scholarly journals Clinically observed deletions in SARS-CoV-2 Nsp1 affect protein stability and its ability to inhibit translation

2021 ◽  
Author(s):  
Pravin Kumar ◽  
Erin Schexnaydre ◽  
Karim Rafie ◽  
Ilya Terenin ◽  
Vasili Hauryliuk ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Ribosomal Subunit ◽  
Fine Tuning ◽  
In Vitro Translation ◽  
Interferon Response ◽  
Type I ◽  
Biochemical Basis ◽  
Translation Inhibition ◽  
Terminal Domain ◽  
Nonstructural Protein 1

Nonstructural protein 1 (Nsp1) is a major pathogenicity factor of SARS-CoV-2. It inhibits host-cell translation, primarily through a direct interaction between its C-terminal domain and the mRNA entry channel of the 40S small ribosomal subunit, with an N-terminal β-barrel domain fine-tuning the inhibition and promoting selective translation of viral mRNA. SARS-CoV-2 nsp1 is a target of recurring deletions, some of which are associated with altered COVID-19 disease progression. To provide the biochemical basis for this, it is essential to characterize the efficiency of translational inhibition by the said protein variants. Here, we use an in vitro translation system to investigate the translation inhibition capacity of a series of clinically observed Nsp1 deletion variants. We find that a frequently observed deletion of residues 79-89 destabilized the N-terminal domain (NTD) and severely reduced the capacity of Nsp1 to inhibit translation. Interestingly, shorter deletions in the same region have been reported to effect the type I interferon response but did not affect translation inhibition, indicating a possible translation-independent role of the Nsp1 NTD in interferon response modulation. Taken together, our data provide a mechanistic basis for understanding how deletions in Nsp1 influence SARS-CoV-2 induction of interferon response and COVID-19 progression.

scholarly journals SARS-CoV-2 Nucleocapsid Protein Targets RIG-I-Like Receptor Pathways to Inhibit the Induction of Interferon Response

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 530
Author(s):  
Soo Jin Oh ◽  
Ok Sarah Shin
Keyword(s):  
Nucleocapsid Protein ◽  
Nuclear Translocation ◽  
Nonstructural Protein ◽  
Poly I:C ◽  
Interferon Response ◽  
Type I ◽  
Antiviral Immune Response ◽  
N Protein ◽  
Nonstructural Protein 1 ◽  
Polycytidylic Acid

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) that has resulted in the current pandemic. The lack of highly efficacious antiviral drugs that can manage this ongoing global emergency gives urgency to establishing a comprehensive understanding of the molecular pathogenesis of SARS-CoV-2. We characterized the role of the nucleocapsid protein (N) of SARS-CoV-2 in modulating antiviral immunity. Overexpression of SARS-CoV-2 N resulted in the attenuation of retinoic acid inducible gene-I (RIG-I)-like receptor-mediated interferon (IFN) production and IFN-induced gene expression. Similar to the SARS-CoV-1 N protein, SARS-CoV-2 N suppressed the interaction between tripartate motif protein 25 (TRIM25) and RIG-I. Furthermore, SARS-CoV-2 N inhibited polyinosinic: polycytidylic acid [poly(I:C)]-mediated IFN signaling at the level of Tank-binding kinase 1 (TBK1) and interfered with the association between TBK1 and interferon regulatory factor 3 (IRF3), subsequently preventing the nuclear translocation of IRF3. We further found that both type I and III IFN production induced by either the influenza virus lacking the nonstructural protein 1 or the Zika virus were suppressed by the SARS-CoV-2 N protein. Our findings provide insights into the molecular function of the SARS-CoV-2 N protein with respect to counteracting the host antiviral immune response.

scholarly journals SARS-CoV-2 Nsp1 suppresses host but not viral translation through a bipartite mechanism

2020 ◽  
Author(s):  
Ming Shi ◽  
Longfei Wang ◽  
Pietro Fontana ◽  
Setu Vora ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Ribosomal Subunit ◽  
Direct Interaction ◽  
Mrna Translation ◽  
Protein Translation ◽  
Host Protein ◽  
Terminal Domain ◽  
Nonstructural Protein 1 ◽  
40S Ribosomal Subunit ◽  
Open Question

