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Major histocompatibility complex class I (MHC-I) plays crucial roles against viral infections not only by initiating CD8+ T cell immunity but also by modulating natural killer (NK) cell cytotoxicity. Understanding how viruses precisely regulate MHC-I to optimize their infection is important; however, the manipulation of MHC-I molecules by porcine epidemic diarrhea virus (PEDV) remains unclear. In this study, we demonstrate that PEDV infection promotes the transcription of NLRC5, a key transactivator of MHC-I, in several porcine cell lines and in vivo. Paradoxically, no increase in MHC-I expression is observed after PEDV infection both in vitro and in vivo. Mechanistic studies revealed that PEDV infection inhibits the translation of PEDV-elicited NLRC5 mRNA and the expression of downstream MHC-I proteins, without affecting the expression of physiological NLRC5 and MHC-I proteins. Through viral protein screening, we identified PEDV nonstructural protein 1 (nsp1) as the critical antagonist that inhibits NLRC5-mediated upregulation of MHC-I, and the nsp1's inhibitory effect on MHC-I requires the motif of 15 amino acids at its C-terminus. Notably, our results revealed that the cytotoxic ability of NK cells against PEDV-infected cells is similar to that against healthy cells. Collectively, our findings uncover an immune evasion mechanism by which PEDV-infected cells masquerade as healthy cells to evade NK and T cell immunity. This is achieved by targeting NLRC5, a key MHC-I transcriptional regulator, via nsp1. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric coronavirus that inflicts substantial financial losses on the swine industry. Major histocompatibility complex class I (MHC-I) is a critical factor influencing both CD8+ T cell and natural killer (NK) cell immunity. However, how PEDV manipulates MHC-I expression to optimize its infection process remains largely unknown. In this study, we demonstrate that PEDV's nonstructural protein 1 (nsp1) inhibits virus-mediated induction of MHC-I expression by directly targeting NLRC5, a key MHC-I transactivator. Intriguingly, nsp1 does not reduce physiological NLRC5 and MHC-I expression. This selective inhibition of virus-elicited NLRC5 mRNA translation allows PEDV-infected cells to masquerade as healthy cells, thereby evading CD8+ T cell and NK cell cytotoxicity. Our findings provide unique insights into the mechanisms by which PEDV evades CD8+ T cell and NK cell immunity. [ABSTRACT FROM AUTHOR]

Background: Dengue virus (DENV) infection, spread by Aedes aegypti mosquitoes, is a significant public health concern in tropical and subtropical regions. Among the four distinct serotypes of DENV (DENV-1 to DENV-4), DENV-2 is associated with the highest number of fatalities worldwide. However, there is no specific treatment available for dengue patients caused by DENV-2. Objective: This study aimed to identify inhibitory phytocompounds in silico in Vernonia cinerea (V. cinerea), a widely used traditional medicinal plant, for treating DENV-2 associated illnesses. Methods: The chemical structures of 17 compounds from V. cinerea were sourced from the Indian Medicinal Plants, Phytochemistry, and Therapeutics (IMPPAT) database. These compounds underwent geometry optimization, were screened against nonstructural protein 1 (NSP1) of DENV-2, and further validated through molecular dynamics simulations (MDS). Baicalein, an established drug against DENV-2, was used for validation in molecular screening, MDS, and MM-GBSA analyses. Results: Among these compounds, Beta-amyrin, Beta-amyrin acetate, Chrysoeriol, Isoorientin, and Luteolin showed promising potential as inhibitors of the NSP1 of DENV-2, supported by the results of thermodynamic properties, molecular orbitals, electrostatic potentials, spectral data and molecular screening. Besides, these compounds adhered to the Lipinski's "rule of 5", showing no hepatotoxicity/cytotoxicity, with mixed mutagenicity, immunotoxicity, and carcinogenicity. Furthermore, final validation through MDS confirmed their potential, demonstrating stable tendencies with significant inhibitory activities against NSP1 of DENV-2 over the control drug Baicalein. Among the screened compounds, Chrysoeriol emerged as the most promising inhibitor of NSP1 of DENV-2, followed by Luteolin and Isoorientin. Conclusion: Taken together, our results suggest that Chrysoeriol is the best inhibitor of NSP1 of DENV-2, which could be evaluated as a therapeutic agent or a lead compound to treat and manage DENV-2 infections. [ABSTRACT FROM AUTHOR]

Alphavirus infection induces dramatic remodeling of host cellular membranes, producing filopodia-like and intercellular extensions. The formation of filopodia-like extensions has been primarily assigned to the replication protein nsP1, which binds and reshapes the host plasma membrane when expressed alone. While reported decades ago, the molecular mechanisms behind nsP1 membrane deformation remain unknown. Using mammalian epithelial cells and Chikungunya virus (CHIKV) as models, we characterized nsP1-induced membrane deformations as highly dynamic actin-rich lamellipodia and filopodia-like extensions. Through pharmacological inhibition and genetic invalidation, we identified the critical contribution of the Rac1 GTPase and its downstream effectors PAK1 and the actin nucleator Arp2 in nsP1-induced membrane deformation. An intact Rac1-PAK1-Arp2 signaling axis was also required for optimal CHIKV genome replication. Therefore, our results designate the Rac1-PAK1-Arp2 pathway as an essential signaling node for CHIKV infection and establish a parallel requirement for host factors involved in nsP1-induced plasma membrane reshaping and assembly of a functional replication complex. [ABSTRACT FROM AUTHOR]

Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes widespread changes in epigenetic modifications and chromatin architecture in the host cell. Recent evidence suggests that SARS-CoV-2 nonstructural protein 1 (nsp1) plays an important role in driving these changes. Previously thought to be primarily involved in host translation shutoff and cellular mRNA degradation, nsp1 has now been shown to be a truly multifunctional protein that affects host gene expression at multiple levels. The functions of nsp1 are surprisingly diverse and include not only the downregulation of cellular mRNA translation and stability, but also the inhibition of mRNA export from the nucleus, the suppression of host immune signaling, and, most recently, the epigenetic regulation of host gene expression. In this review, we first summarize the current knowledge on SARS-CoV-2-induced changes in epigenetic modifications and chromatin structure. We then focus on the role of nsp1 in epigenetic reprogramming, with a particular emphasis on the silencing of immune-related genes. Finally, we discuss potential molecular mechanisms underlying the epigenetic functions of nsp1 based on evidence from SARS-CoV-2 interactome studies. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculoskeletal disease in humans. Replication of the viral RNA genome occurs in specialized membranous replication organelles (ROs) or spherules, which contain the viral replication complex. Initially generated by RNA synthesis-associated plasma membrane deformation, alphavirus ROs are generally rapidly endocytosed to produce type I cytopathic vacuoles (CPV-I), from which nascent RNAs are extruded for cytoplasmic translation. By contrast, CHIKV ROs are poorly internalized, raising the question of their fate and functionality at the late stage of infection. Here, using in situ cryogenic-electron microscopy approaches, we investigate the outcome of CHIKV ROs and associated replication machinery in infected human cells. We evidence the late persistence of CHIKV ROs at the plasma membrane with a crowned protein complex at the spherule neck similar to the recently resolved replication complex. The unexpectedly heterogeneous and large diameter of these compartments suggests a continuous, dynamic growth of these organelles beyond the replication of a single RNA genome. Ultrastructural analysis of surrounding cytoplasmic regions supports that outgrown CHIKV ROs remain dynamically active in viral RNA synthesis and export to the cell cytosol for protein translation. Interestingly, rare ROs with a homogeneous diameter are also marginally internalized in CPV-I near honeycomb-like arrangements of unknown function, which are absent in uninfected controls, thereby suggesting a temporal regulation of this internalization. Altogether, this study sheds new light on the dynamic pattern of CHIKV ROs and associated viral replication at the interface with cell membranes in infected cells. IMPORTANCE The Chikungunya virus (CHIKV) is a positive-stranded RNA virus that requires specialized membranous replication organelles (ROs) for its genome replication. Our knowledge of this viral cycle stage is still incomplete, notably regarding the fate and functional dynamics of CHIKV ROs in infected cells. Here, we show that CHIKV ROs are maintained at the plasma membrane beyond the first viral cycle, continuing to grow and be dynamically active both in viral RNA replication and in its export to the cell cytosol, where translation occurs in proximity to ROs. This contrasts with the homogeneous diameter of ROs during internalization in cytoplasmic vacuoles, which are often associated with honeycomb-like arrangements of unknown function, suggesting a regulated mechanism. This study sheds new light on the dynamics and fate of CHIKV ROs in human cells and, consequently, on our understanding of the Chikungunya viral cycle. [ABSTRACT FROM AUTHOR]

