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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.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.

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Competing interests statement:A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Castlevax, Amovir, Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories, Pharmamar, CureLab Oncology, CureLab Veterinary, Synairgen, Paratus, and Pfizer, outside of the reported work. A.G.-S. has been an invited speaker in meeting events organized by Seqirus, Janssen, Abbott, and Astrazeneca. A.G.-S. is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York.

The intrinsically disordered C-terminal peptide region of severe acute respiratory syndrome coronavirus 2 nonstructural protein-1 (Nsp1-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-CT 10 ), 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., Competing Interests: Competing interests statement:The authors declare no competing interest.

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.

SARS-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., Importance: Cyclosporine 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., Competing Interests: The authors declare no conflict of interest.

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.

Natural killer (NK) cells are cytotoxic effector cells that target and lyse virally infected cells; many viruses therefore encode mechanisms to escape such NK cell killing. Here, we interrogate the ability of SARS-CoV-2 to modulate NK cell recognition and lysis of infected cells. We find that NK cells exhibit poor cytotoxic responses against SARS-CoV-2-infected targets, preferentially killing uninfected bystander cells. We demonstrate that this escape is driven by downregulation of ligands for the activating receptor NKG2D (NKG2D-L). Indeed, early in viral infection, prior to NKG2D-L downregulation, NK cells are able to target and kill infected cells; however, this ability is lost as viral proteins are expressed. Finally, we find that SARS-CoV-2 non-structural protein 1 (Nsp1) mediates downregulation of NKG2D-L and that Nsp1 alone is sufficient to confer resistance to NK cell killing. Collectively, our work demonstrates that SARS-CoV-2 evades direct NK cell cytotoxicity and describes a mechanism by which this occurs., Competing Interests: Declaration of interests C.A.B. reports compensation for consulting and/or SAB membership from Catamaran Bio, DeepCell, Immunebridge, Sangamo Therapeutics, Bicycle Tx, and Revelation Biosciences on topics unrelated to this study. L.Q. is a founder and scientific advisor of Epic Bio, and a scientific advisor of Laboratory of Genomics Research. These roles are unrelated to the work., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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)).

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 age-related neurodegenerative diseases that are affecting millions of people worldwide and currently without effective treatment.

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. IMPORTANCE The complement system acts as a vital link between the innate and the adaptive immunity and has the ability to recognize and neutralize various pathogens. Activation of the complement system acts as a double-edged sword, as appropriate levels of activation protect against pathogenic infections, but excessive responses can provoke a dramatic inflammatory response and cause tissue damage, leading to pathological processes, which often appear in COVID-19 patients. However, how PEDV, as the most severe coronavirus causing diarrhea in piglets, regulates the complement system has not been previously reported. In this study, for the first time, we identified a novel mechanism of a PEDV variant in the suppression of C3 expression, showing that different coronaviruses and even different subtype strains differ in regulation of C3 expression. In addition, this study provides a deeper understanding of the mechanism of the PEDV variant in immune escape and enhanced virulence.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

The 5'UTR part of coronavirus genomes plays key roles in the viral replication cycle and translation of viral mRNAs. The first 75-80 nt, also called the leader sequence, are identical for genomic mRNA and subgenomic mRNAs. Recently, it was shown that cooperative actions of a 5'UTR segment and the nonstructural protein NSP1 are essential for both the inhibition of host mRNAs and for specific translation of viral mRNAs. Here, sequence analyses of both the 5'UTR RNA segment and the NSP1 protein have been done for several coronaviruses, with special attention to the betacoronaviruses. The conclusions are: (i) precise specific molecular signatures can be found in both the RNA and the NSP1 protein; (ii) both types of signatures correlate between each other. Indeed, definite sequence motifs in the RNA correlate with sequence motifs in the protein, indicating a coevolution between the 5'UTR and NSP1 in betacoronaviruses. Experimental mutational data on 5'UTR and NSP1 from SARS-CoV-2 using cell-free translation extracts support these conclusions and show that some conserved key residues in the amino-terminal half of the NSP1 protein are essential for evasion to the inhibitory effect of NSP1 on translation., (© 2022 Sosnowski et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Ubiquitination plays a major role in immune regulation after viral infection. An alternatively spliced porcine E3 ubiquitin ligase RNF122 promoted PRRSV infection and upregulated in PRRSV-infected PAM cells was identified. We characterized the core promoter of RNF122, located between -550 to -470 bp upstream of the transcription start site (TSS), which displayed significant differential transcriptional activities in regulating the transcription and expression of RNF122. The transcription factor HLTF was inhibited by nsp1α and nsp7 of PRRSV, and the transcription factor E2F complex regulated by nsp9. Together, they modulated the transcription and expression of RNF122. RNF122 could mediate K63-linked ubiquitination to raise stability of PRRSV nsp4 protein and thus promote virus replication. Moreover, RNF122 also performed K27-linked and K48-linked ubiquitination of MDA5 to degrade MDA5 and inhibit IFN production, ultimately promoted virus proliferation. In this study, we illustrate a new immune escape mechanism of PRRSV that enhances self-stability and function of viral nsp4, thus, regulating RNF122 expression to antagonize IFNα/β production. The present study broadens our knowledge of PRRSV-coding protein modulating transcription, expression and modification of host protein to counteract innate immune signaling, and may provide novel insights for the development of antiviral drugs.

