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

doi:10.1128/JVI.00902-18

. 2018 Sep 26;92(20):e00902-18.

doi: 10.1128/JVI.00902-18. Print 2018 Oct 15.

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

Keisuke Nakagawa¹, Krishna Narayanan¹, Masami Wada¹, Shinji Makino^{2

3

4

5

6}

Affiliations

¹ Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas, USA.
² Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas, USA shmakino@utmb.edu.
³ Center for Biodefense and Emerging Infectious Diseases, The University of Texas Medical Branch, Galveston, Texas, USA.
⁴ UTMB Center for Tropical Diseases, The University of Texas Medical Branch, Galveston, Texas, USA.
⁵ Sealy Center for Vaccine Development, The University of Texas Medical Branch, Galveston, Texas, USA.
⁶ The Institute for Human Infections and Immunity, The University of Texas Medical Branch, Galveston, Texas, USA.

PMID: 30068649
PMCID: PMC6158436
DOI: 10.1128/JVI.00902-18

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

Keisuke Nakagawa et al. J Virol. 2018.

. 2018 Sep 26;92(20):e00902-18.

doi: 10.1128/JVI.00902-18. Print 2018 Oct 15.

Authors

Keisuke Nakagawa¹, Krishna Narayanan¹, Masami Wada¹, Shinji Makino^{2

3

4

5

6}

Affiliations

¹ Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas, USA.
² Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas, USA shmakino@utmb.edu.
³ Center for Biodefense and Emerging Infectious Diseases, The University of Texas Medical Branch, Galveston, Texas, USA.
⁴ UTMB Center for Tropical Diseases, The University of Texas Medical Branch, Galveston, Texas, USA.
⁵ Sealy Center for Vaccine Development, The University of Texas Medical Branch, Galveston, Texas, USA.
⁶ The Institute for Human Infections and Immunity, The University of Texas Medical Branch, Galveston, Texas, USA.

PMID: 30068649
PMCID: PMC6158436
DOI: 10.1128/JVI.00902-18

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.IMPORTANCE 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.

Keywords: MERS coronavirus; accessory protein; stress granules.

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Figures

**FIG 1**
Induction of SGs in MERS-CoV-Δp4-infected cells. (A) Schematic diagrams of genomes of MERS-CoV-WT (WT) and MERS-CoV-Δp4 (Δp4). The 5′ and 3′ untranslated regions (bars) and viral open reading frames (boxes) are not drawn according to their lengths. (B to D) HeLa/CD26 cells were infected with MERS-CoV-WT or MERS-CoV-Δp4 at an MOI of 3. At 9 h p.i., the infected cells were fixed with 4% formaldehyde and stained for TIA-1 (B), G3BP (C), or eIF4A (D), shown in green, together with the MERS-CoV N protein, shown in red. Right panels show enlarged images of the regions shown in white boxes in the merged image panels. (E) HeLa/CD26 cells were infected with MERS-CoV-Δp4 at an MOI of 3. At 8 h p.i., the infected cells were treated with 100 μg/ml of CHX or DMSO for 1 h. After fixing, the cells were stained for TIA-1 (green) and N protein (red). (F) HeLa/CD26 cells (left) or Vero cells (right) were infected with MERS-CoV-WT or MERS-CoV-Δp4 at an MOI of 3. At the indicated times p.i., the cells were fixed and stained for TIA-1 and MERS-CoV N protein. Among the N protein-positive cells, those carrying at least a single TIA-1-postive granule were counted as SG positive, while SG-negative calls lacked any TIA-1-postive granules. The percentage of SG-positive cells was calculated by counting the number of SG-positive cells out of 25 to 34 N protein-positive cells per field. A total of 20 fields were counted for each sample. Each bar represents the mean (±standard deviation). hpi, hours postinfection. (G, H) Vero cells (G) or 293/CD26 cells (H) were mock infected (Mock) or infected with MERS-CoV-WT or MERS-CoV-Δp4 at an MOI of 3. At 24 h p.i., the infected cells were fixed with 4% formaldehyde and were stained for G3BP (green), together with MERS-CoV N protein (red).