BackgroundDengue virus (DENV) infection, spread by Aedes aegypti mosquitoes, is a significant public health concern in tropical and subtropical regions. Among the four distinct serotypes of DENV (DENV-1 to DENV-4), DENV-2 is associated with the highest number of fatalities worldwide. However, there is no specific treatment available for dengue patients caused by DENV-2.ObjectiveThis study aimed to identify inhibitory phytocompounds in silico in Vernonia cinerea (V. cinerea), a widely used traditional medicinal plant, for treating DENV-2 associated illnesses.MethodsThe chemical structures of 17 compounds from V. cinerea were sourced from the Indian Medicinal Plants, Phytochemistry, and Therapeutics (IMPPAT) database. These compounds underwent geometry optimization, were screened against nonstructural protein 1 (NSP1) of DENV-2, and further validated through molecular dynamics simulations (MDS). Baicalein, an established drug against DENV-2, was used for validation in molecular screening, MDS, and MM-GBSA analyses.ResultsAmong these compounds, Beta-amyrin, Beta-amyrin acetate, Chrysoeriol, Isoorientin, and Luteolin showed promising potential as inhibitors of the NSP1 of DENV-2, supported by the results of thermodynamic properties, molecular orbitals, electrostatic potentials, spectral data and molecular screening. Besides, these compounds adhered to the Lipinski’s “rule of 5”, showing no hepatotoxicity/cytotoxicity, with mixed mutagenicity, immunotoxicity, and carcinogenicity. Furthermore, final validation through MDS confirmed their potential, demonstrating stable tendencies with significant inhibitory activities against NSP1 of DENV-2 over the control drug Baicalein. Among the screened compounds, Chrysoeriol emerged as the most promising inhibitor of NSP1 of DENV-2, followed by Luteolin and Isoorientin.ConclusionTaken together, our results suggest that Chrysoeriol is the best inhibitor of NSP1 of DENV-2, which could be evaluated as a therapeutic agent or a lead compound to treat and manage DENV-2 infections.

Abstract Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel porcine enteric coronavirus that causes acute watery diarrhea, vomiting, and dehydration in newborn piglets. The type III interferon (IFN-λ) response serves as the primary defense against viruses that replicate in intestinal epithelial cells. However, there is currently no information available on how SADS-CoV modulates the production of IFN-λ. In this study, we utilized IPI-FX cells (a cell line of porcine ileum epithelium) as an in vitro model to investigate the potential immune evasion strategies employed by SADS-CoV against the IFN-λ response. Our results showed that SADS-CoV infection suppressed the production of IFN-λ1 induced by poly(I:C). Through screening SADS-CoV-encoded proteins, nsp1, nsp5, nsp10, nsp12, nsp16, E, S1, and S2 were identified as antagonists of IFN-λ1 production. Specifically, SADS-CoV nsp1 impeded the activation of the IFN-λ1 promoter mediated by MAVS, TBK1, IKKε, and IRF1. Both SADS-CoV and nsp1 obstructed poly(I:C)-induced nuclear translocation of IRF1. Moreover, SADS-CoV nsp1 degraded IRF1 via the ubiquitin-mediated proteasome pathway without interacting with it. Overall, our study provides the first evidence that SADS-CoV inhibits the type III IFN response, shedding light on the molecular mechanisms employed by SADS-CoV to evade the host immune response.

The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

The intrinsically disordered C-terminal peptide region of severe acute respiratory syndrome Coronavirus 2 nonstructural protein-1 (Nspl-CT) inhibits host protein synthesis by blocking messenger RNA (mRNA) access to the 40S ribosome entrance tunnel. Aqueous copper(II) ions bind to the disordered peptide with micromolar affinity, creating a possible strategy to restore protein synthesis during host infection. Electron paramagnetic resonance (EPR) and tryptophan fluorescence measurements on a 10-residue model of the disordered protein region (Nsp1-CT10), combined with advanced quantum mechanics calculations, suggest that the peptide binds to copper(II) as a multidentate ligand. Two optimized computational models of the copper(II)-peptide complexes were derived: One corresponding to pH 6.5 and the other describing the complex at pH 7.5 to 8.5. Simulated EPR spectra based on the calculated model structures are in good agreement with experimental spectra. [ABSTRACT FROM AUTHOR]

Non-structural protein 1 (Nsp1) represents one of the most crucial SARS-CoV-2 virulence factors by inhibiting the translation of host mRNAs and promoting their degradation. We selected naturally occurring virus lineages with specific Nsp1 deletions located at both the N- and C-terminus of the protein. Our data provide new insights into how Nsp1 coordinates these functions on host and viral mRNA recognition. Residues 82–85 in the N-terminal part of Nsp1 likely play a role in docking the 40S mRNA entry channel, preserving the inhibition of host gene expression without affecting cellular mRNA decay. Furthermore, this domain prevents viral mRNAs containing the 5′-leader sequence to escape translational repression. These findings support the presence of distinct domains within the Nsp1 protein that differentially modulate mRNA recognition, translation and turnover. These insights have implications for the development of drugs targeting viral proteins and provides new evidences of how specific mutations in SARS-CoV-2 Nsp1 could attenuate the virus. [ABSTRACT FROM AUTHOR]

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel porcine enteric coronavirus that causes acute watery diarrhea, vomiting, and dehydration in newborn piglets. The type III interferon (IFN-λ) response serves as the primary defense against viruses that replicate in intestinal epithelial cells. However, there is currently no information available on how SADS-CoV modulates the production of IFN-λ. In this study, we utilized IPI-FX cells (a cell line of porcine ileum epithelium) as an in vitro model to investigate the potential immune evasion strategies employed by SADS-CoV against the IFN-λ response. Our results showed that SADS-CoV infection suppressed the production of IFN-λ1 induced by poly(I:C). Through screening SADS-CoV-encoded proteins, nsp1, nsp5, nsp10, nsp12, nsp16, E, S1, and S2 were identified as antagonists of IFN-λ1 production. Specifically, SADS-CoV nsp1 impeded the activation of the IFN-λ1 promoter mediated by MAVS, TBK1, IKKε, and IRF1. Both SADS-CoV and nsp1 obstructed poly(I:C)-induced nuclear translocation of IRF1. Moreover, SADS-CoV nsp1 degraded IRF1 via the ubiquitin-mediated proteasome pathway without interacting with it. Overall, our study provides the first evidence that SADS-CoV inhibits the type III IFN response, shedding light on the molecular mechanisms employed by SADS-CoV to evade the host immune response. [ABSTRACT FROM AUTHOR]

Vanadium (V) pollution potentially threatens human health. Here, it is found that nsp1 and nsp2, Rhizobium symbiosis defective mutants of Medicago truncatula, are sensitive to V. Concentrations of phosphorus (P), iron (Fe), and sulfur (S) with V are negatively correlated in the shoots of wild‐type R108, but not in mutant nsp1 and nsp2 shoots. Mutations in the P transporter PHT1, PHO1, and VPT families, Fe transporter IRT1, and S transporter SULTR1/3/4 family confer varying degrees of V tolerance on plants. Among these gene families, MtPT1, MtZIP6, MtZIP9, and MtSULTR1; 1 in R108 roots are significantly inhibited by V stress, while MtPHO1; 2, MtVPT2, and MtVPT3 are significantly induced. Overexpression of Arabidopsis thaliana VPT1 or M. truncatula MtVPT3 increases plant V tolerance. However, the response of these genes to V is weakened in nsp1 or nsp2 and influenced by soil microorganisms. Mutations in NSPs reduce rhizobacterial diversity under V stress and simplify the V‐responsive operational taxonomic unit modules in co‐occurrence networks. Furthermore, R108 recruits more beneficial rhizobacteria related to V, P, Fe, and S than does nsp1 or nsp2. Thus, NSPs can modulate the accumulation and tolerance of legumes to V through P, Fe, and S transporters, ion homeostasis, and rhizobacterial community responses. [ABSTRACT FROM AUTHOR]

