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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.

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 ? subunit of eukaryotic initiation factor 2 (eIF2?). Replication of MERS-CoV lacking the genes for 4a and 4b (MERS-CoV-?p4), 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-?p4-induced SG formation. MERS-CoV-?p4 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-?p4 replication, demonstrating that SG formation was detrimental for MERS-CoV replication. Inefficient MERS-CoV-?p4 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 eIF2? 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.

SUBMITTER: Nakagawa K

PROVIDER: S-EPMC6158436 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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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.

Nakagawa Keisuke K Narayanan Krishna K Wada Masami M Makino Shinji S

Journal of virology 20180926 20

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 ...[more]

PMID: 30068649

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Project description:Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infections that can be life-threatening. To establish an infection and spread, MERS-CoV, like most other viruses, must navigate through an intricate network of antiviral host responses. Besides the well-known type I interferon (IFN-?/?) response, the protein kinase R (PKR)-mediated stress response is being recognized as an important innate response pathway. Upon detecting viral dsRNA, PKR phosphorylates eIF2?, leading to the inhibition of cellular and viral translation and the formation of stress granules (SGs), which are increasingly recognized as platforms for antiviral signaling pathways. It is unknown whether cellular infection by MERS-CoV activates the stress response pathway or whether the virus has evolved strategies to suppress this infection-limiting pathway. Here, we show that cellular infection with MERS-CoV does not lead to the formation of SGs. By transiently expressing the MERS-CoV accessory proteins individually, we identified a role of protein 4a (p4a) in preventing activation of the stress response pathway. Expression of MERS-CoV p4a impeded dsRNA-mediated PKR activation, thereby rescuing translation inhibition and preventing SG formation. In contrast, p4a failed to suppress stress response pathway activation that is independent of PKR and dsRNA. MERS-CoV p4a is a dsRNA binding protein. Mutation of the dsRNA binding motif in p4a disrupted its PKR antagonistic activity. By inserting p4a in a picornavirus lacking its natural PKR antagonist, we showed that p4a exerts PKR antagonistic activity also under infection conditions. However, a recombinant MERS-CoV deficient in p4a expression still suppressed SG formation, indicating the expression of at least one other stress response antagonist. This virus also suppressed the dsRNA-independent stress response pathway. Thus, MERS-CoV interferes with antiviral stress responses using at least two different mechanisms, with p4a suppressing the PKR-dependent stress response pathway, probably by sequestering dsRNA. MERS-CoV p4a represents the first coronavirus stress response antagonist described.

Project description:Middle East respiratory syndrome coronavirus (MERS-CoV) is a beta coronavirus that emerged in 2012, causing severe pneumonia and renal failure. MERS-CoV encodes five accessory proteins. Some of them have been shown to interfere with host antiviral immune response. However, the roles of protein 8b in innate immunity and viral virulence was rarely studied. Here, we introduced individual MERS-CoV accessory protein genes into the genome of an attenuated murine coronavirus (Mouse hepatitis virus, MHV), respectively, and found accessory protein 8b could enhance viral replication in vivo and in vitro and increase the lethality of infected mice. RNA-seq analysis revealed that protein 8b could significantly inhibit type I interferon production (IFN-I) and innate immune response in mice infected with MHV expressing protein 8b. We also found that MERS-CoV protein 8b could initiate from multiple internal methionine sites and at least three protein variants were identified. Residues 1-23 of protein 8b was demonstrated to be responsible for increased virulence in vivo. In addition, the inhibitory effect on IFN-I of protein 8b might not contribute to its virulence enhancement as aa1-23 deletion did not affect IFN-I production in vitro and in vivo. Next, we also found that protein 8b was localized to the endoplasmic reticulum (ER)/Golgi membrane in infected cells, which was disrupted by C-terminal region aa 88-112 deletion. This study will provide new insight into the pathogenesis of MERS-CoV infection. IMPORTANCE Multiple coronaviruses (CoV) cause severe respiratory infections and become global public health threats such as SARS-CoV, MERS-CoV, and SARS-CoV-2. Each coronavirus contains different numbers of accessory proteins which show high variability among different CoVs. Accessory proteins are demonstrated to play essential roles in pathogenesis of CoVs. MERS-CoV contains 5 accessory proteins (protein 3, 4a, 4b, 5, 8b), and deletion of all four accessory proteins (protein 3, 4a, 4b, 5), significantly affects MERS-CoV replication and pathogenesis. However, whether ORF8b also regulates MERS-CoV infection is unknown. Here, we constructed mouse hepatitis virus (MHV) recombinant virus expressing MERS-CoV protein 8b and demonstrated protein 8b could significantly enhance the virulence of MHV, which is mediated by N-terminal domain of protein 8b. This study will shed light on the understanding of pathogenesis of MERS-CoV infection.

Project description:UNLABELLED:Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-?E) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-?E genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein in trans, indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-?E mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection. IMPORTANCE:Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans, whereas it would only survive a single virus infection cycle in vivo. This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection.

Dataset Information

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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