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., (© 2021 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Rotavirus (RV)-encoded nonstructural protein 1 (NSP1), the product of gene segment 5, effectively antagonizes host interferon (IFN) signaling via multiple mechanisms. Recent studies with the newly established RV reverse genetics system indicate that NSP1 is not essential for the replication of the simian RV SA11 strain in cell culture. However, the role of NSP1 in RV infection in vivo remains poorly characterized due to the limited replication of heterologous simian RVs in the suckling mouse model. Here, we used an optimized reverse genetics system and successfully recovered recombinant murine RVs with or without NSP1 expression. While the NSP1-null virus replicated comparably with the parental murine RV in IFN-deficient and IFN-competent cell lines in vitro , it was highly attenuated in 5-day-old wild-type suckling pups in both the 129sv and C57BL/6 backgrounds. In the absence of NSP1 expression, murine RV had significantly reduced replication in the ileum, systemic spread to mesenteric lymph nodes, fecal shedding, diarrhea occurrence, and transmission to uninoculated littermates. The defective replication of the NSP1-null RV in small intestinal tissues occurred as early as 1 day postinfection. Of interest, the replication and pathogenesis defects of NSP1-null RV were only minimally rescued in Stat1 knockout pups, suggesting that NSP1 facilitates RV replication in an IFN-independent manner. Our findings highlight a pivotal function of NSP1 during homologous RV infections in vivo and identify NSP1 as an ideal viral protein for targeted attenuation for future vaccine development. IMPORTANCE Rotavirus remains one of the most important causes of severe diarrhea and dehydration in young children worldwide. Although NSP1 is dispensable for rotavirus replication in cell culture, its exact role in virus infection in vivo remains unclear. In this study, we demonstrate, for the first time in a pathologically valid homologous small animal model, that in the context of a fully replication-competent, pathogenic, and transmissible murine rotavirus, loss of NSP1 expression substantially attenuated virus replication in the gastrointestinal tract, diarrheal disease, and virus transmission. Notably, the NSP1-deficient murine rotavirus also replicated poorly in mice lacking host interferon or inflammasome signaling. Our data provide the first piece of evidence that NSP1 is essential for murine rotavirus replication in vivo , making it an attractive target for developing improved next-generation rotavirus vaccines better suited for socioeconomically disadvantaged and immunocompromised individuals.

The nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 is a critical viral protein that suppresses host gene expression by blocking the assembly of the ribosome on host mRNAs. To understand the mechanism of inhibition of host gene expression, we sought to identify cellular proteins that interact with nsp1. Using proximity-dependent biotinylation followed by proteomic analyses of biotinylated proteins, here we captured multiple dynamic interactions of nsp1 with host cell proteins. In addition to ribosomal proteins, we identified several pre-mRNA processing proteins that interact with nsp1, including splicing factors and transcription termination proteins, as well as exosome, and stress granule (SG)-associated proteins. We found that the interactions with transcription termination factors are primarily governed by the C-terminal region of nsp1 and are disrupted by the mutation of amino acids K164 and H165 that are essential for its host shutoff function. We further show that nsp1 interacts with Ras GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) and colocalizes with G3BP1 in SGs under sodium arsenite-induced stress. Finally, we observe that the presence of nsp1 disrupts the maturation of SGs over a long period. Isolation of SG core at different times shows a gradual loss of G3BP1 in the presence of nsp1., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Nsp1 of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of the Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1, eRF1 and ABCE1. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates peptide release and formation of termination complexes. Detailed analysis of Nsp1 activity during translation termination stages reveals that Nsp1 facilitates stop codon recognition. We demonstrate that Nsp1 stimulation targets eRF1 and does not affect eRF3. Moreover, Nsp1 increases amount of the termination complexes at all three stop codons. The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that the biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and remove them from the pool of the active ribosomes.

The coronavirus disease COVID-19 has been the cause of millions of deaths worldwide. Among the SARS-CoV-2 proteins, the non-structural protein 1 (NSP1) has great importance during the virus infection process and is present in both alpha and beta-CoVs. Therefore, monitoring of NSP1 polymorphisms is crucial in order to understand their role during infection and virus-induced pathogenicity. Herein, we analyzed how mutations detected in the circulating SARS-CoV-2 in the population of the city of Manaus, Amazonas state, Brazil could modify the tertiary structure of the NSP1 protein. Three mutations were detected in the SARS-CoV-2 NSP1 gene: deletion of the amino acids KSF from positions 141 to 143 (delKSF), SARS-CoV-2, lineage B.1.195; and two substitutions, R29H and R43C, SARS-CoV-2 lineage B.1.1.28 and B.1.1.33, respectively. The delKSF was found in 47 samples, whereas R29H and R43C were found in two samples, one for each mutation. The NSP1 structures carrying the mutations R43C and R29H on the N-terminal portion (e.g. residues 10 to 127) showed minor backbone divergence compared to the Wuhan model. However, the NSP1 C-terminal region (residues 145 to 180) was severely affected in the delKSF and R29H mutants. The intermediate variable region (residues 144 to 148) leads to changes in the C-terminal region, particularly in the delKSF structure. New investigations must be carried out to analyze how these changes affect NSP1 activity during the infection. Our results reinforce the need for continuous genomic surveillance of SARS-CoV-2 to better understand virus evolution and assess the potential impact of the viral mutations on the approved vaccines and future therapies.

