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

Abstract

11Flaviviruses translate their genomes as multi-pass transmembrane proteins at the endoplasmic 12 reticulum (ER) membrane. Here, we show that the ER membrane protein complex (EMC) is 13 indispensable for the expression of viral polyproteins. We demonstrated that EMC was essential 14 for accurate folding and post-translational stability rather than translation efficiency. Specifically, 15we revealed degradation of NS4A-NS4B, a region rich in transmembrane domains, in absence of 16 EMC. Orthogonally, by serial passaging of virus on EMC-deficient cells, we identified two non-17 synonymous point mutations in NS4A and NS4B, which rescued viral replication. Finally, we 18 showed a physical interaction between EMC and viral NS4B and that the NS4A-4B region adopts 19 an aberrant topology in the absence of the EMC leading to degradation. Together, our data 20 highlight how flaviviruses hijack the EMC for transmembrane protein biogenesis to achieve 21 optimal expression of their polyproteins, which reinforces a role for the EMC in stabilizing 22 challenging transmembrane proteins during synthesis.Subsequently, the polyprotein is cleaved by the viral NS2B-NS3 protease and the cellular signal 27 peptidase into the individual structural (C, prM, E) and non-structural (NS1-5) proteins required 28 for replication and assembly into new virions (Neufeldt et al., 2018). Similar to cellular 29 transmembrane proteins, the viral polyprotein relies on the host cell machinery for targeting to the 30 ER, translocation across the membrane and insertion of transmembrane domains (TMDs) into 31 the lipid bilayer during its translation. These processes are facilitated by the signal recognition 32 particle (SRP), the translocon-associated protein (TRAP) complex (also known as signal-33 sequence receptor complex), the Sec61 translocon and the signal peptidase complex. These cell 34 components were all identified as essential host factors for flavivirus infection in genetic screens 35 underscoring their importance for virus replication (Krishnan et al., 2008; Marceau et al., 2016; 36 Sessions et al., 2009; Zhang et al., 2016). For most transmembrane proteins, recognition of a 37 hydrophobic TMD or signal sequence by the SRP followed by transfer to the Sec61 translocation 38 channel are sufficient for membrane targeting and accurate topogenesis (Shao and Hegde, 2011). 39Recently, it was shown that the ER membrane protein complex (EMC) plays a role in the insertion 40 and/or stabilization of certain transmembrane proteins (Chitwood et al., 2018; Guna et al., 2018; 41 Shurtleff et al., 2018). Intriguingly, several genome-wide CRISPR knockout (KO) screens for 42 flavivirus dependency factors showed strong enrichment of the EMC (Ma et al., 2015; Marceau 43 et al., 2016; Savidis et al., 2016; Zhang et al., 2016). However, its role in the virus life cycle is not 44 yet understood. 45The EMC was originally discovered in a genetic screen for yeast mutants modulating the unfolded 46 protein response (UPR) (Jonikas et al., 2009). Genetic interac...

Membrane protein biogenesis at the ER: the highways and byways

“…The role of the EMC during the co-translational biogenesis of multi-span TMPs is not strictly limited to protein clients whose first TMD is type III-like (Fig. 4A), additionally extending to the stabilisation and/or insertion of downstream TMDs [99][100][101]112,113] irrespective of the orientation of the first TMD [89,90]. The decisive feature for the EMC-dependence of these multi-span TMPs is reduced hydrophobicity and/or increased polarity or charge, as further evidenced by the ability to create artificial EMC dependency by introducing polar/charged residues into a TMD [90,99,100].…”

WNK1 is an assembly factor for the human ER membrane protein complex

“…The strategies reported here lay out a roadmap for their identification and validation. Characterization of these assembly factors will reveal how the cell ensures the stoichiometric assembly of intricate molecular machines in the crowded cellular milieu, and provide insight into how these processes may fail leading to disease (Harper and Bennett, 2016;Oromendia and Amon, 2014). (A) FLAG-tagged WNK1 and its truncations were tested for binding to EMC2 (see methods).…”

“…Detailed biochemical reconstitution experiments have shown that the EMC can both cotranslationally insert TMDs in the Nexo-topology, and post-translationally insert tailanchored proteins with TMDs of moderate hydrophobicity (Chitwood et al, 2018;Guna et al, 2018). Proteomic interrogation of EMC function also suggests it plays a role in stabilizing multi-pass membrane proteins, including many viral coat proteins (Barrows et al, 2019;Chitwood and Hegde, 2019;Ngo et al, 2019;Shurtleff et al, 2018;Tian et al, 2019).…”

Regulated assembly of the ER membrane protein complex

“…The strategies reported here lay out a roadmap for their identification and validation. Characterization of these assembly factors will reveal how the cell ensures the stoichiometric assembly of intricate molecular machines in the crowded cellular milieu, and provide insight into how these processes may fail leading to disease (Harper and Bennett, 2016;Oromendia and Amon, 2014).…”