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

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Keisuke Nakagawa ◽  
Krishna Narayanan ◽  
Masami Wada ◽  
Shinji Makino
Keyword(s):  
Middle East ◽  
Virus Replication ◽  
Stress Responses ◽  
Middle East Respiratory Syndrome ◽  
Stress Granule ◽  
Accessory Protein ◽  
Viral Mrnas ◽  
Translation Arrest ◽  
Viral Translation ◽  
Infected Cells

ABSTRACTStress 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.IMPORTANCEMiddle 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.

scholarly journals Disruption of Stress Granule Formation by the Multifunctional Cricket Paralysis Virus 1A Protein

2016 ◽  
Vol 91 (5) ◽  
Author(s):  
Anthony Khong ◽  
Craig H. Kerr ◽  
Clarence H. L. Yeung ◽  
Kathleen Keatings ◽  
Arabinda Nayak ◽  
...  
Keyword(s):  
Virus Infection ◽  
Stress Granule ◽  
Granule Formation ◽  
Viral Translation ◽  
Content Type ◽  
Cellular Processes ◽  
Cellular Environment ◽  
Viral Yield ◽  
Infected Cells ◽  
Paralysis Virus

ABSTRACT Stress granules (SGs) are cytosolic ribonucleoprotein aggregates that are induced during cellular stress. Several viruses modulate SG formation, suggesting that SGs have an impact on virus infection. However, the mechanisms and impact of modulating SG assembly in infected cells are not completely understood. In this study, we identify the dicistrovirus cricket paralysis virus 1A (CrPV-1A) protein that functions to inhibit SG assembly during infection. Moreover, besides inhibiting RNA interference, CrPV-1A also inhibits host transcription, which indirectly modulates SG assembly. Thus, CrPV-1A is a multifunctional protein. We identify a key R146A residue that is responsible for these effects, and mutant CrPV(R146A) virus infection is attenuated in Drosophila melanogaster S2 cells and adult fruit flies and results in increased SG formation. Treatment of CrPV(R146A)-infected cells with actinomycin D, which represses transcription, restores SG assembly suppression and viral yield. In summary, CrPV-1A modulates several cellular processes to generate a cellular environment that promotes viral translation and replication. IMPORTANCE RNA viruses encode a limited set of viral proteins to modulate an array of cellular processes in order to facilitate viral replication and inhibit antiviral defenses. In this study, we identified a viral protein, called CrPV-1A, within the dicistrovirus cricket paralysis virus that can inhibit host transcription, modulate viral translation, and block a cellular process called stress granule assembly. We also identified a specific amino acid within CrPV-1A that is important for these cellular processes and that mutant viruses containing mutations of CrPV-1A attenuate virus infection. We also demonstrate that the CrPV-1A protein can also modulate cellular processes in human cells, suggesting that the mode of action of CrPV-1A is conserved. We propose that CrPV-1A is a multifunctional, versatile protein that creates a cellular environment in virus-infected cells that permits productive virus infection.

scholarly journals Viral Translation Is Coupled to Transcription in Sindbis Virus-Infected Cells

2007 ◽  
Vol 81 (13) ◽  
pp. 7061-7068 ◽  
Author(s):  
Miguel A. Sanz ◽  
Alfredo Castelló ◽  
Luis Carrasco
Keyword(s):  
Sindbis Virus ◽  
Late Phase ◽  
Mrna Translation ◽  
Encephalomyocarditis Virus ◽  
Host Protein ◽  
Viral Mrnas ◽  
Viral Translation ◽  
Bhk Cells ◽  
Infected Cells

ABSTRACT During the late phase of Sindbis virus infection, the viral subgenomic mRNA is translated efficiently in BHK cells, whereas host protein synthesis is inhibited. However, transfection of in vitro-generated Sindbis virus subgenomic mRNA leads to efficient translation in uninfected BHK cells, whereas it is a poor substrate in infected cells. Therefore, the structure of the subgenomic mRNA itself is not sufficient to confer its translatability in infected cells. In this regard, translation of the subgenomic mRNA requires synthesis from the viral transcription machinery. The lack of translation of transfected viral mRNAs in infected cells is not due to their degradation nor is it a consequence of competition between viral transcripts and transfected mRNAs, because a replicon that cannot produce subgenomic mRNA also interferes with exogenous mRNA translation. Interestingly, subgenomic mRNA is translated more efficiently when it is transfected into uninfected cells than when it is transcribed from a transfected replicon. Finally, a similar behavior was observed for other RNA viruses, such as vesicular stomatitis virus and encephalomyocarditis virus. These findings support the notion that translation is coupled to transcription in cells infected with different animal viruses.

scholarly journals Middle East Respiratory Syndrome Coronavirus Accessory Protein 4a Is a Type I Interferon Antagonist

2013 ◽  
Vol 87 (22) ◽  
pp. 12489-12495 ◽  
Author(s):  
D. Niemeyer ◽  
T. Zillinger ◽  
D. Muth ◽  
F. Zielecki ◽  
G. Horvath ◽  
...  
Keyword(s):  
Middle East ◽  
Type I Interferon ◽  
Middle East Respiratory Syndrome ◽  
Accessory Protein ◽  
Type I

scholarly journals Inhibition of anti-viral stress granule formation by coronavirus endoribonuclease nsp15 ensures efficient virus replication