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., Competing Interests: Declaration of interests B.A.G. is a member of the Vaccine Advisory Board for Celsion Corporation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The current COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has become a worldwide health crisis, necessitating coordinated scientific research and urgent identification of new drug targets for treatment of COVID-19 lung disease. The covid19-nmr consortium seeks to support drug development by providing publicly accessible NMR data on the viral RNA elements and proteins. The SARS-CoV-2 genome comprises a single RNA of about 30 kb in length, in which 14 open reading frames (ORFs) have been annotated, and encodes approximately 30 proteins. The first two-thirds of the SARS-CoV-2 genome is made up of two large overlapping open-reading-frames (ORF1a and ORF1b) encoding a replicase polyprotein, which is subsequently cleaved to yield 16 so-called non-structural proteins. The non-structural protein 1 (Nsp1), which is considered to be a major virulence factor, suppresses host immune functions by associating with host ribosomal complexes at the very end of its C-terminus. Furthermore, Nsp1 facilitates initiation of viral RNA translation via an interaction of its N-terminal domain with the 5' untranslated region (UTR) of the viral RNA. Here, we report the near-complete backbone chemical-shift assignments of full-length SARS-CoV-2 Nsp1 (19.8 kDa), which reveal the domain organization, secondary structure and backbone dynamics of Nsp1, and which will be of value to further NMR-based investigations of both the biochemical and physiological functions of Nsp1., (© 2021. The Author(s).)

Nonstructural protein 1 (Nsp1) of severe acute respiratory syndrome coronaviruses (SARS-CoVs) is an important pathogenic factor that inhibits host protein translation by means of its C terminus. However, its N-terminal function remains elusive. Here, we determined the crystal structure of the N terminus (amino acids [aa] 11 to 125) of SARS-CoV-2 Nsp1 at a 1.25-Å resolution. Further functional assays showed that the N terminus of SARS-CoVs Nsp1 alone loses the ability to colocalize with ribosomes and inhibit protein translation. The C terminus of Nsp1 can colocalize with ribosomes, but its protein translation inhibition ability is significantly weakened. Interestingly, fusing the C terminus of Nsp1 with enhanced green fluorescent protein (EGFP) or other proteins in place of its N terminus restored the protein translation inhibitory ability to a level equivalent to that of full-length Nsp1. Thus, our results suggest that the N terminus of Nsp1 is able to stabilize the binding of the Nsp1 C terminus to ribosomes and act as a nonspecific barrier to block the mRNA channel, thus abrogating host mRNA translation.

Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a nonstructural protein, Nsp1, for shutting down host translation. However, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing Nsp1. We perform RNA-seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation levels. We discover that a functionally coherent subset of human genes is preferentially translated in the context of Nsp1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we uncovered a remarkable enrichment of 5' terminal oligo-pyrimidine (TOP) tracts among preferentially translated genes. Using reporter assays, we validated that 5' UTRs from TOP transcripts can drive preferential expression in the presence of Nsp1. Finally, we found that LARP1, a key effector protein in the mTOR pathway, may contribute to preferential translation of TOP transcripts in response to Nsp1 expression. Collectively, our study suggests fine-tuning of host gene expression and translation by Nsp1 despite its global repressive effect on host protein synthesis., (© 2021 Rao et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Background: Non-structural protein 1 (Nsp1), a virulence agent of SARS-CoV-2, has emerged as an important target for drug discovery. Nsp1 shuts down the host gene function by associating with the 40S ribosomal subunit., Methods: Molecular interactions, drug-likeness, physiochemical property predictions, and robust molecular dynamics (MD) simulations were employed to discover novel Nsp1 inhibitors. In this study, we evaluated a series of molecules based on the plant (Cedrus deodara) derived α,β,γ-Himachalenes scaffolds., Results: The results obtained from estimated affinity and ligand efficiency suggested that BCH10, BCH15, BCH16, and BCH17 could act as potential inhibitors of Nsp1. Moreover, MD simulations comprising various MD driven time-dependent analyses and thermodynamic free energy calculations also suggested stable protein-ligand complexes and strong interactions with the binding site. Furthermore, the selected molecules passed drug likeliness parameters and the physiochemical property analysis showed acceptable bioactivity scores., Conclusion: The structural parameters of dynamic simulations revealed that the reported molecules could act as lead compounds against SARS-CoV-2 Nsp1 protein., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Chikungunya virus (CHIKV) nonstructural protein 1 (nsP1) harbors the methyltransferase (MTase) and guanylyltransferase (GTase) activities needed for viral RNA capping and represents a promising antiviral drug target. We compared the antiviral efficacies of nsP1 inhibitors belonging to the MADTP, CHVB, and FHNA series (6'-fluoro-homoneplanocin A [FHNA], its 3'-keto form, and 6'-β-fluoro-homoaristeromycin). Cell-based phenotypic cross-resistance assays revealed that the CHVB and MADTP series had similar modes of action that differed from that of the FHNA series. In biochemical assays with purified Semliki Forest virus and CHIKV nsP1, CHVB compounds strongly inhibited MTase and GTase activities, while MADTP-372 had a moderate inhibitory effect. FHNA did not directly inhibit the enzymatic activity of CHIKV nsP1. The first-of-their-kind molecular-docking studies with the cryo-electron microscopy (cryo-EM) structure of CHIKV nsP1, which is assembled into a dodecameric ring, revealed that the MADTP and CHVB series bind at the S -adenosylmethionine (SAM)-binding site in the capping domain, where they would function as competitive or noncompetitive inhibitors. The FHNA series was predicted to bind at the secondary binding pocket in the r ing- a perture m embrane- b inding and o ligomerization (RAMBO) domain, potentially interfering with the membrane binding and oligomerization of nsP1. Our cell-based and enzymatic assays, in combination with molecular docking and mapping of compound resistance mutations to the nsP1 structure, allowed us to group nsP1 inhibitors into functionally distinct classes. This study identified druggable pockets in the nsP1 dodecameric structure and provides a basis for the rational design, optimization, and combination of inhibitors of this unique and promising antiviral drug target.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic. While previous studies have shown that several SARS-CoV-2 proteins can antagonize the interferon (IFN) response, some of the mechanisms by which they do so are not well understood. In this study, we describe two novel mechanisms by which SARS-CoV-2 blocks the IFN pathway. Type I IFNs and IFN-stimulated genes (ISGs) were poorly induced during SARS-CoV-2 infection, and once infection was established, cells were highly resistant to ectopic induction of IFNs and ISGs. Levels of two key IFN signaling pathway components, Tyk2 and STAT2, were significantly lower in SARS-CoV-2-infected cells. Expression of nonstructural protein 1 (NSP1) or nucleocapsid in the absence of other viral proteins was sufficient to block IFN induction, but only NSP1 was able to inhibit IFN signaling. Mapping studies suggest that NSP1 prevents IFN induction in part by blocking IRF3 phosphorylation. In addition, NSP1-induced depletion of Tyk2 and STAT2 dampened ISG induction. Together, our data provide new insights into how SARS-CoV-2 successfully evades the IFN system to establish infection. IMPORTANCE SARS-CoV-2 is the causative agent of COVID-19, a serious disease that can have a myriad of symptoms from loss of taste and smell to pneumonia and hypercoagulation. The rapid spread of SARS-CoV-2 can be attributed in part to asymptomatic transmission, where infected individuals shed large amounts of virus before the onset of disease. This is likely due to the ability of SARS-CoV-2 to effectively suppress the innate immune system, including the IFN response. Indeed, we show that the IFN response is efficiently blocked during SARS-CoV-2 infection, a process that is mediated in large part by nonstructural protein 1 and nucleocapsid. Our study provides new insights on how SARS-CoV-2 evades the IFN response to successfully establish infection. These findings should be considered for the development and administration of therapeutics against SARS-CoV-2.