Following virus recognition of host cell receptors and viral particle/genome internalization, viruses replicate in the host via hijacking essential host cell machinery components to evade the provoked antiviral innate immunity against the invading pathogen. Respiratory viral infections are usually acute with the ability to activate pattern recognition receptors (PRRs) in/on host cells, resulting in the production and release of interferons (IFNs), proinflammatory cytokines, chemokines, and IFN-stimulated genes (ISGs) to reduce virus fitness and mitigate infection. Nevertheless, the game between viruses and the host is a complicated and dynamic process, in which they restrict each other via specific factors to maintain their own advantages and win this game. The primary role of the non-structural protein 1 (NS1 and Nsp1) of influenza A viruses (IAV) and the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respectively, is to control antiviral host-induced innate immune responses. This review provides a comprehensive overview of the genesis, spatial structure, viral and cellular interactors, and the mechanisms underlying the unique biological functions of IAV NS1 and SARS-CoV-2 Nsp1 in infected host cells. We also highlight the role of both non-structural proteins in modulating viral replication and pathogenicity. Eventually, and because of their important role during viral infection, we also describe their promising potential as targets for antiviral therapy and the development of live attenuated vaccines (LAV). Conclusively, both IAV NS1 and SARS-CoV-2 Nsp1 play an important role in virus–host interactions, viral replication, and pathogenesis, and pave the way to develop novel prophylactic and/or therapeutic interventions for the treatment of these important human respiratory viral pathogens. [ABSTRACT FROM AUTHOR]

ABSTRACTSARS-CoV-2, the causative agent of COVID-19, has been intensely studied in search of effective antiviral treatments. The immunosuppressant cyclosporine A (CsA) has been suggested to be a pan-coronavirus inhibitor, yet its underlying mechanism remained largely unknown. Here, we found that non-structural protein 1 (Nsp1) of SARS-CoV-2 usurped CsA-suppressed nuclear factor of activated T cells (NFAT) signaling to drive the expression of cellular DEAD-box helicase 5 (DDX5), which facilitates viral replication. Nsp1 interacted with calcineurin A (CnA) to displace the regulatory protein regulator of calcineurin 3 (RCAN3) of CnA for NFAT activation. The influence of NFAT activation on SARS-CoV-2 replication was also validated by using the Nsp1-deficient mutant virus. Calcineurin inhibitors, such as CsA and VIVIT, inhibited SARS-CoV-2 replication and exhibited synergistic antiviral effects when used in combination with nirmatrelvir. Our study delineated the molecular mechanism of CsA-mediated inhibition of SARS-CoV-2 replication and the anti-SARS-CoV-2 action of calcineurin inhibitors.IMPORTANCECyclosporine A (CsA), commonly used to inhibit immune responses, is also known to have anti-SARS-CoV-2 activity, but its mode of action remains elusive. Here, we provide a model to explain how CsA antagonizes SARS-CoV-2 through three critical proteins: DDX5, NFAT1, and Nsp1. DDX5 is a cellular facilitator of SARS-CoV-2 replication, and NFAT1 controls the production of DDX5. Nsp1 is a viral protein absent from the mature viral particle and capable of activating the function of NFAT1 and DDX5. CsA and similar agents suppress Nsp1, NFAT1, and DDX5 to exert their anti-SARS-CoV-2 activity either alone or in combination with Paxlovid.

Nsp1 is a SARS-CoV-2 host shutoff factor that both represses cellular translation and promotes host RNA decay. However, it is unclear how these two activities are connected and interact with normal translation processes. Here, we performed mutational analyses of Nsp1, and these revealed that both the N and C terminal domains of Nsp1 are important for translational repression. Furthermore, we demonstrate that specific residues in the N terminal domain are required for cellular RNA degradation but not bulk translation shutoff of host mRNAs, thereby separating RNA degradation from translation repression. We also present evidence that Nsp1 mediated RNA degradation requires engagement of the ribosome with mRNA. First, we observe that cytosolic lncRNAs, which are not translated, escape Nsp1 mediated degradation. Second, inhibition of translation elongation with emetine does not prevent Nsp1 mediated degradation, while blocking translation initiation before 48S ribosome loading reduces mRNA degradation. Taken together, we suggest that Nsp1 represses translation and promotes mRNA degradation only after ribosome engagement with the mRNA. This raises the possibility that Nsp1 may trigger RNA degradation through pathways that recognize stalled ribosomes.

Nonstructural protein 1 (nsp1) is a coronavirus (CoV) virulence factor that restricts cellular gene expression by inhibiting translation through blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We perform a detailed structure-guided mutational analysis of severe acute respiratory syndrome (SARS)-CoV-2 nsp1, revealing insights into how it coordinates these activities against host but not viral mRNA. We find that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, both enhancing its restriction of host gene expression and enabling mRNA containing the SARS-CoV-2 leader sequence to escape translational repression. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in SARS-CoV-2 nsp1 could attenuate the virus.

Abstract Vanadium (V) pollution potentially threatens human health. Here, it is found that nsp1 and nsp2, Rhizobium symbiosis defective mutants of Medicago truncatula, are sensitive to V. Concentrations of phosphorus (P), iron (Fe), and sulfur (S) with V are negatively correlated in the shoots of wild‐type R108, but not in mutant nsp1 and nsp2 shoots. Mutations in the P transporter PHT1, PHO1, and VPT families, Fe transporter IRT1, and S transporter SULTR1/3/4 family confer varying degrees of V tolerance on plants. Among these gene families, MtPT1, MtZIP6, MtZIP9, and MtSULTR1; 1 in R108 roots are significantly inhibited by V stress, while MtPHO1; 2, MtVPT2, and MtVPT3 are significantly induced. Overexpression of Arabidopsis thaliana VPT1 or M. truncatula MtVPT3 increases plant V tolerance. However, the response of these genes to V is weakened in nsp1 or nsp2 and influenced by soil microorganisms. Mutations in NSPs reduce rhizobacterial diversity under V stress and simplify the V‐responsive operational taxonomic unit modules in co‐occurrence networks. Furthermore, R108 recruits more beneficial rhizobacteria related to V, P, Fe, and S than does nsp1 or nsp2. Thus, NSPs can modulate the accumulation and tolerance of legumes to V through P, Fe, and S transporters, ion homeostasis, and rhizobacterial community responses.

Following virus recognition of host cell receptors and viral particle/genome internalization, viruses replicate in the host via hijacking essential host cell machinery components to evade the provoked antiviral innate immunity against the invading pathogen. Respiratory viral infections are usually acute with the ability to activate pattern recognition receptors (PRRs) in/on host cells, resulting in the production and release of interferons (IFNs), proinflammatory cytokines, chemokines, and IFN-stimulated genes (ISGs) to reduce virus fitness and mitigate infection. Nevertheless, the game between viruses and the host is a complicated and dynamic process, in which they restrict each other via specific factors to maintain their own advantages and win this game. The primary role of the non-structural protein 1 (NS1 and Nsp1) of influenza A viruses (IAV) and the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respectively, is to control antiviral host-induced innate immune responses. This review provides a comprehensive overview of the genesis, spatial structure, viral and cellular interactors, and the mechanisms underlying the unique biological functions of IAV NS1 and SARS-CoV-2 Nsp1 in infected host cells. We also highlight the role of both non-structural proteins in modulating viral replication and pathogenicity. Eventually, and because of their important role during viral infection, we also describe their promising potential as targets for antiviral therapy and the development of live attenuated vaccines (LAV). Conclusively, both IAV NS1 and SARS-CoV-2 Nsp1 play an important role in virus–host interactions, viral replication, and pathogenesis, and pave the way to develop novel prophylactic and/or therapeutic interventions for the treatment of these important human respiratory viral pathogens.

Tripartite motif (TRIM)-containing proteins are a family of regulatory proteins that can participate in the induction of antiviral cytokines and antagonize viral replication. Promyelocytic leukemia (PML) protein is known as TRIM19 and is a major scaffold protein organizing the PML nuclear bodies (NBs). PML NBs are membrane-less organelles in the nucleus and play a diverse role in maintaining cellular homeostasis including antiviral response. Porcine reproductive and respiratory syndrome virus (PRRSV), a member virus of the family Arteriviridae, inhibits type I interferon (IFN) response during infection, and nonstructural protein 1 (nsp1) of the virus has been identified as a potent IFN antagonist. We report that the numbers of PML NBs per nucleus were significantly downregulated during infection of PRRSV. The overexpression of all six isoforms of PML suppressed the PRRSV replication, and conversely, the silencing of PML gene expression enhanced the PRRSV replication. The suppression of PML NBs by the nsp1 protein was common in other member viruses of the family, represented by equine arteritis virus, lactate dehydrogenase elevating virus of mice, and simian hemorrhagic fever virus. Our study unveils a conserved viral strategy in arteriviruses for innate immune evasion.