2021 ◽  
Vol 17 (2) ◽  
pp. e1008690 ◽  
Author(s):  
Bo Gao ◽  
Xiaoqian Gong ◽  
Shouguo Fang ◽  
Wenlian Weng ◽  
Huan Wang ◽  
...  
Keyword(s):  
Virus Replication ◽  
Animal Health ◽  
Viral Dsrna ◽  
Granule Formation ◽  
Translation Arrest ◽  
Infectious Bronchitis ◽  
Innate Immune Signaling ◽  
Chemically Induced ◽  
Infected Cells ◽  
Coronavirus Infection

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-β which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.

scholarly journals Middle East Respiratory Syndrome Coronavirus ORF8b Accessory Protein Suppresses Type I IFN Expression by Impeding HSP70-Dependent Activation of IRF3 Kinase IKKε

2020 ◽  
Vol 205 (6) ◽  
pp. 1564-1579
Author(s):  
Lok-Yin Roy Wong ◽  
Zi-Wei Ye ◽  
Pak-Yin Lui ◽  
Xuyang Zheng ◽  
Shuofeng Yuan ◽  
...  
Keyword(s):  
Middle East ◽  
Middle East Respiratory Syndrome ◽  
Accessory Protein ◽  
Type I ◽  
Type I Ifn

scholarly journals The N-terminal Region of Middle East Respiratory Syndrome Coronavirus Accessory Protein 8b is Essential for Enhanced Virulence of an Attenuated Murine Coronavirus

2021 ◽  
Author(s):  
Yuming Li ◽  
Yingkang Jin ◽  
Lijun Kuang ◽  
Zhenhua Luo ◽  
Fang Li ◽  
...  
Keyword(s):  
Immune Response ◽  
Middle East ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Terminal Region ◽  
Middle East Respiratory Syndrome ◽  
Accessory Protein ◽  
Accessory Proteins

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.

scholarly journals The Decision to Publish Gutierrez-Alvarez et al., “Middle East Respiratory Syndrome Coronavirus Gene 5 Modulates Pathogenesis in Mice”

2020 ◽  
Author(s):  
Mark Heise ◽  
Terence S. Dermody ◽  
Arturo Casadevall ◽  
Rozanne M Sandri-Goldin ◽  
Patrick D. Schloss
Keyword(s):  
Middle East ◽  
Protein Gene ◽  
Middle East Respiratory Syndrome ◽  
Accessory Protein

The paper “Middle East respiratory syndrome coronavirus Gene 5 modulates pathogenesis in mice” by Gutierrez-Alvarez et al., in this issue of the Journal of Virology (1), demonstrates that the MERS-CoV accessory protein, Gene 5, also known as ORF5, plays a major role in MERS-CoV pathogenesis.…

scholarly journals Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses

2016 ◽  
Vol 12 (10) ◽  
pp. e1005982 ◽  
Author(s):  
Huib H. Rabouw ◽  
Martijn A. Langereis ◽  
Robert C. M. Knaap ◽  
Tim J. Dalebout ◽  
Javier Canton ◽  
...  
Keyword(s):  
Middle East ◽  
Stress Responses ◽  
Accessory Protein ◽  
Respiratory Coronavirus

scholarly journals The Accessory Protein ORF3 Contributes to Porcine Epidemic Diarrhea Virus Replication by Direct Binding to the Spike Protein

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 399 ◽  
Author(s):  
Challika Kaewborisuth ◽  
Qigai He ◽  
Anan Jongkaewwattana
Keyword(s):  
Virus Replication ◽  
Porcine Epidemic Diarrhea Virus ◽  
Watery Diarrhea ◽  
Accessory Protein ◽  
Structural Protein ◽  
S Protein ◽  
Diarrhea Virus ◽  
Neonatal Piglets ◽  
Infected Cells ◽  
Porcine Epidemic Diarrhea

The porcine epidemic diarrhea virus (PEDV) is an important swine pathogen responsible for severe watery diarrhea, particularly in neonatal piglets. Despite extensive studies performed to elucidate the function of several viral proteins, the contribution of an accessory protein ORF3 in PEDV replication is still largely unknown. Here, we constructed expression plasmids as well as recombinant PEDV carrying myc-tagged ORF3 to assess their expression and subcellular localization in both transfected and infected cells. In PEDV-infected cells, ORF3 was predominantly localized in the cytoplasm, partially in the endoplasmic reticulum (ER) and the Golgi apparatus (Golgi). Interestingly, ORF3 with the N-terminal Flag tag was also detected on the cell surface concomitant with the spike (S) protein as determined by flow cytometry and confocal microscopy. ORF3 and S proteins were also co-localized at perinuclear compartments and in the vesicle-like structures in transfected and infected cells. We also demonstrated that both full-length and naturally truncated ORF3 proteins could interact with the S protein but with different binding affinity, which correlate with the ability of the protein to regulate virus replication in cell culture. Collectively, our results underscore the unprecedented role of the ORF3, which involves the interaction of ORF3 with S and, possibly, other structural protein during PEDV replication.

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