**FIG 2**
Growth kinetics of MERS-CoV-WT and MERS-CoV-Δp4 in HeLa/CD26 and Vero cells. HeLa/CD26 cells (A) or Vero cells (B) were infected with MERS-CoV-WT (WT) or MERS-CoV-Δp4 (Δp4) at an MOI of 0.01 (left) or 3 (right). Culture supernatants were collected at the indicated times p.i., and virus titers were determined by plaque assay. Each dot represents the mean virus titer (±standard deviation) for three wells. Asterisks represent statistically significant differences in virus titers (P < 0.05).

**FIG 3**
Phosphorylation statuses of PKR and eIF2α and efficiencies of host and viral protein synthesis in infected cells. HeLa/CD26 cells or Vero cells were either mock infected (Mock) or infected with MERS-CoV-WT (WT) or MERS-CoV-Δp4 (Δp4) at an MOI of 3. (A and B) Whole-cell lysates were prepared at 9 h p.i. for HeLa/CD26 cells (A) and 24 h p.i. for Vero cells (B) and subjected to Western blot analysis to detect PKR, phosphorylated PKR (p-PKR), eIF2α, phosphorylated eIF2α (p-eIF2α), the MERS-CoV 4a protein, the MERS-CoV 4b protein, and tubulin. (C and D) HeLa/CD26 cells (C) or Vero cells (D) were radiolabeled for 1 h with 100 μCi of Tran³⁵S-label, and cell lysates were prepared at the indicated times p.i. Cell lysates were subjected to SDS-PAGE analysis, followed by autoradiography (top) and colloid Coomassie brilliant blue staining (bottom). Arrows, virus-specific proteins.

**FIG 4**
Accumulation of viral mRNAs and proteins in infected HeLa/CD26 cells. HeLa/CD26 cells were mock infected (Mock) or infected with MERS-CoV-WT (WT) or MERS-CoV-Δp4 (Δp4) at an MOI of 3. At the indicated times p.i., total intracellular RNAs and proteins were prepared. (A) Northern blot analysis of viral mRNAs using a riboprobe that binds to all viral mRNAs. The numbers 1 to 8 represent viral mRNA species. The 28S and 18S rRNAs were detected by ethidium bromide staining (rRNA). (B and C) The amounts of mRNA 1 (B) and subgenomic mRNA 8 (C) were quantified by qRT-PCR. The expression levels of mRNAs were normalized to the levels of 18S rRNA. Each bar represents the mean (±standard deviation) for three independent samples. ns, not significant (P > 0.05). (D) Western blot analysis of intracellular accumulation of the MERS-CoV S, M, and N proteins and tubulin.

**FIG 5**
Expression of host mRNAs involved in innate immune responses in infected HeLa/CD26 cells. HeLa/CD26 cells were mock infected (Mock) or infected with MERS-CoV-WT (WT) or MERS-CoV-Δp4 (Δp4) at an MOI of 3. SeV was used as a positive control. Total intracellular RNAs were extracted at the indicated times p.i., and the amounts of endogenous IFN-β (A), IFN-λ1 (B), OAS (C), and ISG56 (D) mRNAs were determined by qRT-PCR analysis. The expression levels of the genes were normalized to the expression levels of 18S rRNA. Each bar represents the mean (±standard deviation) for three independent samples. ns, not significant (P > 0.05).

**FIG 6**
Subcellular localization of viral mRNAs and an SG marker, eIF4A, in MERS-CoV-Δp4-infected cells. HeLa/CD26 cells were mock infected (top) or infected with MERS-CoV-WT (middle) or MERS-CoV-Δp4 (bottom) at an MOI of 3. At 9 h p.i., viral mRNAs were detected by riboprobe binding to all viral mRNAs (green), and SGs were detected by anti-eIF4A antibody (red). Samples were subjected to fluorescence microscopic examination.