Type I interferons (IFNs) are produced by most cells in response to virus infection and stimulate a program of anti-viral gene expression in neighboring cells to suppress virus replication. Type III IFNs have similar properties, however their effects are limited to epithelial cells at mucosal surfaces due to restricted expression of the type III IFN receptor. Rotavirus (RV) replicates in intestinal epithelial cells that respond predominantly to type III IFNs, and it has been shown that type III rather than type I IFNs are important for controlling RV infections in vivo. The RV NSP1 protein antagonizes the host type I IFN response by targeting IRF-3, IRF-5, IRF-7, or β-TrCP for proteasome-mediated degradation in a strain-specific manner. Here we provide the first demonstration that NSP1 proteins from several human and animal RV strains antagonize type III as well as type I IFN induction. We also show that NSP1 is a potent inhibitor of IRF-1, a previously undescribed property of NSP1 which is conserved among human and animal RVs. Interestingly, all NSP1 proteins were substantially more effective inhibitors of IRF-1 than either IRF-3 or IRF-7 which has significance for evasion of basal anti-viral immunity and type III IFN induction in the intestinal epithelium.

The SARS-CoV-2 virus, the causative agent of COVID-19, is undergoing constant mutation. Here, we utilized an integrative approach combining epidemiology, virus genome sequencing, clinical phenotyping, and experimental validation to locate mutations of clinical importance. We identified 35 recurrent variants, some of which are associated with clinical phenotypes related to severity. One variant, containing a deletion in the Nsp1-coding region (Δ500-532), was found in more than 20% of our sequenced samples and associates with higher RT-PCR cycle thresholds and lower serum IFN-β levels of infected patients. Deletion variants in this locus were found in 37 countries worldwide, and viruses isolated from clinical samples or engineered by reverse genetics with related deletions in Nsp1 also induce lower IFN-β responses in infected Calu-3 cells. Taken together, our virologic surveillance characterizes recurrent genetic diversity and identified mutations in Nsp1 of biological and clinical importance, which collectively may aid molecular diagnostics and drug design., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. We demonstrate biochemically that NSP1 inhibits translation of model human and SARS-CoV-2 messenger RNAs (mRNAs). NSP1 specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1-40S subunit binding in real time, we determine that eukaryotic translation initiation factors (eIFs) allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of mRNA. We further elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 proteins from viruses in at least two subgenera of beta-CoVs associate with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Many viruses disrupt host gene expression by degrading host mRNAs and/or manipulating translation activities to create a cellular environment favorable for viral replication. Often, virus-induced suppression of host gene expression, including those involved in antiviral responses, contributes to viral pathogenicity. Accordingly, clarifying the mechanisms of virus-induced disruption of host gene expression is important for understanding virus-host cell interactions and virus pathogenesis. Three highly pathogenic human coronaviruses (CoVs), including severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2, have emerged in the past two decades. All of them encode nonstructural protein 1 (nsp1) in their genomes. Nsp1 of SARS-CoV and MERS-CoV exhibit common biological functions for inducing endonucleolytic cleavage of host mRNAs and inhibition of host translation, while viral mRNAs evade the nsp1-induced mRNA cleavage. SARS-CoV nsp1 is a major pathogenic determinant for this virus, supporting the notion that a viral protein that suppresses host gene expression can be a virulence factor, and further suggesting the possibility that SARS-CoV-2 nsp1, which has high amino acid identity with SARS-CoV nsp1, may serve as a major virulence factor. This review summarizes the gene expression suppression functions of nsp1 of CoVs, with a primary focus on SARS-CoV nsp1 and MERS-CoV nsp1.

Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs -1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical -1 and -2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1β (nsp1β) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1β and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate -1 and -2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1β and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1β:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1β within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1β and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1β and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism., Competing Interests: Conflict of interest—E. J. S. and E. E. T. have a patent that relates to aspects of this work (patent number: US9623103)., (© 2020 Patel et al.)

The causative virus of the COVID-19 pandemic, SARS-CoV-2, uses its nonstructural protein 1 (Nsp1) to suppress cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the cellular transcriptome. Our cryo-EM structure of the Nsp1-40S ribosome complex shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the structure of the 48S preinitiation complex formed by Nsp1, 40S, and the cricket paralysis virus internal ribosome entry site (IRES) RNA, which shows that it is nonfunctional because of the incorrect position of the mRNA 3' region. Our results elucidate the mechanism of host translation inhibition by SARS-CoV-2 and advance understanding of the impacts from a major pathogenicity factor of SARS-CoV-2., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the coronavirus disease 2019 (COVID-19) pandemic. Accurate detection of SARS-CoV-2 using molecular assays is critical for patient management and the control of the COVID-19 pandemic. However, there is an increasing number of SARS-CoV-2 viruses with mutations at the primer or probe binding sites, and these mutations may affect the sensitivity of currently available real-time reverse transcription-polymerase chain reaction (RT-PCR) assays targeting the nucleocapsid (N), envelope (E), and open reading frame 1a or 1b genes. Using sequence-independent single-primer amplification and nanopore whole-genome sequencing, we have found that the nonstructural protein 1 (nsp1) gene, located at the 5' end of the SARS-CoV-2 genome, was highly expressed in the nasopharyngeal or saliva specimens of 9 COVID-19 patients of different clinical severity. Based on this finding, we have developed a novel nsp1 real-time RT-PCR assay. The primers and probes are highly specific for SARS-CoV-2. Validation with 101 clinical specimens showed that our nsp1 RT-PCR assay has a sensitivity of 93.1% (95% confidence interval [CI]: 86.2%-97.2%), which was similar to those of N and E gene RT-PCR assays. The diagnostic specificity was 100% (95% CI: 92.9%-100%). The addition of nsp1 for multitarget detection of SARS-CoV-2 can avoid false-negative results due to mutations at the primers/probes binding sites of currently available RT-PCR assays., (© 2020 Wiley Periodicals LLC.)

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.

Coronaviruses (CoVs) have repeatedly emerged from wildlife hosts and infected humans and livestock animals to cause epidemics with significant morbidity and mortality. CoVs infect various organs, including respiratory and enteric systems, as exemplified by newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The constellation of viral factors that contribute to developing enteric disease remains elusive. Here, we investigated CoV interferon antagonists for their contribution to enteric pathogenesis. Using an infectious clone of an enteric CoV, porcine epidemic diarrhea virus (icPEDV), we generated viruses with inactive versions of interferon antagonist nonstructural protein 1 (nsp1), nsp15, and nsp16 individually or combined into one virus designated icPEDV-mut4. Interferon-responsive PK1 cells were infected with these viruses and produced higher levels of interferon responses than were seen with wild-type icPEDV infection. icPEDV-mut4 elicited robust interferon responses and was severely impaired for replication in PK1 cells. To evaluate viral pathogenesis, piglets were infected with either icPEDV or icPEDV-mut4. While the icPEDV-infected piglets exhibited clinical disease, the icPEDV-mut4-infected piglets showed no clinical symptoms and exhibited normal intestinal pathology at day 2 postinfection. icPEDV-mut4 replicated in the intestinal tract, as revealed by detection of viral RNA in fecal swabs, with sequence analysis documenting genetic stability of the input strain. Importantly, icPEDV-mut4 infection elicited IgG and neutralizing antibody responses to PEDV. These results identify nsp1, nsp15, and nsp16 as virulence factors that contribute to the development of PEDV-induced diarrhea in swine. Inactivation of these CoV interferon antagonists is a rational approach for generating candidate vaccines to prevent disease and spread of enteric CoVs, including SARS-CoV-2. IMPORTANCE Emerging coronaviruses, including SARS-CoV-2 and porcine CoVs, can infect enterocytes, cause diarrhea, and be shed in the feces. New approaches are needed to understand enteric pathogenesis and to develop vaccines and therapeutics to prevent the spread of these viruses. Here, we exploited a reverse genetic system for an enteric CoV, porcine epidemic diarrhea virus (PEDV), and outline an approach of genetically inactivating highly conserved viral factors known to limit the host innate immune response to infection. Our report reveals that generating PEDV with inactive versions of three viral interferon antagonists, nonstructural proteins 1, 15, and 16, results in a highly attenuated virus that does not cause diarrhea in animals and elicits a neutralizing antibody response in virus-infected animals. This strategy may be useful for generating live attenuated vaccine candidates that prevent disease and fecal spread of enteric CoVs, including SARS-CoV-2., (Copyright © 2020 American Society for Microbiology.)

