Abstract
ABSTRACTDengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year. We employed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen to identify host dependency factors required for DENV propagation and identified the oligosaccharyltransferase (OST) complex as an essential host factor for DENV infection. Mammalian cells express two OSTs containing either STT3A or STT3B. We found that the canonical catalytic function of the OSTs as oligosaccharyltransferases is not necessary for DENV infection, as cells expressing catalytically inactive STT3A or STT3B are able to support DENV propagation. However, the OST subunit MAGT1, which associates with STT3B, is also required for DENV propagation. MAGT1 expression requires STT3B, and a catalytically inactive STT3B also rescues MAGT1 expression, supporting the hypothesis that STT3B serves to stabilize MAGT1 in the context of DENV infection. We found that the oxidoreductase CXXC active site motif of MAGT1 was necessary for DENV propagation, as cells expressing an AXXA MAGT1 mutant were unable to support DENV infection. Interestingly, cells expressing single-cysteine CXXA or AXXC mutants of MAGT1 were able to support DENV propagation. Utilizing the engineered peroxidase APEX2, we demonstrate the close proximity between MAGT1 and NS1 or NS4B during DENV infection. These results reveal that the oxidoreductase activity of the STT3B-containing OST is necessary for DENV infection, which may guide the development of antiviral agents targeting DENV.
Highlights
Dengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year
We provide evidence that DENV NS4B interacts with STT3B/MAGT1 OST complexes based on NS4B glycosylation and proximity labeling experiments and that NS4B synthesis is reduced in STT3B knockout cells
We assessed luciferase activity and saw a significant and marked decrease in luciferase activity in STT3A, STT3B, and MAGT1 knockout cells compared to control cells transduced with a control green fluorescent protein (GFP)-targeting Single-guide RNAs (sgRNAs), demonstrating that protection from DENV-mediated cell death in these knockout cells is mediated by inhibition of DENV infection rather than by a block of cell death signaling pathways (Fig. 2A)
Summary
Dengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year. We found that the canonical catalytic function of the OSTs as oligosaccharyltransferases is not necessary for DENV infection, as cells expressing catalytically inactive STT3A or STT3B are able to support DENV propagation. Utilizing the engineered peroxidase APEX2, we demonstrate the close proximity between MAGT1 and NS1 or NS4B during DENV infection These results reveal that the oxidoreductase activity of the STT3B-containing OST is necessary for DENV infection, which may guide the development of antiviral agents targeting DENV. We show that MAGT1 associates with DENV NS1 and NS4B during viral infection, suggesting that these nonstructural proteins may be targets of MAGT1 oxidoreductase activity These results provide insight into the cell biology of DENV infection, which may guide the development of antivirals against DENV. While STT3A is likely responsible for cotranslational glycosylation of nascent proteins entering the ER lumen, STT3B is responsible for posttranslational glycosylation of proteins with sequons skipped by STT3A [7]
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