**FIG 7**
SG formation interferes with efficient MERS-CoV replication. HeLa/CD26 cells were transfected with control siRNA (siCont) or siRNA targeting TIA-1 (siTIA-1) or G3BP1/2 (siG3BP1/2). (A) At 24 h after siRNA transfection, cells were infected with MERS-CoV-Δp4 at an MOI of 3. At 9 h p.i., the cells were fixed and stained for G3BP or TIA-1, together with N protein. The numbers of SGs in each N protein-positive cell were counted, and the average numbers of SGs per cell were calculated. Each bar represents the mean (±standard deviation) for 20 cells infected with MERS-CoV-Δp4. Whole-cell lysates were prepared at 24 h posttransfection and subjected to Western blot analysis to detect TIA-1, G3BP1, G3BP2, or tubulin. (B) At 24 h after siRNA transfection, cells were infected with MERS-CoV-WT or MERS-CoV-Δp4 at an MOI of 3. The titers of the released viruses at the indicated times p.i. were determined by plaque assay. Filled symbols represent virus titers in control siRNA-transfected cells, while empty symbols represent virus titers in TIA-1-specific siRNA-transfected cells or G3BP1/2-specific siRNA-transfected cells. Each box represents the mean (±standard deviation) for three wells. Asterisks represent statistically significant differences (P < 0.05). (C and D) At 24 h after siRNA transfection, cells were mock infected (Mock) or infected with MERS-CoV-WT (WT) or MERS-CoV-Δp4 (Δp4) at an MOI of 3. These cells were radiolabeled for 1 h with 100 μCi of Tran³⁵S-label, and cell lysates were prepared at the indicated times p.i. Cell lysates were subjected to SDS-PAGE analysis, followed by autoradiography (left) and colloid Coomassie brilliant blue staining (right). (E and F) At 24 h after siRNA transfection, cells were infected with MERS-CoV-Δp4 at an MOI of 3, and total cell lysates and RNAs were prepared at 9 h p.i. Western blot analysis of viral S protein, M protein, N protein, and tubulin (E). The amounts of mRNA 1 and subgenomic mRNA 8 were quantified by qRT-PCR (F). Expression levels of mRNAs were normalized to the expression levels of 18S rRNA. Each bar represents the mean (±standard deviation) for three independent samples. ns, not significant (P > 0.05). (G and H) At 24 h siRNA after transfection, cells were mock infected or infected with MERS-CoV-WT or MERS-CoV-Δp4 at an MOI of 3. Cell lysates were prepared at 9 h p.i. and subjected to Western blot analysis to detect eIF2α and phosphorylated eIF2α (p-eIF2α).

**FIG 8**
MERS-CoV 4a accessory protein alone is sufficient for inhibiting SG formation in infected cells. (A) Schematic diagrams of the genomes of MERS-CoV-WT (WT) and MERS-CoV-Δ4a (Δ4a). Boxes represent open reading frames derived from MERS-CoV-WT. The 5′ and 3′ untranslated regions (bars) and viral open reading frames (boxes) are not drawn according to their lengths. (B and C) HeLa/CD26 cells were infected with MERS-CoV-WT or MERS-CoV-Δ4a at an MOI of 3. At 9 h p.i., the infected cells were fixed and stained for TIA-1 (C) or G3BP (B) (green), together with MERS-CoV N protein (red).