Alphavirus genome replication is carried out by the viral replication complex inside modified membrane structures called spherules. The viral nonstructural protein 1 (nsP1) is the only membrane-associated protein that anchors the replication complex to the cellular membranes. Although an internal amphipathic helix of nsP1 is critical for membrane association, the mechanism of nsP1 interaction with membranes and subsequent membrane reorganization is not well understood. We studied the membrane interaction of chikungunya virus (CHIKV) nsP1 and show that both the CHIKV nsP1 protein and the amphipathic peptide specifically bind to negatively charged phospholipid vesicles. Using cryo-electron microscopy, we further show that nsP1 forms a contiguous coat on lipid vesicles and induces structural reorganization, while the amphipathic peptide alone failed to deform the membrane bilayer. This suggests that although amphipathic helix of nsP1 is required for initial membrane binding, the remaining cytoplasmic domain of nsP1 is involved in the subsequent membrane reorganization., Competing Interests: Declaration of competing interest None., (Copyright © 2020. Published by Elsevier Inc.)

Two type 2 field porcine reproductive and respiratory syndrome viruses (PRRSV) isolated from PRRS-affected swine farms were attenuated by de-optimization of codon pair bias in NSP1. In 3-week-old pigs infection, the attenuated viruses showed significantly lower replication ability than the original viruses without distinct clinical sign and pathological lesions, which were observed in pig infected with the original viruses. Regarding induction of PRRSV specific immunity, the level of the neutralizing antibodies as well as secretion of IFN-γ-SCs in PBMCs was not different between the attenuated viruses and the original viruses. More importantly, pigs infected with the attenuated viruses exhibited significant reduction in respiratory scores, viremia, macroscopic and microscopic lung lesion scores, and PRRSV-antigen with interstitial pneumonia against a heterologous challenge with a type 2 virulent strain. Conclusively, the viruses attenuated by CPD in this study demonstrated potential usefulness as vaccine strains to provide protective immunity against diverse virulent PRRSVs., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome coronavirus nonstructural protein 1 (nsp1) is a key factor in virus-induced down-regulation of host gene expression. In infected cells, nsp1 engages in a multipronged mechanism to inhibit host gene expression by binding to the 40S ribosome to block the assembly of translationally competent ribosome, and then inducing endonucleolytic cleavage and the degradation of host mRNAs. Here, we report a previously undetected mechanism by which nsp1 exploits the nuclear pore complex and disrupts the nuclear-cytoplasmic transport of biomolecules. We identified members of the nuclear pore complex from the nsp1-associated protein assembly and found that the expression of nsp1 in HEK cells disrupts Nup93 localization around the nuclear envelope without triggering proteolytic degradation, while the nuclear lamina remains unperturbed. Consistent with its role in host shutoff, nsp1 alters the nuclear-cytoplasmic distribution of an RNA binding protein, nucleolin. Our results suggest that nsp1, alone, can regulate multiple steps of gene expression including nuclear-cytoplasmic transport.

Porcine reproductive and respiratory syndrome virus (PRRSV) blocks host mRNA nuclear export to the cytoplasm, and nonstructural protein 1 beta (nsp1β) of PRRSV has been identified as the protein that disintegrates the nuclear pore complex. In the present study, the molecular basis for the inhibition of host mRNA nuclear export was investigated. Nucleoporin 62 (Nup62) was found to bind to nsp1β, and the region representing the C-terminal residues 328 to 522 of Nup62 was determined to be the binding domain for nsp1β. The nsp1β L126A mutant in the SAP domain did not bind to Nup62, and in L126A-expressing cells, host mRNA nuclear export occurred normally. The vL126A mutant PRRSV generated by reverse genetics replicated at a lower rate, and the titer was lower than for wild-type virus. In nsp1β-overexpressing cells or small interfering RNA (siRNA)-mediated Nup62 knockdown cells, viral protein synthesis increased. Notably, the production of type I interferons (IFN-α/β), IFN-stimulated genes (PKR, OAS, Mx1, and ISG15 genes), IFN-induced proteins with tetratricopeptide repeats (IFITs) 1 and 2, and IFN regulatory factor 3 decreased in these cells. As a consequence, the growth of vL126A mutant PRRSV was rescued to the level of wild-type PRRSV. These findings are attributed to nuclear pore complex (NPC) disintegration by nsp1β, resulting in increased viral protein production and decreased host protein production, including antiviral proteins in the cytoplasm. Our study reveals a new strategy of PRRSV for immune evasion and enhanced replication during infection. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS and is known to effectively suppress host innate immunity. The PRRSV nsp1β protein blocks host mRNA nuclear export, which has been shown to be one of the viral mechanisms for inhibition of antiviral protein production. nsp1β binds to the cellular protein nucleoporin 62 (Nup62), and as a consequence, the nuclear pore complex (NPC) is disintegrated and the nucleocytoplasmic trafficking of host mRNAs and host proteins is blocked. We show the dual benefits of Nup62 and nsp1β binding for PRRSV replication: the inhibition of host antiviral protein expression and the exclusive use of host translation machinery by the virus. Our study unveils a novel strategy of PRRSV for immune evasion and enhanced replication during infection., (Copyright © 2019 American Society for Microbiology.)