We describe an investigation using structural mass spectrometry (MS) of the impact of two antibodies, 15497 and 15498, binding the highly flexible SARS-CoV-2 Nsp1 protein. We determined the epitopes and paratopes involved in the antibody–protein interactions by using hydrogen–deuterium exchange MS (HDX-MS). Notably, the Fab (Fragment antigen binding) for antibody 15498 captured a high energy form of the antigen exhibiting significant conformational changes that added flexibility over most of the Nsp1 protein. The Fab for antibody 15497, however, showed usual antigen binding behavior, revealing local changes presumably including the binding site. These findings illustrate an unusual antibody effect on an antigen and are consistent with the dynamic nature of the Nsp1 protein. Our studies suggest that this interaction capitalizes on the high flexibility of Nsp1 to undergo conformational change and be trapped in a higher energy state by binding with a specific antibody. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea (PED) is an enterophilic infectious disease caused by the porcine epidemic diarrhea virus (PEDV), which can lead to dehydrationlike diarrhea in piglets with a mortality rate of up to 100%, causing huge economic losses to the global pig industry. In this study, we isolated two PEDV strains, FS202201 and JY202201, from diarrheal samples collected from two new PED outbreak farms in 2022. We performed phylogenetic analysis of the S gene and whole gene sequence. The effects of the different mutations on viral pathogenicity were investigated using piglet challenge experiments. The results showed that both strains belong to the G2c subtype, a widely prevalent virulent strain. Compared with FS202201, JY202201 harbored substitution and deletion mutations in nsp1. Both FS202201 and JY202201 infected piglets showed severe diarrhea and significant intestinal tissue lesions at an infection dose of 104 TCID50/mL, with a mortality rate of 50%; however, JY202201 required an additional day to reach mortality stabilization. An infection dose of 10³ TCID50/mL reduced diarrhea and intestinal tissue lesions in piglets, with mortality rates of the two strains at 16.7% and 0%, respectively. In addition, PEDV was detected in the heart, liver, spleen, lungs, kidneys, mesenteric lymph nodes, stomach, large intestine, duodenum, jejunum, and ileum, with the highest levels in the intestinal tissues. In conclusion, this study enriches the epidemiology of PEDV and provides a theoretical basis for the study of its pathogenic mechanism and prevention through virus isolation, identification, and pathogenicity research on newly identified PED in the main transmission hub area of PEDV in China (Guangdong). [ABSTRACT FROM AUTHOR]

Non-structural protein 1 (Nsp1) represents one of the most crucial SARS-CoV-2 virulence factors by inhibiting the translation of host mRNAs and promoting their degradation. We selected naturally occurring virus lineages with specific Nsp1 deletions located at both the N- and C-terminus of the protein. Our data provide new insights into how Nsp1 coordinates these functions on host and viral mRNA recognition. Residues 82–85 in the N-terminal part of Nsp1 likely play a role in docking the 40S mRNA entry channel, preserving the inhibition of host gene expression without affecting cellular mRNA decay. Furthermore, this domain prevents viral mRNAs containing the 5′-leader sequence to escape translational repression. These findings support the presence of distinct domains within the Nsp1 protein that differentially modulate mRNA recognition, translation and turnover. These insights have implications for the development of drugs targeting viral proteins and provides new evidences of how specific mutations in SARS-CoV-2 Nsp1 could attenuate the virus.

Swine acute diarrhea syndrome (SADS) is first reported in January 2017 in Southern China. It subsequently causes widespread outbreaks in multiple pig farms, leading to economic losses. Therefore, it is an urgent to understand the molecular mechanisms underlying the pathogenesis and immune evasion of Swine acute diarrhea syndrome coronavirus (SADS‐CoV). Our research discovered that SADS‐CoV inhibited the production of interferon‐β (IFN‐β) during viral infection. The nonstructural protein 1 (nsp1) prevented the phosphorylation of TBK1 by obstructing the interaction between TBK1 and Ub protein. Moreover, nsp1 induced the degradation of CREB‐binding protein (CBP) through the proteasome‐dependent pathway, thereby disrupting the IFN‐β enhancer and inhibiting IFN transcription. Finally, we identified nsp1‐Phe39 as the critical amino acid that downregulated IFN production. In conclusion, our findings described two mechanisms in nsp1 that inhibited IFN production and provided new insights into the evasion strategy adopted by SADS‐CoV to evade host antiviral immunity. [ABSTRACT FROM AUTHOR]

The identification of multiple simultaneous orientations of small molecule inhibitors binding to a protein target is a common challenge. It has recently been reported that the conformational heterogeneity of ligands is widely underreported in the Protein Data Bank, which is likely to impede optimal exploitation to improve affinity of these ligands. Significantly less is even known about multiple binding orientations for fragments (<300 Da), although this information would be essential for subsequent fragment optimisation using growing, linking or merging and rational structure-based design. Here, we use recently reported fragment hits for the SARS-CoV-2 non-structural protein 1 (nsp1) N-terminal domain to propose a general procedure for unambiguously identifying binding orientations of 2-dimensional fragments containing either sulphur or chloro substituents within the wavelength range of most tunable beamlines. By measuring datasets at two energies, using a tunable beamline operating in vacuum and optimised for data collection at very low X-ray energies, we show that the anomalous signal can be used to identify multiple orientations in small fragments containing sulphur and/or chloro substituents or to verify recently reported conformations. Although in this specific case we identified the positions of sulphur and chlorine in fragments bound to their protein target, we are confident that this work can be further expanded to additional atoms or ions which often occur in fragments. Finally, our improvements in the understanding of binding orientations will also serve to improve the rational optimisation of SARS-CoV-2 nsp1 fragment hits [ABSTRACT FROM AUTHOR]

Since its successful isolation in China in 1995, the porcine reproductive and respiratory syndrome virus (PRRSV) has been mutating into highly pathogenic strains by constantly changing pathogenicity and genetic makeup. In this study, we investigated the prevalence and genetic variation of nonstructural protein 1 (NSP1) in PRRSV-2, the main strain prevalent in China. After formulating hypotheses regarding the biology of the NSP1 protein, the nucleotide and amino acid similarity of NSP1 were analyzed and compared in 193 PRRSV-2 strains. The results showed that NSP1 has a stable hydrophobic protein with a molecular weight of 43,060.76 Da. Although NSP1 lacked signal peptides, it could regulate host cell signaling. Furthermore, NSP1 of different strains had high nucleotide (79.6–100%) and amino acid similarity (78.6–100%). In the amino acid sequence comparison of 15 representative strains of PRRSV-2, multiple amino acid substitution sites were found in NSP1. Phylogenetic tree analysis showed that lineages 1 and 8 had different evolutionary branches with long genetic distances. This study lays the foundation for an in-depth understanding of the nature and genetic variation of NSP1 and the development of a safe and effective vaccine in the future. [ABSTRACT FROM AUTHOR]

Autophagy, a cellular surveillance mechanism, plays an important role in combating invading pathogens. However, viruses have evolved various strategies to disrupt autophagy and even hijack it for replication and release. Here, we demonstrated that Middle East respiratory syndrome coronavirus (MERS-CoV) non-structural protein 1(nsp1) induces autophagy but inhibits autophagic activity. MERS-CoV nsp1 expression increased ROS and reduced ATP levels in cells, which activated AMPK and inhibited the mTOR signalling pathway, resulting in autophagy induction. Meanwhile, as an endonuclease, MERS-CoV nsp1 downregulated the mRNA of lysosome-related genes that were enriched in nsp1-located granules, which diminished lysosomal biogenesis and acidification, and inhibited autophagic flux. Importantly, MERS-CoV nsp1-induced autophagy can lead to cell death in vitro and in vivo. These findings clarify the mechanism by which MERS-CoV nsp1-mediated autophagy regulation, providing new insights for the prevention and treatment of the coronavirus.