SUMMARYThe Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a highly contagious virus that underlies the current COVID-19 pandemic. SARS-CoV-2 is thought to disable various features of host immunity and cellular defense. The SARS-CoV-2 nonstructural protein 1 (Nsp1) is known to inhibit host protein translation and could be a target for antiviral therapy against COVID-19. However, how SARS-CoV-2 circumvents this translational blockage for the production of its own proteins is an open question. Here, we report a bipartite mechanism of SARS-CoV-2 Nsp1 which operates by: (1) hijacking the host ribosome via direct interaction of its C-terminal domain (CT) with the 40S ribosomal subunit and (2) specifically lifting this inhibition for SARS-CoV-2 via a direct interaction of its N-terminal domain (NT) with the 5’ untranslated region (5’ UTR) of SARS-CoV-2 mRNA. We show that while Nsp1-CT is sufficient for binding to 40S and inhibition of host protein translation, the 5’ UTR of SARS-CoV-2 mRNA removes this inhibition by binding to Nsp1-NT, suggesting that the Nsp1-NT-UTR interaction is incompatible with the Nsp1-CT-40S interaction. Indeed, lengthening the linker between Nsp1-NT and Nsp1-CT of Nsp1 progressively reduced the ability of SARS-CoV-2 5’ UTR to escape the translational inhibition, supporting that the incompatibility is likely steric in nature. The short SL1 region of the 5’ UTR is required for viral mRNA translation in the presence of Nsp1. Thus, our data provide a comprehensive view on how Nsp1 switches infected cells from host mRNA translation to SARS-CoV-2 mRNA translation, and that Nsp1 and 5’ UTR may be targeted for anti-COVID-19 therapeutics.

scholarly journals Fine-Tuning of Type I Interferon Response by STAT3

2019 ◽  
Vol 10 ◽  
Author(s):  
Ming-Hsun Tsai ◽  
Li-Mei Pai ◽  
Chien-Kuo Lee
Keyword(s):  
Type I Interferon ◽  
Fine Tuning ◽  
Interferon Response ◽  
Type I

scholarly journals Nonstructural Proteins 1 and 2 of Respiratory Syncytial Virus Suppress Maturation of Human Dendritic Cells

2008 ◽  
Vol 82 (17) ◽  
pp. 8780-8796 ◽  
Author(s):  
Shirin Munir ◽  
Cyril Le Nouen ◽  
Cindy Luongo ◽  
Ursula J. Buchholz ◽  
Peter L. Collins ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Respiratory Syncytial Virus ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Type I ◽  
Ns1 Protein ◽  
Myeloid Dendritic Cells ◽  
Nonstructural Protein 1 ◽  
Syncytial Virus ◽  
Dc Maturation

ABSTRACT Human respiratory syncytial virus (RSV) is the most important agent of serious pediatric respiratory tract disease worldwide. One of the main characteristics of RSV is that it readily reinfects and causes disease throughout life without the need for significant antigenic change. The virus encodes nonstructural protein 1 (NS1) and NS2, which are known to suppress type I interferon (IFN) production and signaling. In the present study, we monitored the maturation of human monocyte-derived myeloid dendritic cells (DC) following inoculation with recombinant RSVs bearing deletions of the NS1 and/or NS2 proteins and expressing enhanced green fluorescent protein. Deletion of the NS1 protein resulted in increased expression of cell surface markers of DC maturation and an increase in the expression of multiple cytokines and chemokines. This effect was enhanced somewhat by further deletion of the NS2 protein, although deletion of NS2 alone did not have a significant effect. The upregulation was largely inhibited by pretreatment with a blocking antibody against the type I IFN receptor, suggesting that suppression of DC maturation by NS1/2 is, at least in part, a result of IFN antagonism mediated by these proteins. Therefore, this study identified another effect of the NS1 and NS2 proteins. The observed suppression of DC maturation may result in decreased antigen presentation and T-lymphocyte activation, leading to incomplete and/or weak immune responses that might contribute to RSV reinfection.

scholarly journals Lysine 164 is critical for SARS-CoV-2 Nsp1 inhibition of host gene expression

2020 ◽  
Author(s):  
Zhou Shen ◽  
Guangxu Zhang ◽  
Yilin Yang ◽  
Mengxia Li ◽  
Siqi Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
Ribosomal Subunit ◽  
Virus Production ◽  
Host Gene ◽  
Host Protein ◽  
Renilla Luciferase ◽  
Host Gene Expression ◽  
Nonstructural Protein 1