Chikungunya virus (CHIKV) is transmitted to people by mosquitoes, and CHIKV infection causes fever and joint pain. Fatty acid synthase (FASN) has been identified as a proviral factor for CHIKV. How FASN participates in CHIKV replication remains to be elucidated. In this study, we demonstrated that palmitic acid (PA) can restore the suppression of CHIKV replication by FASN inhibitors. The palmitoylation and plasma membrane localization of CHIKV nsP1 were reduced by FASN inhibitors. Triple mutation of Cys417, Cys418, and Cys419 in nsP1 blocked its palmitoylation and severely disrupted CHIKV replication. Furthermore, two zinc finger DHHC domain-containing palmitoyltransferases (ZDHHCs), ZDHHC2 and ZDHHC19, promoted nsP1 palmitoylation and CHIKV replication. Our results not only identified the key enzymes for the palmitoylation of nsP1 but also provided mechanistic insights into the roles of FASN in CHIKV replication. IMPORTANCE S-palmitoylation is an important form of lipid posttranslational modification, which affects the function of proteins by regulating their transport, stability, and localization. Previous studies have shown that FASN is critical for CHIKV replication; however, the mechanism for this function of FASN remains unknown. The key zinc finger DHHC domain-containing palmitoyltransferases involved in the palmitoylation of nsP1 are not clear. We demonstrated that FASN promoted CHIKV replication through nsP1 palmitoylation. ZDHHC2 and ZDHHC19 were identified as the major enzymes for nsP1 palmitoylation. Since nsP1 proteins are conserved in alphaviruses, our results highlight the mechanisms by which alphavirus nsP1 is palmitoylated., (Copyright © 2019 American Society for Microbiology.)

Middle East respiratory syndrome coronavirus (MERS-CoV) nsp1 suppresses host gene expression in expressed cells by inhibiting translation and inducing endonucleolytic cleavage of host mRNAs, the latter of which leads to mRNA decay. We examined the biological functions of nsp1 in infected cells and its role in virus replication by using wild-type MERS-CoV and two mutant viruses with specific mutations in the nsp1; one mutant lacked both biological functions, while the other lacked the RNA cleavage function but retained the translation inhibition function. In Vero cells, all three viruses replicated efficiently with similar replication kinetics, while wild-type virus induced stronger host translational suppression and host mRNA degradation than the mutants, demonstrating that nsp1 suppressed host gene expression in infected cells. The mutant viruses replicated less efficiently than wild-type virus in Huh-7 cells, HeLa-derived cells, and 293-derived cells, the latter two of which stably expressed a viral receptor protein. In 293-derived cells, the three viruses accumulated similar levels of nsp1 and major viral structural proteins and did not induce IFN -β and IFN -λ mRNAs; however, both mutants were unable to generate intracellular virus particles as efficiently as wild-type virus, leading to inefficient production of infectious viruses. These data strongly suggest that the endonucleolytic RNA cleavage function of the nsp1 promoted MERS-CoV assembly and/or budding in a 293-derived cell line. MERS-CoV nsp1 represents the first CoV gene 1 protein that plays an important role in virus assembly/budding and is the first identified viral protein whose RNA cleavage-inducing function promotes virus assembly/budding. IMPORTANCE MERS-CoV represents a high public health threat. Because CoV nsp1 is a major viral virulence factor, uncovering the biological functions of MERS-CoV nsp1 could contribute to our understanding of MERS-CoV pathogenicity and spur development of medical countermeasures. Expressed MERS-CoV nsp1 suppresses host gene expression, but its biological functions for virus replication and effects on host gene expression in infected cells are largely unexplored. We found that nsp1 suppressed host gene expression in infected cells. Our data further demonstrated that nsp1, which was not detected in virus particles, promoted virus assembly or budding in a 293-derived cell line, leading to efficient virus replication. These data suggest that nsp1 plays an important role in MERS-CoV replication and possibly affects virus-induced diseases by promoting virus particle production in infected hosts. Our data, which uncovered an unexpected novel biological function of nsp1 in virus replication, contribute to further understanding of the MERS-CoV replication strategies., (Copyright © 2018 American Society for Microbiology.)

Chikungunya virus (CHIKV) is an arthropod-borne alphavirus. Alphaviruses are positive strand RNA viruses that require a 5' cap structure to direct translation of the viral polyprotein and prevent degradation of the viral RNA genome by host cell nucleases. Formation of the 5' RNA cap is orchestrated by the viral protein nsP1, which binds GTP and provides the N-7 methyltransferase and guanylyltransferase activities that are necessary for cap formation. Viruses with aberrant nsP1 activity are unable to replicate effectively suggesting that nsP1 is a promising target for antiviral drug discovery. Given the absence of commercially available antiviral therapies for CHIKV, it is imperative to identify compounds that could be developed as potential therapeutics. This study details a high-throughput screen of 3051 compounds from libraries containing FDA-approved drugs, natural products, and known bioactives against CHIKV nsP1 using a fluorescence polarization-based GTP competition assay. Several small molecule hits from this screen were able to compete with GTP for the CHIKV nsP1 GTP binding site at low molar concentrations. Compounds were also evaluated with an orthogonal assay that measured the ability of nsP1 to perform the guanylation step of the capping reaction in the presence of inhibitor. In addition, live virus assays with CHIKV and closely related alphavirus, Sindbis virus, were used in conjunction with cell toxicity assays to determine the antiviral activity of compounds in cell culture. The naturally derived compound lobaric acid was found to inhibit CHIKV nsP1 GTP binding and guanylation as well as attenuate viral growth in vitro at both 24 hpi and 48 hpi in hamster BHK21 and human Huh 7 cell lines. These data indicate that development of lobaric acid and further exploration of CHIKV nsP1 as a drug target may aid in the progress of anti-alphaviral drug development strategies., (Copyright © 2018. Published by Elsevier B.V.)