**FIG 9**
Characterization of MERS-CoV-Δ4a replication in HeLa/CD26 cells. HeLa/CD26 cells were either mock infected (Mock) or infected with MERS-CoV-WT (WT) or MERS-CoV-Δ4a (Δ4a) at an MOI of 3. (A) Whole-cell lysates were prepared at 9 h p.i. and subjected to Western blot analysis to detect PKR, phosphorylated PKR (p-PKR), eIF2α, phosphorylated eIF2α (p-eIF2α), the MERS-CoV 4a protein, the MERS-CoV 4b protein, and tubulin. (B) HeLa/CD26 cells were radiolabeled for 1 h with 100 μCi of Tran³⁵S-label, and cell lysates were prepared at the indicated times p.i. Cell lysates were subjected to SDS-PAGE analysis, followed by autoradiography (top) and colloid Coomassie brilliant blue staining (bottom). Arrows depict virus-specific proteins. (C) Northern blot analysis of viral mRNAs using a riboprobe that binds to all viral mRNAs. The numbers 1 to 8 represent viral mRNA species. The 28S and 18S rRNAs were detected by ethidium bromide staining (rRNA). (D) Western blot analysis of intracellular accumulation of the MERS-CoV S, M, and N proteins and tubulin. (E and F) HeLa/CD26 (E) or Vero cells (F) were infected with MERS-CoV-WT or MERS-CoV-Δ4a at an MOI of 0.01 (left) or 3 (right). Culture supernatants were collected at the indicated times p.i., and virus titers were determined by plaque assay. Each dot represents the mean virus titer (±standard deviation) for three wells. Asterisks represent statically significant differences in virus titers (P < 0.05).

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References

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[1] Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. 2012. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 367:1814–1820. doi:10.1056/NEJMoa1211721. - DOI - PubMed

[2] Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. 2012. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 367:1814–1820. doi:10.1056/NEJMoa1211721. - DOI - PubMed

[3] Alagaili AN, Briese T, Mishra N, Kapoor V, Sameroff SC, Burbelo PD, de Wit E, Munster VJ, Hensley LE, Zalmout IS, Kapoor A, Epstein JH, Karesh WB, Daszak P, Mohammed OB, Lipkin WI. 2014. Middle East respiratory syndrome coronavirus infection in dromedary camels in Saudi Arabia. mBio 5:e00884-. doi:10.1128/mBio.00884-14. - DOI - PMC - PubMed

[4] Alagaili AN, Briese T, Mishra N, Kapoor V, Sameroff SC, Burbelo PD, de Wit E, Munster VJ, Hensley LE, Zalmout IS, Kapoor A, Epstein JH, Karesh WB, Daszak P, Mohammed OB, Lipkin WI. 2014. Middle East respiratory syndrome coronavirus infection in dromedary camels in Saudi Arabia. mBio 5:e00884-. doi:10.1128/mBio.00884-14. - DOI - PMC - PubMed

[5] Wernery U, Lau SK, Woo PC. 2017. Middle East respiratory syndrome (MERS) coronavirus and dromedaries. Vet J 220:75–79. doi:10.1016/j.tvjl.2016.12.020. - DOI - PMC - PubMed

[6] Wernery U, Lau SK, Woo PC. 2017. Middle East respiratory syndrome (MERS) coronavirus and dromedaries. Vet J 220:75–79. doi:10.1016/j.tvjl.2016.12.020. - DOI - PMC - PubMed

[7] Chu DK, Poon LL, Gomaa MM, Shehata MM, Perera RA, Abu Zeid D, El Rifay AS, Siu LY, Guan Y, Webby RJ, Ali MA, Peiris M, Kayali G. 2014. MERS coronaviruses in dromedary camels, Egypt. Emerg Infect Dis 20:1049–1053. doi:10.3201/eid2006.140299. - DOI - PMC - PubMed

[8] Chu DK, Poon LL, Gomaa MM, Shehata MM, Perera RA, Abu Zeid D, El Rifay AS, Siu LY, Guan Y, Webby RJ, Ali MA, Peiris M, Kayali G. 2014. MERS coronaviruses in dromedary camels, Egypt. Emerg Infect Dis 20:1049–1053. doi:10.3201/eid2006.140299. - DOI - PMC - PubMed

[9] Drosten C, Kellam P, Memish ZA. 2014. Evidence for camel-to-human transmission of MERS coronavirus. N Engl J Med 371:1359–1360. doi:10.1056/NEJMc1409847. - DOI - PubMed

[10] Drosten C, Kellam P, Memish ZA. 2014. Evidence for camel-to-human transmission of MERS coronavirus. N Engl J Med 371:1359–1360. doi:10.1056/NEJMc1409847. - DOI - PubMed

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

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

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