The emerging pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused social and economic disruption worldwide, infecting over 9.0 million people and killing over 469 000 by 24 June 2020. Unfortunately, no vaccine or antiviral drug that completely eliminates the transmissible disease coronavirus disease 2019 (COVID-19) has been developed to date. Given that coronavirus nonstructural protein 1 (nsp1) is a good target for attenuated vaccines, it is of great significance to explore the detailed characteristics of SARS-CoV-2 nsp1. Here, we first confirmed that SARS-CoV-2 nsp1 had a conserved function similar to that of SARS-CoV nsp1 in inhibiting host-protein synthesis and showed greater inhibition efficiency, as revealed by ribopuromycylation and Renilla luciferase (Rluc) reporter assays. Specifically, bioinformatics and biochemical experiments showed that by interacting with 40S ribosomal subunit, the lysine located at amino acid 164 (K164) was the key residue that enabled SARS-CoV-2 nsp1 to suppress host gene expression. Furthermore, as an inhibitor of host-protein expression, SARS-CoV-2 nsp1 contributed to cell-cycle arrest in G0/G1 phase, which might provide a favourable environment for virus production. Taken together, this research uncovered the detailed mechanism by which SARS-CoV-2 nsp1 K164 inhibited host gene expression, laying the foundation for the development of attenuated vaccines based on nsp1 modification.

scholarly journals Equine Arteritis Virus Does Not Induce Interferon Production in Equine Endothelial Cells: Identification of Nonstructural Protein 1 as a Main Interferon Antagonist

2014 ◽  
Vol 2014 ◽  
pp. 1-13 ◽  
Author(s):  
Yun Young Go ◽  
Yanhua Li ◽  
Zhenhai Chen ◽  
Mingyuan Han ◽  
Dongwan Yoo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Nonstructural Protein ◽  
Critical Role ◽  
Equine Arteritis Virus ◽  
Luciferase Reporter ◽  
Type I ◽  
Mrna Levels ◽  
Type I Ifn ◽  
Rt Pcr ◽  
Nonstructural Protein 1

The objective of this study was to investigate the effect of equine arteritis virus (EAV) on type I interferon (IFN) production. Equine endothelial cells (EECs) were infected with the virulent Bucyrus strain (VBS) of EAV and expression of IFN-βwas measured at mRNA and protein levels by quantitative real-time RT-PCR and IFN bioassay using vesicular stomatitis virus expressing the green fluorescence protein (VSV-GFP), respectively. Quantitative RT-PCR results showed that IFN-βmRNA levels in EECs infected with EAV VBS were not increased compared to those in mock-infected cells. Consistent with quantitative RT-PCR, Sendai virus- (SeV-) induced type I IFN production was inhibited by EAV infection. Using an IFN-βpromoter-luciferase reporter assay, we subsequently demonstrated that EAV nsps 1, 2, and 11 had the capability to inhibit type I IFN activation. Of these three nsps, nsp1 exhibited the strongest inhibitory effect. Taken together, these data demonstrate that EAV has the ability to suppress the type I IFN production in EECs and nsp1 may play a critical role to subvert the equine innate immune response.

scholarly journals Interaction between NS1 and Cellular MAVS Contributes to NS1 Mitochondria Targeting

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1909
Author(s):  
Yeu-Yang Tseng ◽  
Chih-Ying Kuan ◽  
Masaki Mibayashi ◽  
Chi-Jene Chen ◽  
Peter Palese ◽  
...  
Keyword(s):  
Influenza A ◽  
Early Stage ◽  
Nonstructural Protein ◽  
Influenza Virus Infection ◽  
Type I ◽  
Mitochondrial Localization ◽  
Independent Manner ◽  
Nonstructural Protein 1 ◽  
Mitochondrial Antiviral Signaling ◽  
Mitochondria Targeting

Influenza A virus nonstructural protein 1 (NS1) plays an important role in evading host innate immunity. NS1 inhibits interferon (IFN) responses via multiple mechanisms, including sequestering dsRNA and suppressing retinoic acid-inducible gene I (RIG-I) signaling by interacting with RIG-I and tripartite motif-containing protein 25 (TRIM25). In the current study, we demonstrated the mitochondrial localization of NS1 at the early stage of influenza virus infection. Since NS1 does not contain mitochondria-targeting signals, we suspected that there is an association between the NS1 and mitochondrial proteins. This hypothesis was tested by demonstrating the interaction of NS1 with mitochondrial antiviral-signaling protein (MAVS) in a RIG-I-independent manner. Importantly, the association with MAVS facilitated the mitochondrial localization of NS1 and thereby significantly impeded MAVS-mediated Type I IFN production.

scholarly journals A Naturally Occurring Deletion in the Effector Domain of H5N1 Swine Influenza Virus Nonstructural Protein 1 Regulates Viral Fitness and Host Innate Immunity

2018 ◽  
Vol 92 (11) ◽  
Author(s):  
Junyong Wang ◽  
Yan Zeng ◽  
Shuai Xu ◽  
Jiayun Yang ◽  
Wanbing Wang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Type I Interferon ◽  
Nonstructural Protein ◽  
Full Length ◽  
Type I ◽  
Effector Domain ◽  
Nonstructural Protein 1 ◽  
Naturally Occurring