MERS-CoV infection can damage the cellular metabolic processes, but the underlying mechanisms are largely unknown. Through screening, we found non-structural protein 1 (nsp1) of MERS-CoV could inhibit cell viability, cell cycle, and cell migration through its endonuclease activity. Transcriptome sequencing revealed that MERS-CoV nsp1 specifically downregulated the mRNAs of ribosomal protein genes, oxidative phosphorylation protein genes, and antigen presentation genes, but upregulated the mRNAs of transcriptional regulatory genes. Further analysis shown nsp1 existed in a novel ribonucleosome complex formed via liquid-liquid phase separation, which did not co-localize with mitochondria, lysosomes, P-bodies, or stress granules. Interestingly, the nsp1-located granules specifically contained mRNAs of ribosomal protein genes and oxidative phosphorylation genes, which may explain why MERS-CoV nsp1 selectively degraded these mRNAs in cells. Finally, MERS-CoV nsp1 transgenic mice showed significant loss of body weight and an increased sensitivity to poly(I:C)-induced inflammatory death. These findings demonstrate a new mechanism by which MERS-CoV impairs cell viability, which serves as a potential novel target for preventing MERS-CoV infection-induced pathological damage. Abbreviations: (Middle East respiratory syndrome coronavirus (MERS-CoV), Actinomycin D (Act D), liquid-liquid phase separation (LLPS), stress granules (SGs), Mass spectrometry (IP-MS), RNA Binding Protein Immunoprecipitation (RIP))

Coronaviruses cause mild to severe respiratory infections. The highly contagious SARS-CoV-2 new coronavirus caused a global outbreak of atypical viral pneumonia in late 2019. Acute respiratory distress syndrome, multiple organ failure, respiratory failure, and death can result from the infection. This study aimed to evaluate the possible inhibition activity from numerous medicinal plants' bioactive compounds against three non-structural proteins, namely Nsp1, Nsp2, and Nsp10 of SARS-CoV-2, through the computational study. Molecular docking was performed on the ligands and the target protein. This study investigated multiple criteria, including binding affinity value, location, and chemical interaction. In this present study, we found the top three highest binding affinity values of bioactive compounds against Nisp1, namely cafestol, crocin, and ledene; the top three highest binding affinity values of bioactive compounds against Nisp2, including cafestol, kahweol, and theaflavin 3,3'-digallate; and top three highest binding affinity value of bioactive compounds against Nisp10 namely cafestol, kahweol, and theaflavin-3,3'-digallate. Interestingly, we also found that cafestol, crocin, and theaflavin-3,3'-digallate binds to all target proteins. [ABSTRACT FROM AUTHOR]

Introduction: With the emergence of SARS-CoV-2 mutant strains, especially the epidemic of Omicron, it continues to evolve to strengthen immune evasion. Omicron BQ. 1 and XBB pose a serious threat to the current COVID-19 vaccine (including bivalent mRNA vaccine for mutant strains) and COVID-19-positive survivors, and all current therapeutic monoclonal antibodies are ineffective against them. Older people, those with multimorbidity, and those with specific underlying health conditions remain at increased risk of COVID-19 hospitalization and death after the initial vaccine booster. However, smallmolecule drugs for conserved targets remain effective and urgently needed. Methods: The non-structural protein of SARS-CoV-2 non-structural protein 1 (Nsp1) can bind to the host 40S ribosomal subunit and activate the nuclease to hydrolyze the host RNA, while the viral RNA is unaffected, thus hijacking the host system. First, the present study analyzed mutations in the Nsp1 protein and then constructed a maximum-likelihood phylogenetic tree. A virtual drug screening method based on the Nsp1 structure (Protein Data Bank ID: 7K5I) was constructed, 7495 compounds from three databases were collected for molecular docking and virtual screening, and the binding free energy was calculated by the MM/GBSA method. Results: Our study shows that Nsp1 is relatively conserved and can be used as a comparatively fixed drug target and that therapies against Nsp1 will target all of these variants. Golvatinib, Gliquidone, and Dihydroergotamine were superior to other compounds in the crystal structure of binding conformation and free energy. All effectively interfered with Nsp1 binding to 40S protein, confirming the potential inhibitory effect of these three compounds on SARS-CoV-2. Discussion: In particular, Golwatinib provides a candidate for treatment and prophylaxis in elderly patients with Omicjon, suggesting further evaluation of the anti-SARS-CoV-2 activity of these compounds in cell culture. Further studies are needed to determine the utility of this finding through prospective clinical trials and identify other meaningful drug combinations. [ABSTRACT FROM AUTHOR]

Replication of the severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2) strongly affects cellular metabolism and results in rapid development of the cytopathic effect (CPE). The hallmarks of virus-induced modifications are inhibition of translation of cellular mRNAs and redirection of the cellular translational machinery to the synthesis of virus-specific proteins. The multifunctional nonstructural protein 1 (nsp1) of SARS-CoV-2 is a major virulence factor and a key contributor to the development of translational shutoff. In this study, we applied a wide range of virological and structural approaches to further analyze nsp1 functions. The expression of this protein alone was found to be sufficient to cause CPE. However, we selected several nsp1 mutants exhibiting noncytopathic phenotypes. The attenuating mutations were detected in three clusters, located in the C-terminal helices, in one of the loops of the structured domain and in the junction of the disordered and structured fragment of nsp1. NMR-based analysis of the wild type nsp1 and its mutants did not confirm the existence of a stable b5-strand that was proposed by the X-ray structure. In solution, this protein appears to be present in a dynamic conformation, which is required for its functions in CPE development and viral replication. The NMR data also suggest a dynamic interaction between the N-terminal and C-terminal domains. The identified nsp1 mutations make this protein noncytotoxic and incapable of inducing translational shutoff, but they do not result in deleterious effects on viral cytopathogenicity. IMPORTANCE The nsp1 of SARS-CoV-2 is a multifunctional protein that modifies the intracellular environment for the needs of viral replication. It is responsible for the development of translational shutoff, and its expression alone is sufficient to cause a cytopathic effect (CPE). In this study, we selected a wide range of nsp1 mutants exhibiting noncytopathic phenotypes. The attenuating mutations, clustered in three different fragments of nsp1, were extensively characterized via virological and structural methods. Our data strongly suggest interactions between the nsp1 domains, which are required for the protein's functions in CPE development. Most of the mutations made nsp1 noncytotoxic and incapable of inducing translational shutoff. Most of them did not affect the viability of the viruses, but they did decrease the rates of replication in cells competent in type I IFN induction and signaling. These mutations, and their combinations, in particular, can be used for the development of SARS-CoV-2 variants with attenuated phenotypes. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the global pig industry, which modulates the host’s innate antiviral immunity to achieve immune evasion. RIG-I-like receptors (RLRs) sense viral RNA and activate the interferon signaling pathway. LGP2, a member of the RLR family, plays an important role in regulating innate immunity. However, the role of LGP2 in virus infection is controversial. Whether LGP2 has a role during infection with PRRSV remains unclear. Here, we found that LGP2 overexpression restrained the replication of PRRSV, while LGP2 silencing facilitated PRRSV replication. LGP2 was prone to interact with MDA5 and enhanced viral RNA enrichment and recognition by MDA5, thus promoting the activation of RIG-I/IRF3 and NF-kB signaling pathways and reinforcing the expression of proinflammatory cytokines and type I interferon during PRRSV infection. Meanwhile, there was a decreased protein expression of LGP2 upon PRRSV infection in vitro. PRRSV Nsp1 and Nsp2 interacted with LGP2 and promoted K63-linked ubiquitination of LGP2, ultimately leading to the degradation of LGP2. These novel findings indicate that LGP2 plays a role in regulating PRRSV replication through synergistic interaction with MDA5. Moreover, targeting LGP2 is responsible for PRRSV immune evasion. Our work describes a novel mechanism of virus-host interaction and provides the basis for preventing and controlling PRRSV. IMPORTANCE LGP2, a member of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), shows higher-affinity binding to RNA and work synergism with RIG-I or MDA5. However, LGP2 has divergent responses to different viruses, which remains controversial in antiviral immune responses. Here, we present the detailed process of LGP2 in positively regulating the anti-PRRSV response. Upon PRRSV infection, LGP2 was prone to bind to MDA5 and enhanced MDA5 signaling, manifesting the enrichment of viral RNA on MDA5 and the activation of downstream IRF3 and NF-kB, which results in increased proinflammatory cytokines and type I interferon expression, ultimately inhibiting PRRSV at the early stage of infection. Moreover, PRRSV Nsp1 and Nsp2 interacted with LGP2 via ubiquitinproteasome pathways, thus blocking LGP2-mediated immune response. This research helps us understand the host recognition and innate antiviral response to PRRSV infection by neglected pattern recognition receptors, which sheds light on the detailed mechanism of virus-host interaction. [ABSTRACT FROM AUTHOR]

