The ER membrane protein complex is important for the biogenesis of flavivirus polyproteins

Abstract

Flaviviruses such as dengue (DENV), Zika (ZIKV) or West Nile virus (WNV) translate their genome as a single multi-pass transmembrane (TM) protein at the endoplasmic reticulum (ER) membrane. Several genetic knockout (KO) screens identified the ER membrane protein complex (EMC) as a critical host component for flavivirus infection. The EMC facilitates accurate insertion, topology and/or stabilization of specific cellular TM proteins including a subset of tail-anchored proteins and G protein-coupled receptors. Here, we show that deletion of EMC in human cell lines decreased infection with DENV, ZIKV and WNV by 100 to 10,000-fold. Using replicon and immunoblotting studies in EMC KO cells, we demonstrated that the EMC was essential for viral protein expression. Ribosome Profiling of DENV-infected wild-type and EMC KO cells revealed no drastic differences in translation efficiency. Instead, absence of EMC led to a large fraction of expressed viral proteins being targeted to the proteasome post-translationally. We detected a decrease in stability in NS4A-NS4B of the viral polyprotein, a region rich in transmembrane domains. Additionally, we identified non-synonymous point mutations in NS4A and NS4B by performing iterative passaging of DENV on EMC KO cells. Adaptive mutations rescued both viral replication and stable expression of the NS4A-NS4B polyprotein segment in EMC KO cells. Lastly, we showed a physical interaction between the EMC and DENV NS4B protein post-cleavage and rapid degradation of processed NS4B in the absence of EMC. Together, our results suggest that the EMC engages with DENV polyproteins to ensure proper biogenesis of the NS4A-NS4B region, and provide further evidence for the cellular function of the EMC in the stable expression of TM proteins.