ABSTRACT Nonstructural protein 1 (NS1) of influenza A virus regulates innate immune responses via various mechanisms. We previously showed that a naturally occurring deletion (the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus impairs the inhibition of type I interferon (IFN) in chicken fibroblasts and attenuates virulence in chickens. Here we found that the virus bearing this deletion in its NS1 effector domain showed diminished inhibition of IFN-related cytokine expression and attenuated virulence in mice. We further showed that deletion of the EALQR motif disrupted NS1 dimerization, impairing double-stranded RNA (dsRNA) sequestration and competitive binding with RIG-I. In addition, the EALQR-deleted NS1 protein could not bind to TRIM25, unlike full-length NS1, and was less able to block TRIM25 oligomerization and self-ubiquitination, further impairing the inhibition of TRIM25-mediated RIG-I ubiquitination compared to that with full-length NS1. Our data demonstrate that the EALQR deletion prevents NS1 from blocking RIG-I-mediated IFN induction via a novel mechanism to attenuate viral replication and virulence in mammalian cells and animals. IMPORTANCE H5 highly pathogenic avian influenza viruses have infected more than 800 individuals across 16 countries, with an overall case fatality rate of 53%. Among viral proteins, nonstructural protein 1 (NS1) of influenza virus is considered a key determinant for type I interferon (IFN) antagonism, pathogenicity, and host range. However, precisely how NS1 modulates virus-host interaction, facilitating virus survival, is not fully understood. Here we report that a naturally occurring deletion (of the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus disrupted NS1 dimerization, which diminished the blockade of IFN induction via the RIG-I signaling pathway, thereby impairing virus replication and virulence in the host. Our study demonstrates that the EALQR motif of NS1 regulates virus fitness to attain a virus-host compromise state in animals and identifies this critical motif as a potential target for the future development of small molecular drugs and attenuated vaccines.

scholarly journals Coronavirus Endoribonuclease and Deubiquitinating Interferon Antagonists Differentially Modulate the Host Response during Replication in Macrophages

2020 ◽  
Vol 94 (11) ◽  
Author(s):  
Aaron Volk ◽  
Matthew Hackbart ◽  
Xufang Deng ◽  
Yazmin Cruz-Pulido ◽  
Amornrat O’Brien ◽  
...  
Keyword(s):  
Host Response ◽  
Nonstructural Protein ◽  
Type I Interferons ◽  
Interferon Response ◽  
Type I ◽  
Wild Type ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Interferon Antagonists

ABSTRACT Coronaviruses (CoVs) encode multiple interferon (IFN) antagonists that modulate the host response to virus replication. Here, we evaluated the host transcriptional response to infection with murine coronaviruses encoding independent mutations in one of two different viral antagonists, the deubiquitinase (DUB) within nonstructural protein 3 or the endoribonuclease (EndoU) within nonstructural protein 15. We used transcriptomics approaches to compare the scope and kinetics of the host response to the wild-type (WT), DUBmut, and EndoUmut viruses in infected macrophages. We found that the EndoUmut virus activates a focused response that predominantly involves type I interferons and interferon-related genes, whereas the WT and DUBmut viruses more broadly stimulate upregulation of over 2,800 genes, including networks associated with activating the unfolded protein response (UPR) and the proinflammatory response associated with viral pathogenesis. This study highlights the role of viral interferon antagonists in shaping the kinetics and magnitude of the host response during virus infection and demonstrates that inactivating a dominant viral antagonist, the coronavirus endoribonuclease, dramatically alters the host response in macrophages. IMPORTANCE Macrophages are an important cell type during coronavirus infections because they “notice” the infection and respond by inducing type I interferons, which limits virus replication. In turn, coronaviruses encode proteins that mitigate the cell’s ability to signal an interferon response. Here, we evaluated the host macrophage response to two independent mutant coronaviruses, one with reduced deubiquitinating activity (DUBmut) and the other containing an inactivated endoribonuclease (EndoUmut). We observed a rapid, robust, and focused response to the EndoUmut virus, which was characterized by enhanced expression of interferon and interferon-related genes. In contrast, wild-type virus and the DUBmut virus elicited a more limited interferon response and ultimately activated over 2,800 genes, including players in the unfolded protein response and proinflammatory pathways associated with progression of significant disease. This study reveals that EndoU activity substantially contributes to the ability of coronaviruses to evade the host innate response and to replicate in macrophages.

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