Porcine reproductive and respiratory syndrome virus (PRRSV) has the ability to suppress the type I interferons (IFNs-α/β) induction to facilitate its survival during infection, and the nsp1 protein of PRRSV has been identified as the potent IFN antagonist. The nsp1β subunit of nsp1 has also been shown to block the host mRNA nuclear export as one of the mechanisms to suppress host antiviral protein production. The SAP motif in nsp1β is the functional motif for both IFN suppression and host mRNA nuclear retention, and using infectious clones, two mutant viruses vL126A and vL135A have been generated. These mutants retain the infectivity, but the phenotype is negative for both IFN suppression and host mRNA nuclear retention due to the loss of the SAP motif. To examine the pathogenic role of IFN suppression in pigs, 40 piglets were allotted to four groups and each group was intramuscularly infected with vL126A, vL135A, wild-type (WT) PRRSV, and placebo. Pigs infected with vL126A or vL135A exhibited mild clinical signs with low viral titers and short duration of viremia. The levels of PRRSV-specific antibody remained comparable in all infected groups but the neutralizing antibody titers were high in vL126A-infected or vL135A-infected pigs. The IFN-α concentration was also high in pigs infected with the SAP mutants. Reversion to WT sequence was observed in the SAP motif in some animals, and the revertants regained the function to suppress IFN production and host mRNA nuclear export, indicating strong selection pressure in the SAP motif of nsp1β. Together, our data demonstrate that the IFN antagonism and host mRNA nuclear retention mediated by nsp1β contributes to viral virulence, and loss of these functions confers PRRSV attenuation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

MERS-CoV is the only lethal human CoV still endemic in the Arabian Peninsula and neither vaccine nor therapeutics against MERS-CoV infection is available. The nsp1 of CoV is thought to be a major virulence factor because it suppresses protein synthesis through the degradation of host mRNA. In contrast, viral RNA circumvents the nsp1-mediated translational shutoff for an efficient propagation. In this study, we identified amino acid residue in MERS-CoV nsp1 that differ from those of SARS-CoV nsp1, and that appear to be crucial for circumventing the translational shutoff. In addition, reverse genetics analysis suggested the presence of a cis-acting element at the 5'-terminus of the nsp1-coding region, which contributes to the specific recognition of viral RNA that is required for an efficient viral replication. Our results suggest the CoVs share a common mechanism for circumventing the nsp1-mediated translational shutoff., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Porcine epidemic diarrhea virus emerged in the US is known to suppress the type I interferons response during infection. In the present study using porcine epithelial cells, we showed that PEDV inhibited both NF-κB and proinflammatory cytokines. PEDV blocked the p65 activation in infected cells and suppressed the PRD II-mediated NF-κB activity. Of the total of 22 viral proteins, nine proteins were identified as NF-κB antagonists, and nsp1 was the most potent suppressor of proinflammatory cytokines. Nsp1 interfered the phosphorylation and degradation of IκBα, and thus blocked the p65 activation. Mutational studies demonstrated the essential requirements of the conserved residues of nsp1 for NF-κB suppression. Our study showed that PEDV inhibited NF-κB activity and nsp1 was a potent NF-κB antagonist for suppression of both IFN and early production of pro-inflammatory cytokines., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Positive-strand RNA genomes function as mRNA for viral protein synthesis which is fully reliant on host cell translation machinery. Competing with cellular protein translation apparatus needs to ensure the production of viral proteins, but this also stifles host innate defense. In the present study, we showed that porcine reproductive and respiratory syndrome virus (PRRSV), whose replication takes place in the cytoplasm, imprisoned host cell mRNA in the nucleus, which suggests a novel mechanism to enhance translation of PRRSV genome. PRRSV nonstructural protein (nsp) 1β was identified as the nuclear protein playing the role for host mRNA nuclear retention and subversion of host protein synthesis. A SAP (SAF-A/B, Acinus, and PIAS) motif was identified in nsp1β with the consensus sequence of 126 -LQxxLxxxGL- 135 . In situ hybridization unveiled that SAP mutants were unable to cause nuclear retention of host cell mRNAs and did not suppress host protein synthesis. In addition, these SAP mutants reverted PRRSV-nsp1β-mediated suppression of interferon (IFN) production, IFN signaling, and TNF-α production pathway. Using reverse genetics, a series of SAP mutant PRRS viruses, vK124A, vL126A, vG134A, and vL135A were generated. No mRNA nuclear retention was observed during vL126A and vL135A infections. Importantly, vL126A and vL135A did not suppress IFN production. For other arteriviruses, mRNA nuclear accumulation was also observed for LDV-nsp1β and SHFV-nsp1β. EAV-nsp1 was exceptional and did not block the host mRNA nuclear export., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Type I interferons (IFN-α/β) are the major components of the innate immune response of hosts, and in turn many viruses have evolved to modulate the host response during infection. We found that the IFN-β production was significantly suppressed during PEDV infection in cells. To identify viral IFN antagonists and to study their suppressive function, viral coding sequences for the entire structural and nonstructural proteins were cloned and expressed. Of 16 PEDV nonstructural proteins (nsps), nsp1, nsp3, nsp7, nsp14, nsp15 and nsp16 were found to inhibit the IFN-β and IRF3 promoter activities. The sole accessory protein ORF3, structure protein envelope (E), membrane (M), and nucleocapsid (N) protein were also shown to inhibit such activities. PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP) by degrading CBP. A further study showed that the CBP degradation by nsp1 was proteasome-dependent. Our data demonstrate that PEDV modulates the host innate immune responses by degrading CBP and suppressing ISGs expression., (Copyright © 2015 Elsevier Inc. All rights reserved.)