Transport through the nuclear pore complex (NPC) relies on intrinsically disordered FG-nucleoporins (FG-Nups) forming a selective barrier. Away from the NPC, FG-Nups readily form condensates and aggregates, and we address how this behavior is surveilled in cells. FG-Nups, including Nsp1, together with the nuclear transport receptor Kap95, form a native daughter cell-specific cytosolic condensate in yeast. In aged cells, this condensate disappears as cytosolic Nsp1 levels decline. Biochemical assays and modeling show that Nsp1 is a modulator of FG-Nup condensates, promoting a liquid-like state. Nsp1's presence in the cytosol and condensates is critical, as a reduction of cytosolic levels in young cells induces NPC defects and a general decline in protein quality control that quantitatively mimics aging phenotypes. These phenotypes can be rescued by a cytosolic form of Nsp1. We conclude that Nsp1 is a phase state regulator that surveils FG-Nups and impacts general protein homeostasis. [Display omitted] • Nups form native cytosolic condensates • Nsp1 reduction mimics nuclear pore complex aging phenotypes • Nsp1 acts as phase-state modulator of FG-Nups • Nsp1 shares surveillance function with classical chaperones Here, Otto et al. address how the reduced expression of the intrinsically disordered FG-nucleoporin Nsp1 in aged cells relates to age-related nuclear pore complex assembly problems. They find that Nsp1 acts as a phase-state modulator, promoting a liquid-like state of FG-nucleoporin condensates and impacting general protein homeostasis. [ABSTRACT FROM AUTHOR]

As one of the most important swine enteropathogenic coronavirus, porcine epidemic diarrhea virus (PEDV) is the causative agent of an acute and devastating enteric disease that causes lethal watery diarrhea in suckling piglets. Recent progress in studying PEDV has revealed many intriguing findings on its prevalence and genetic evolution, rapid diagnosis, suppression of host gene expression, and suppression of the host innate immune system. Due to the continuous mutation of the PEDV genome, viral evasions from innate immune defenses and mixed infection with other coronaviruses, the spread of the virus is becoming wider and faster, making it even more necessary to prevent the infections caused by wild-type PEDV variants. It has also been reported that PEDV nsp1 is an essential virulence determinant and is critical for inhibiting host gene expression by structural and biochemical analyses. The inhibition of host protein synthesis employed by PEDV nsp1 may contribute to the regulation of host cell proliferation and immune evasion-related biological functions. In this review, we critically evaluate the recent studies on these aspects of PEDV and assess prospects in understanding the function of PEDV proteins in regulating host innate immune response and viral virulence. • PEDV is an important pathogenic microorganism that has a devastating impact on the swine industry. • The development of rapid diagnostic methods is important to curb the spread of PEDV and reduce economic losses. • PEDV nsp1 is an essential virulence determinant and is critical for inhibiting host gene expression. • PEDV M protein may contribute to evade the host innnate immune response and promote its replication. [ABSTRACT FROM AUTHOR]

An overarching goal of aging and age-related neurodegenerative disease research is to discover effective therapeutic strategies applicable to a broad spectrum of neurodegenerative diseases. Little is known about the extent to which targetable pathogenic mechanisms are shared among these seemingly diverse diseases. Translational control is critical for maintaining proteostasis during aging. Gaining control of the translation machinery is also crucial in the battle between viruses and their hosts. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic. Here, we show that overexpression of SARS-CoV-2–encoded nonstructural protein 1 (Nsp1) robustly rescued neuromuscular degeneration and behavioral phenotypes in Drosophila models of Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. These diseases share a common mechanism: the accumulation of aberrant protein species due to the stalling and collision of translating ribosomes, leading to proteostasis failure. Our genetic and biochemical analyses revealed that Nsp1 acted in a multipronged manner to resolve collided ribosomes, abort stalled translation, and remove faulty translation products causative of disease in these models, at least in part through the ribosome recycling factor ABCE1, ribosome-associated quality-control factors, autophagy, and AKT signaling. Nsp1 exhibited exquisite specificity in its action, as it did not modify other neurodegenerative conditions not known to be associated with ribosome stalling. These findings uncover a previously unrecognized mechanism of Nsp1 in manipulating host translation, which can be leveraged for combating agerelated neurodegenerative diseases that are affecting millions of people worldwide and currently without effective treatment. [ABSTRACT FROM AUTHOR]

Alongshan virus (ALSV) is a newly discovered pathogen in the Flaviviridae family, characterized by a unique multi-segmented genome that is distantly related to the canonical flaviviruses. Understanding the pathogenic mechanism of this emerging segmented flavivirus is crucial for the development of effective intervention strategies. In this study, we demonstrate that ALSV can infect various mammalian cells and induce the expression of antiviral genes. Furthermore, ALSV is sensitive to IFN-β, but it has developed strategies to counteract the host's type I IFN response. Mechanistically, ALSV's nonstructural protein NSP1 interacts with and degrades human STAT2 through an autophagy pathway, with species-dependent effects. This degradation directly inhibits the expression of interferon-stimulated genes (ISGs). Additionally, NSP1-mediated degradation of STAT2 disrupts mitochondrial dynamics, leading to mitophagy and inhibition of mitochondrial biogenesis. This, in turn, suppresses the host's innate immune response. Interestingly, we found that inhibiting mitophagy using 3-methyladenine and enhancing mitochondrial biogenesis with the PPARγ agonist pioglitazone can reverse NSP1-mediated inhibition of ISGs, suggesting that promoting mitochondrial mass could serve as an effective antiviral strategy. Specifically, the NSP1 methyltransferase domain binds to the key sites of F175/R176 located in the coiled-coil domain of STAT2. Our findings provide valuable insights into the intricate regulatory cross talk between ALSV and the host's innate immune response, shedding light on the pathogenesis of this emerging segmented flavivirus and offering potential intervention strategies.IMPORTANCEAlongshan virus (ALSV), a segmented flavivirus belonging to the Flaviviridae family, was first identified in individuals who had been bitten by ticks in Northeastern China. ALSV infection is responsible for causing Alongshan fever, a condition characterized by various clinical symptoms, including fever, headache, skin rash, myalgia, arthralgia, depression, and coma. There is an urgent need for effective antiviral therapies. Here, we demonstrate that ALSV is susceptible to IFN-β but has developed mechanisms to counteract the host's innate immune response. Specifically, the ALSV nonstructural protein NSP1 interacts with STAT2, leading to its degradation via an autophagy pathway that exhibits species-dependent effects. Additionally, NSP1 disrupts mitochondrial dynamics and suppresses mitochondrial biogenesis, resulting in a reduction in mitochondrial mass, which ultimately contributes to the inhibition of the host's innate immune response. Interestingly, we found that inhibiting mitophagy and promoting mitochondrial biogenesis can reverse NSP1-mediated suppression of innate immune response by increasing mitochondrial mass. These findings provide valuable insights into the molecular mechanisms of ALSV pathogenesis and suggest potential therapeutic targets against ALSV infection.

Major histocompatibility complex class I (MHC-I) plays crucial roles against viral infections not only by initiating CD8 + T cell immunity but also by modulating natural killer (NK) cell cytotoxicity. Understanding how viruses precisely regulate MHC-I to optimize their infection is important; however, the manipulation of MHC-I molecules by porcine epidemic diarrhea virus (PEDV) remains unclear. In this study, we demonstrate that PEDV infection promotes the transcription of NLRC5, a key transactivator of MHC-I, in several porcine cell lines and in vivo . Paradoxically, no increase in MHC-I expression is observed after PEDV infection both in vitro and in vivo . Mechanistic studies revealed that PEDV infection inhibits the translation of PEDV-elicited NLRC5 mRNA and the expression of downstream MHC-I proteins, without affecting the expression of physiological NLRC5 and MHC-I proteins. Through viral protein screening, we identified PEDV nonstructural protein 1 (nsp1) as the critical antagonist that inhibits NLRC5-mediated upregulation of MHC-I, and the nsp1's inhibitory effect on MHC-I requires the motif of 15 amino acids at its C-terminus. Notably, our results revealed that the cytotoxic ability of NK cells against PEDV-infected cells is similar to that against healthy cells. Collectively, our findings uncover an immune evasion mechanism by which PEDV-infected cells masquerade as healthy cells to evade NK and T cell immunity. This is achieved by targeting NLRC5, a key MHC-I transcriptional regulator, via nsp1.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) is a highly contagious enteric coronavirus that inflicts substantial financial losses on the swine industry. Major histocompatibility complex class I (MHC-I) is a critical factor influencing both CD8 + T cell and natural killer (NK) cell immunity. However, how PEDV manipulates MHC-I expression to optimize its infection process remains largely unknown. In this study, we demonstrate that PEDV's nonstructural protein 1 (nsp1) inhibits virus-mediated induction of MHC-I expression by directly targeting NLRC5, a key MHC-I transactivator. Intriguingly, nsp1 does not reduce physiological NLRC5 and MHC-I expression. This selective inhibition of virus-elicited NLRC5 mRNA translation allows PEDV-infected cells to masquerade as healthy cells, thereby evading CD8 + T cell and NK cell cytotoxicity. Our findings provide unique insights into the mechanisms by which PEDV evades CD8 + T cell and NK cell immunity., Competing Interests: The authors declare no conflict of interest.

