The ER membrane protein complex is required to ensure correct topology and stable expression of flavivirus polyproteins

Abstract

Flaviviruses translate their genomes as multi-pass transmembrane proteins at the endoplasmic reticulum (ER) membrane. Here, we show that the ER membrane protein complex (EMC) is indispensable for the expression of viral polyproteins. We demonstrated that EMC was essential for accurate folding and post-translational stability rather than translation efficiency. Specifically, we revealed degradation of NS4A-NS4B, a region rich in transmembrane domains, in absence of EMC. Orthogonally, by serial passaging of virus on EMC-deficient cells, we identified two non-synonymous point mutations in NS4A and NS4B, which rescued viral replication. Finally, we showed a physical interaction between EMC and viral NS4B and that the NS4A-4B region adopts an aberrant topology in the absence of the EMC leading to degradation. Together, our data highlight how flaviviruses hijack the EMC for transmembrane protein biogenesis to achieve optimal expression of their polyproteins, which reinforces a role for the EMC in stabilizing challenging transmembrane proteins during synthesis.

Squaring the EMC – how promoting membrane protein biogenesis impacts cellular functions and organismal homeostasis

“…Abnormalities appearing at early stages of DENV replication have been described in EMC-deficient cell lines. Although these defects were first attributed to impaired virus entry (Savidis et al, 2016), more recent reports demonstrate aberrant biogenesis and proteasome-mediated degradation of the non-structural polytopic DENV proteins NS4A and NS4Ban observation consistent with the insertase function of the EMC (Lin et al, 2019;Ngo et al, 2019). However, given the complexity of viral entry and replication, it might be premature to rule out a role for the EMC in the accurate biogenesis of cell-surface proteins that mediate viral entry.…”

“…Even though other EMC subunits were not investigated, the position of EMC2 and EMC3 as 'core' subunits required for EMC assembly can reasonably be extrapolated to reflect involvement of the mature EMC. Other studies that implicate different EMC subunits in replication of DENV, ZV and YFV (Le Sommer et al, 2012;Marceau et al, 2016;Ngo et al, 2019;Savidis et al, 2016) may be viewed in the same manner.…”

Sphingolipid Biosynthesis Inhibition As A Host Strategy Against Diverse Pathogens

“…Though understudied, this would seem to modulate the membranes of inclusion bodies, which are used by many pathogens to evade their destruction (67). Also, some pathogens need the ER to make more of themselves (68). Considering CADs associate with membranes (69) potentially in the ER, this could explain why CADs are effective against these types of pathogens; 3) CADs potentiate the effects of many anti-pathogen drugs.…”

Dengue and Zika virus infection are impaired by small molecule ER proteostasis regulator 147 in an ATF6-independent manner

“…The proteostasis network is comprised of chaperones, co-chaperones and other protein quality control factors that control protein folding, assembly, post-translational modification, trafficking, and degradation. Genetic screens have identified many proteostasis factors as essential for DENV replication, including several components of the oligosaccharyl transferase (OST) complex, as well as the ER membrane protein complex (Lin et al, 2019;Marceau et al, 2016;Ngo et al, 2019;Savidis et al, 2016). Aside from promoting biogenesis of viral proteins, these components also have additional roles in organizing viral replication centers at the ER membrane (Marceau et al, 2016;Ngo et al, 2019;Rothan and Kumar, 2019).…”

“…Genetic screens have identified many proteostasis factors as essential for DENV replication, including several components of the oligosaccharyl transferase (OST) complex, as well as the ER membrane protein complex (Lin et al, 2019;Marceau et al, 2016;Ngo et al, 2019;Savidis et al, 2016). Aside from promoting biogenesis of viral proteins, these components also have additional roles in organizing viral replication centers at the ER membrane (Marceau et al, 2016;Ngo et al, 2019;Rothan and Kumar, 2019). Additionally, diverse cellular chaperone and co-chaperone systems are required for all stages of the viral life cycle, including virus entry and disassembly, folding of individual viral proteins, as well as assembly and egress of new virions (Fischl and Bartenschlager, 2011;Heaton et al, 2016;Taguwa et al, 2019).…”