Abstract
Hundreds of cellular host factors are required to support dengue virus infection, but their identity and roles are incompletely characterized. Here, we identify human host dependency factors required for efficient dengue virus-2 (DENV2) infection of human cells. We focused on two, TTC35 and TMEM111, which we previously demonstrated to be required for yellow fever virus (YFV) infection and others subsequently showed were also required by other flaviviruses. These proteins are components of the human endoplasmic reticulum membrane protein complex (EMC), which has roles in ER-associated protein biogenesis and lipid metabolism. We report that DENV, YFV and Zika virus (ZIKV) infections were strikingly inhibited, while West Nile virus infection was unchanged, in cells that lack EMC subunit 4. Furthermore, targeted depletion of EMC subunits in live mosquitoes significantly reduced DENV2 propagation in vivo. Using a novel uncoating assay, which measures interactions between host RNA-binding proteins and incoming viral RNA, we show that EMC is required at or prior to virus uncoating. Importantly, we uncovered a second and important role for the EMC. The complex is required for viral protein accumulation in a cell line harboring a ZIKV replicon, indicating that EMC participates in the complex process of viral protein biogenesis.
Highlights
The Flavivirus genus includes three arboviruses transmitted by Aedes species of mosquitoes that are important human pathogens: dengue (DENV 1–4), yellow fever (YFV) and Zika (ZIKV) viruses
HuH-7 cells were reverse-transfected with small interfering RNAs (siRNA), incubated for 52 hours and inoculated with dengue virus-2 (DENV2) at low multiplicity of infection (MOI)
We screened a library of siRNA pools for the ability to reduce DENV2 infection of a human cell line at a time-point that permitted at least one complete viral lifecycle
Summary
The Flavivirus genus includes three arboviruses transmitted by Aedes species of mosquitoes that are important human pathogens: dengue (DENV 1–4), yellow fever (YFV) and Zika (ZIKV) viruses. Among the proviral factors identified by these screens were subunits of the ER membrane protein complex (EMC). Three more teams identified the EMC as an important proviral factor for DENV, YFV and ZIKV infection[18,19,20]. The EMC was shown to direct the insertion of transmembrane domains consistent with a role in protein biogenesis[25,26] These studies suggest that the diverse phenotypes observed after experimental modulation of the EMC are due to altered accumulation, maturation and/or folding of proteins that transit through or are associated with the ER
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