Getah virus (GETV) is a mosquito-borne, single-stranded, positive-sense RNA virus belonging to the genus Alphavirus of the family Togaviridae. Natural infections of GETV have been identified in a variety of vertebrate species, with pathogenicity mainly in swine, horses, bovines, and foxes. The increasing spectrum of infection and the characteristic causing abortions in pregnant animals pose a serious threat to public health and the livestock economy. Therefore, there is an urgent need to establish a method that can be used for epidemiological investigation in multiple animals. In this study, a realtime reverse transcription fluorescent quantitative PCR (RT-qPCR) method combined with plaque assay was established for GETV with specific primers designed for the highly conserved region of GETV Nsp1 gene. The results showed that after optimizing the condition of RT-qPCR reaction, the minimum detection limit of the assay established in this study was 7.73 PFU/mL, and there was a good linear relationship between viral load and Cq value with a correlation coefficient (R2 ) of 0.998. Moreover, the method has good specificity, sensitivity, and repeatability. The established RT-qPCR is 100-fold more sensitive than the conventional RT-PCR. The best cutoff value for the method was determined to be 37.59 by receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) was 0.956. Meanwhile, we collected 2,847 serum specimens from swine, horses, bovines, sheep, and 17,080 mosquito specimens in Shandong Province in 2022. The positive detection rates by RT-qPCR were 1%, 1%, 0.2%, 0%, and 3%, respectively. In conclusion, the method was used for epidemiological investigation, which has extensive application prospects. [ABSTRACT FROM AUTHOR]

Zoonotic coronaviruses represent an ongoing threat to public health. The classical porcine epidemic diarrhea virus (PEDV) first appeared in the early 1970s. Since 2010, outbreaks of highly virulent PEDV variants have caused great economic losses to the swine industry worldwide. However, the strategies by which PEDV variants escape host immune responses are not fully understood. Complement component 3 (C3) is considered a central component of the three complement activation pathways and plays a crucial role in preventing viral infection. In this study, we found that C3 significantly inhibited PEDV replication in vitro, and both variant and classical PEDV strains induced high levels of interleukin-1β (IL-1β) in Huh7 cells. However, the PEDV variant strain reduces C3 transcript and protein levels induced by IL-1β compared with the PEDV classical strain. Examination of key molecules of the C3 transcriptional signaling pathway revealed that variant PEDV reduced C3 by inhibiting CCAAT/enhancer-binding protein β (C/EBP-β) phosphorylation. Mechanistically, PEDV nonstructural protein 1 (NSP1) inhibited C/EBP-β phosphorylation via amino acid residue 50. Finally, we constructed recombinant PEDVs to verify the critical role of amino acid 50 of NSP1 in the regulation of C3 expression. In summary, we identified a novel antiviral role of C3 in inhibiting PEDV replication and the viral immune evasion strategies of PEDV variants. Our study reveals new information on PEDV-host interactions and furthers our understanding of the pathogenic mechanism of this virus. [ABSTRACT FROM AUTHOR]

The rapid emergence and worldwide detection of the SARS-CoV-2 Omicron variant underscore the importance of robust genomic surveillance systems and prompt information sharing among global public health partners. The Omicron variant has rapidly replaced the Delta variant as a dominating SARS-CoV-2 variant because of natural selection, favoring the variant with higher infectivity and stronger vaccine breakthrough capability. The Omicron variant is also known as B.1.1.529. It has four sub-variants, indicated as BA.1, BA.2, BA.3 and BA.4. Among them, BA.1 is the currently prevailing sub-variant, and BA.2 has been found to be able to alarmingly re-infect patients initially infected by Omicron BA.1. The BA.3 sub-variant is a combination of mutations of BA.1 and BA.2, especially in the spike protein. Today, the BA.4 variant is emerging, which is herein described, and it was the first detected in Italy. Via bioinformatic analysis, we are reporting that the BA.4 that was identified harbors a new mutation, specifically a deletion in the ORF1ab gene, corresponding to KSF141_del in non-structural protein 1 (nsp1), a critical virulence factor able to suppress host translation. The bioinformatics comparison analysis with the other three sub-variants reveals that the deletion was not present before and was never reported until now. Therefore, we can speculate that Omicron BA.4 will become a new dominating "variant of concern" and may also break vaccine protection. Moreover, we show that other proteins are mutated in the BA.4. In particular, seven mutations are recognized in the nucleocapsid (N) protein, and the capability of five different types of rapid antigenic tests are used to identify it. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV), transmitted by mosquitoes, poses a significant global health threat. Presently, no effective treatment options are available to reduce the disease burden. The lack of approved therapeutics against CHIKV and the complex spectrum of chronic musculoskeletal and neurological manifestations raise significant concerns, and repurposing drugs could offer swift avenues in the development of effective treatment strategies. RNA capping is a crucial step meditated by non-structural protein 1 (nsP1) in CHIKV replication. In this study, FDA-approved antivirals targeting CHIKV nsP1 methyltransferase (MTase) have been identified by structure-based virtual screening. Berbamine Hydrochloride (BH), ABT199/Venetoclax (ABT), and Ponatinib (PT) were the top-hits, which exhibited robust binding energies. Tryptophan fluorescence spectroscopy-based assay confirmed binding of BH-, ABT-, and PT to purified nsP1 with K D values ∼5.45 μM, ∼161.3 μM, and ∼3.83 μM, respectively. In a capillary electrophoresis-based assay, a decrease in CHIKV nsP1 MTase activity was observed in a dose-dependent manner. Treatment with BH, ABT, and PT lead to a dose-dependent reduction in the virus titer with IC 50 < 100, ∼6.75, and <3.9 nM, respectively, and reduced viral mRNA levels. The nsP1 MTases are highly conserved among alphaviruses; therefore, BH, ABT, and PT, as expected, inhibited replication machinery in Sindbis virus (SINV) replicon assay with IC 50 ∼1.94, ∼0.23, and >1.25 μM, respectively. These results highlight the potential of repurposing drugs as rapid and effective antiviral therapeutics against CHIKV. [Display omitted] • Berbamine Hydrochloride (BH), ABT199/Venetoclax (ABT), and Ponatinib (PT) identified as inhibitors of CHIKV nsP1. • Molecules BH, ABT and PT make molecular interactions with nsP1 and effectively inhibit methyltransferase activity. • BH, ABT, and PT exhibited potent antiviral activity against CHIKV in cell-based assays, with IC 50 < 100, ∼6.75, and <3.9 nM. • BH, ABT, and PT also inhibit SINV replication machinery demonstrating broad spectrum antiviral efficacy. [ABSTRACT FROM AUTHOR]

Coronavirus disease 19 is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) enveloped virus with a single-stranded positive-sense ribonucleic acid (RNA) genome. The CoV non-structural protein (nsp) 1 is a multifunctional protein that undergoes translation shutoff, messenger RNA (mRNA) cleavage, and RNA binding. The C-terminal region is involved in translational shutoff and RNA cleavage. The N-terminal region of SARS-CoV-2 nsp1 is highly conserved among isolated SARS-CoV-2 variants. However, the I-004 variant, isolated during the early SARS-CoV-2 pandemic, lost eight amino acids in the nsp1 region. In this study, we showed that the eight amino acids are important for viral replication in infected interferon-incompetent cells and that the recombinant virus that lost these amino acids had low pathogenicity in the lungs of hamster models. The loss of eight amino acids-induced mutations occurred in the 5′ untranslated region (UTR), suggesting that nsp1 contributes to the stability of the viral genome during replication. • GDSVEEVL loss in SARS-CoV-2 nsp1 does not affect nsp1-mediated RNA cleavage & shutoff. • Loss of GDSVEEVL in SARS-CoV-2 nsp1 induced low viral replication and pathogenesis. • GDSVEEVL in SARS-CoV-2 nsp1 protects nucleotide deletion in its own UTR. [ABSTRACT FROM AUTHOR]

