Inhibition of Stress Granule Formation by Middle East Respiratory Syndrome Coronavirus 4a Accessory Protein Facilitates Viral Translation, Leading to Efficient Virus Replication

Abstract

Stress granule (SG) formation is generally triggered as a result of stress-induced translation arrest. The impact of SG formation on virus replication varies among different viruses, and the significance of SGs in coronavirus (CoV) replication is largely unknown. The present study examined the biological role of SGs in Middle East respiratory syndrome (MERS)-CoV replication. The MERS-CoV 4a accessory protein is known to inhibit SG formation in cells in which it was expressed by binding to double-stranded RNAs and inhibiting protein kinase R (PKR)-mediated phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Replication of MERS-CoV lacking the genes for 4a and 4b (MERS-CoV-Δp4), but not MERS-CoV, induced SG accumulation in MERS-CoV-susceptible HeLa/CD26 cells, while replication of both viruses failed to induce SGs in Vero cells, demonstrating cell type-specific differences in MERS-CoV-Δp4-induced SG formation. MERS-CoV-Δp4 replicated less efficiently than MERS-CoV in HeLa/CD26 cells, and inhibition of SG formation by small interfering RNA-mediated depletion of the SG components promoted MERS-CoV-Δp4 replication, demonstrating that SG formation was detrimental for MERS-CoV replication. Inefficient MERS-CoV-Δp4 replication was not due to either the induction of type I and type III interferons or the accumulation of viral mRNAs in the SGs. Rather, it was due to the inefficient translation of viral proteins, which was caused by high levels of PKR-mediated eIF2α phosphorylation and likely by the confinement of various factors that are required for translation in the SGs. Finally, we established that deletion of the 4a gene alone was sufficient for inducing SGs in infected cells. Our study revealed that 4a-mediated inhibition of SG formation facilitates viral translation, leading to efficient MERS-CoV replication. Middle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory failure with a high case fatality rate in patients, yet effective antivirals and vaccines are currently not available. Stress granule (SG) formation is one of the cellular stress responses to virus infection and is generally triggered as a result of stress-induced translation arrest. SGs can be beneficial or detrimental for virus replication, and the biological role of SGs in CoV infection is unclear. The present study showed that the MERS-CoV 4a accessory protein, which was reported to block SG formation in cells in which it was expressed, inhibited SG formation in infected cells. Our data suggest that 4a-mediated inhibition of SG formation facilitates the translation of viral mRNAs, resulting in efficient virus replication. To our knowledge, this report is the first to show the biological significance of SG in CoV replication and provides insight into the interplay between MERS-CoV and antiviral stress responses.

Innate Immune Evasion by Human Respiratory RNA Viruses

“…Indeed, CoV nsp1 with its host-shut-off activities (see above) is a likely candidate viral protein that could play a role. A later report by Nakagawa et al [101] however showed that the ORF4 MERS-CoV mutant virus did induce stress granules in another cell line (Hela/ CD26), and also a virus mutant in which 4a alone was removed was not able to suppress SG formation in these cells. This suggests that the activity of 4a, and possibly other stress granule-inhibiting MERS-CoV proteins, may differ per cell line, or that cell lines differ in the activity of their antiviral pathways.…”

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

“…4b i ). Manipulation of SG and related RNA biology is common among coronaviridae 34 – 36 and stress granule formation is thought to be a primarily antiviral response. The promotion of G3BP aggregation by eIF4A inhibitors 28 , 37 may partially explain their antiviral activity (see below).…”

Typical Stress Granule Proteins Interact with the 3′ Untranslated Region of Enterovirus D68 To Inhibit Viral Replication

“…classical being the protein kinase R (PKR)-triggered pathway. PKR regulates the transcription and translation of cells through its phosphorylation initiation factor eIF2α, which plays an important role in cell growth (14). After viral infection, PKR binds to viral double-stranded RNA (dsRNA), which activates the autophosphorylation of the kinase.…”