Endoplasmic reticulum: a focal point of Zika virus infection

Mohd Ropidi, Muhammad Izzuddin; Khazali, Ahmad Suhail; Nor Rashid, Nurshamimi; Yusof, Rohana

ZIKV has been reported to be dependent on several ER proteins including ER-associated signal peptidase complex (SPC) proteins, ER translocon, and ER membrane complex (EMC) proteins. SPC, especially SPC subunit 1 (SPCS1), is crucial for ZIKV pathogenesis as knocking out SPCS1 in 293 T cells significantly reduced ZIKV infection and drastically impaired the production of infectious ZIKV particles [24] . A study using pooled CRISPR/Cas9 cell survival enrichment assay identified ZIKV strongly depends on ER membrane complex (EMC) during early-stage ZIKV replication [25] . EMC is a highly conserved oligomeric complex residing on the ER membrane and is crucial for transmembrane protein folding and lipid trafficking [25] . The dependency on six EMC subunits (EMC1-EMC6) was verified with siRNA assays, where depletion of these proteins significantly impaired the replication of several ZIKV strains [25] . Another study reported that ZIKV NS4B physically interact with EMC1 subunit and depletion of this subunit markedly reduced the level of ZIKV NS4A and NS4B protein [26] , indicating that EMC proteins are required for viral protein biogenesis. Further investigation using DENV NS4B identified two marginally hydrophobic domains at the N-terminal of NS4B to be crucial for NS4B dependence on EMC [26] . Since ZIKV NS4B shares moderate sequence identity, high sequence similarity, and similar topology with other Flaviviruses [27] , it is plausible that the two weak hydrophobic domains of ZIKV NS4B are the specific determinants for ZIKV dependency on EMC proteins. In addition to its direct interaction with viral proteins, EMC proteins also associate with ER Sec61 translocon and oligosaccharyltransferase (OST) complex proteins, both of which are also important for ZIKV infection [24, 25] . Sec61 is a major component of the ER translocon that facilitates the entry of nascent polypeptides into the ER lumen for protein processing. ZIKV dependency on Sec61 translocon was validated in a separate study wherein myolactone treatment, a Sec61α inhibitor, dramatically reduced ZIKV expression [28] . ZIKV replication was restored in cells expressing mutant Sec61α that conferred resistance against myolactone inhibition [28] . ZIKV dependency on OST complex, an integral part of the translocon consisting of eight ER-transmembrane protein subunits that catalyzes co-translational N-glycosylation, is based on EMC1, EMC2, EMC4 and EMC5 interaction with OST complex subunits namely STT3A/B, RPN1/2, and DDOST [25] . Additionally, ZIKV proteins including NS4B have been reported to directly interact with these OST subunits [29] . ZIKV dependency on OST complex is corroborated by Marceau et al. that reported a significant abrogation of ZIKV RNA replication in STT3A knockout cells [30] . Importantly, treatment with NGI-1, a small molecule OST complex inhibitor, significantly reduced ZIKV RNA replication with an EC 50 value of 2.2 μM [31] . The inhibitory activity of this non-toxic molecule (CC 50 = 34.9 μM) is independent of N-linked glycosylation activity of the OST complex as ZIKV replication was drastically impaired in HAP1 cells containing wild-type or catalyticinactive STT3A [31] . The specific mechanism of ZIKV dependency on STT3A is unclear, but immunoprecipitation and electron microscopy studies showed physical interactions between DENV proteins and STT3A/B, forming viral RNA replication complexes that reside within the ER in close proximity to DENV2-vesicle packets [30] . Si