Coronavirus (CoV) nonstructural protein 1 (nsp1) inhibits cellular gene expression and antagonizes interferon (IFN) response. Porcine epidemic diarrhea virus (PEDV) infects pigs and causes high mortality in neonatal piglets. We hypothesized that a recombinant PEDV carrying mutations at the conserved residues N93 and N95 of nsp1 induces higher IFN responses and is more sensitive to IFN responses, leading to virus attenuation. We mutated PEDV nsp1 N93 and N95 to A93 and A95 to generate the recombinant N93/95A virus using the infectious clone of a highly virulent PEDV strain, PC22A (icPC22A), and evaluated N93/95A virus in vitro and in vivo. Compared with icPC22A, the N93/95A mutant replicated to significantly lower infectious titers, triggered stronger type I and III IFN responses, and was more sensitive to IFN treatment in vitro. To evaluate the pathogenicity and immunogenicity, 5-day-old gnotobiotic piglets were orally inoculated with the N93/95A or icPC22A strain or mock inoculated and then challenged at 22 days postinoculation (dpi) with icPC22A. icPC22A in all pigs (100% [5/5]) caused severe diarrhea and death within 6 dpi. Only one pig (25% [1/4]) died in the N93/95A group. Compared with the icPC22A group, significantly delayed and diminished fecal PEDV shedding was detected in the N93/95A group. Postchallenge, all piglets in N93/95A group were protected from severe diarrhea and death, whereas all pigs in the mock-challenged group developed severe diarrhea, and 25% (1/4) of them died. In summary, nsp1 N93A and N95A mutations attenuated PEDV but retained viral immunogenicity and can be targets for the development of live attenuated vaccines for PEDV. IMPORTANCE PEDV causes porcine epidemic diarrhea (PED) and remains a great threat to the swine industry worldwide because no effective vaccines are available yet. Safe and effective live attenuated vaccines can be designed using reverse genetics to induce lactogenic immunity in pregnant sows to protect piglets from the deadly PED. We found that an engineered PEDV mutant carrying N93A and N95A mutations of nsp1 was partially attenuated and remained immunogenic in neonatal pigs. Our study suggested that nsp1 N93 and N95 can be good targets for the rational design of live attenuated vaccines for PEDV using reverse genetics. Because CoV nsp1 is conserved among alphacoronaviruses (α-CoVs) and betacoronaviruses (β-CoVs), it may be a good target for vaccine development for other α-CoVs or β-CoVs. [ABSTRACT FROM AUTHOR]

Nonstructural protein 1 (Nsp1) of SARS-CoV-2 inhibits host cell translation through an interaction between its C-terminal domain and the 40S ribosome. The N-terminal domain (NTD) of Nsp1 is a target of recurring deletions, some of which are associated with altered COVID-19 disease progression. Here, we characterize the efficiency of translational inhibition by clinically observed Nsp1 deletion variants. We show that a frequent deletion of residues 79–89 severely reduces the ability of Nsp1 to inhibit translation while not abrogating Nsp1 binding to the 40S. Notably, while the SARS-CoV-2 50 untranslated region enhances translation of mRNA, it does not protect from Nsp1-mediated inhibition. Finally, thermal stability measurements and structure predictions reveal a correlation between stability of the NTD and the efficiency of translation inhibition. [ABSTRACT FROM AUTHOR]

Summary: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to shutoff of protein synthesis, and nsp1, a central shutoff factor in coronaviruses, inhibits cellular mRNA translation. However, the diverse molecular mechanisms employed by nsp1 as well as its functional importance are unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant, we show that nsp1, through inhibition of translation and induction of mRNA degradation, targets translated cellular mRNA and is the main driver of host shutoff during infection. The propagation of nsp1 mutant virus is inhibited exclusively in cells with intact interferon (IFN) pathway as well as in vivo, in hamsters, and this attenuation is associated with stronger induction of type I IFN response. Therefore, although nsp1’s shutoff activity is broad, it plays an essential role, specifically in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover nsp1’s explicit role in blocking the IFN response.

The molecular mechanisms that drive the infection by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)—the causative agent of coronavirus disease 2019 (COVID‐19)—are under intense current scrutiny to understand how the virus operates and to uncover ways in which the disease can be prevented or alleviated. Recent proteomic screens of the interactions between viral and host proteins have identified the human proteins targeted by SARS‐CoV‐2. The DNA polymerase α (Pol α)–primase complex or primosome—responsible for initiating DNA synthesis during genomic duplication—was identified as a target of nonstructural protein 1 (nsp1), a major virulence factor in the SARS‐CoV‐2 infection. Here, we validate the published reports of the interaction of nsp1 with the primosome by demonstrating direct binding with purified recombinant components and providing a biochemical characterization of their interaction. Furthermore, we provide a structural basis for the interaction by elucidating the cryo‐electron microscopy structure of nsp1 bound to the primosome. Our findings provide biochemical evidence for the reported targeting of Pol α by the virulence factor nsp1 and suggest that SARS‐CoV‐2 interferes with Pol α's putative role in the immune response during the viral infection. PDB Code(s): 7OPL; EMDB codes: 13020, 13021 [ABSTRACT FROM AUTHOR]

Abstract Background The new Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was first detected in Wuhan (China) in December of 2019 is responsible for the current global pandemic. Phylogenetic analysis revealed that it is similar to other betacoronaviruses, such as SARS-CoV and Middle-Eastern Respiratory Syndrome, MERS-CoV. Its genome is ∼ 30 kb in length and contains two large overlapping polyproteins, ORF1a and ORF1ab that encode for several structural and non-structural proteins. The non-structural protein 1 (nsp1) is arguably the most important pathogenic determinant, and previous studies on SARS-CoV indicate that it is both involved in viral replication and hampering the innate immune system response. Detailed experiments of site-specific mutagenesis and in vitro reconstitution studies determined that the mechanisms of action are mediated by (a) the presence of specific amino acid residues of nsp1 and (b) the interaction between the protein and the host’s small ribosomal unit. In fact, substitution of certain amino acids resulted in reduction of its negative effects. Methods A total of 17,928 genome sequences were obtained from the GISAID database (December 2019 to July 2020) from patients infected by SARS-CoV-2 from different areas around the world. Genomes alignment was performed using MAFFT (REFF) and the nsp1 genomic regions were identified using BioEdit and verified using BLAST. Nsp1 protein of SARS-CoV-2 with and without deletion have been subsequently modelled using I-TASSER. Results We identified SARS-CoV-2 genome sequences, from several Countries, carrying a previously unknown deletion of 9 nucleotides in position 686-694, corresponding to the AA position 241-243 (KSF). This deletion was found in different geographical areas. Structural prediction modelling suggests an effect on the C-terminal tail structure. Conclusions Modelling analysis of a newly identified deletion of 3 amino acids (KSF) of SARS-CoV-2 nsp1 suggests that this deletion could affect the structure of the C-terminal region of the protein, important for regulation of viral replication and negative effect on host’s gene expression. In addition, substitution of the two amino acids (KS) from nsp1 of SARS-CoV was previously reported to revert loss of interferon-alpha expression. The deletion that we describe indicates that SARS-CoV-2 is undergoing profound genomic changes. It is important to: (i) confirm the spreading of this particular viral strain, and potentially of strains with other deletions in the nsp1 protein, both in the population of asymptomatic and pauci-symptomatic subjects, and (ii) correlate these changes in nsp1 with potential decreased viral pathogenicity.

Abstract Background Coronavirus disease 2019 (COVID-19) is an emerging zoonotic viral infection, which was started in Wuhan, China, in December 2019 and transmitted to other countries worldwide as a pandemic outbreak. Iran is one of the top ranked countries in the tables of COVID-19-infected and -mortality cases that make the Iranian patients as the potential targets for diversity of studies including epidemiology, biomedical, biodata, and viral proteins computational modelling studies. Results In this study, we applied bioinformatic biodata mining methods to detect CDS and protein sequences of ORF1ab polyprotein of SARS-CoV-2 isolated from oronasopharynx of an Iranian patient. Then through the computational modelling and antigenicity prediction approaches, the identified polyprotein sequence was analyzed. The results revealed that the identified ORF1ab polyprotein belongs to a part of nonstructural protein 1 (nsp1) with the high antigenicity residues in a glycine-proline or hydrophobic amino acid rich domain. Conclusions The results revealed that nsp1 as a virulence factor and crucial agent in spreading of the COVID-19 among the society can be a potential target for the future epidemiology, drug, and vaccine studies.