project, we found that exogenous expression of a hypoglycosylated N207A mutant of DENV NS1 blocked DENV infection in a dominant-negative fashion. Similarly, we found that expression of an N130A mutant of ZIKV NS1 inhibited both DENV and ZIKV infection. Immunofluorescence microscopy revealed that these NS1 mutants exhibited a filamentous phenotype upon flavivirus infection, rather than the punctate distribution of wild-type NS1. We further designed NS1 mutants that were able to inhibit DENV, YFV, and ZIKV infection, and we speculate that they may also prevent infection by other flaviviruses. While the mechanism of inhibition is still unclear, these mutants may be useful tools to study the role of NS1 in flavivirus replication, and potentially in preventing flavivirus transmission. Our novel findings demonstrate cross-flavivirus dominant-negative inhibition of replication by NS1 mutants.In summary, these findings provide a deeper understanding of flavivirus biology and help elucidate the roles of these cellular complexes in viral replication.

[发布机构] University of Michigan

[效力级别] host factor

[关键词] flavivirus;host factor;whole-genome screen;dengue;endoplasmic reticulum;Microbiology and Immunology;Molecular;Cellular and Developmental Biology;Health Sciences;Microbiology & Immunology