Porcine Epidemic Diarrhea Virus nsp7 Inhibits MDA5 Dephosphorylation to Antagonize Type I Interferon Production.

Porcine epidemic diarrhea virus (PEDV) is a reemerging enteropathogenic coronavirus that causes high mortality in piglets and has catastrophic effects on the global pig industry. PEDV-encoded nonstructural protein 7 (nsp7) is an important component of the viral replication and transcription complex, and a previous study reported that it inhibits poly(I:C)-induced type I interferon (IFN) production, but the mechanism by which this occurs remains unclear. Here, we demonstrated that ectopic expression of PEDV nsp7 antagonized Sendai virus (SeV)-induced interferon beta (IFN-ß) production, as well as the activation of transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor-kappa B (NF-κB) in both HEK-293T and LLC-PK1 cells. Mechanistically, PEDV nsp7 targets melanoma differentiation-associated gene 5 (MDA5) and interacts with its caspase activation and recruitment domains (CARDs), which sequester the interactions between MDA5 and the protein phosphatase 1 (PP1) catalytic subunits (PP1α and PP1γ), thereby suppressing MDA5 S828 dephosphorylation and keeping MDA5 inactive. Furthermore, PEDV infection attenuated MDA5 multimerization and MDA5-PP1α/-γ interactions. We also tested the nsp7 orthologs of five other mammalian coronaviruses and found that all of them except severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp7 inhibited MDA5 multimerization and SeV- or MDA5-induced IFN-ß production. Collectively, these results suggest that the inhibition of MDA5 dephosphorylation and multimerization may be a common strategy employed by PEDV and some other coronaviruses to antagonize MDA5-mediated IFN production. IMPORTANCE Since late 2010, a reemerging porcine epidemic diarrhea virus variant with high pathogenesis has swept through most pig farms in many countries, resulting in significant economic losses. Coronavirus nonstructural protein 7 (nsp7), conserved within the family Coronaviridae, combines with nsp8 and nsp12 to form the viral replication and transcription complex that is indispensable for viral replication. However, the function of nsp7 in the infection and pathogenesis of coronaviruses remains largely unknown. Our present study demonstrates that PEDV nsp7 specifically competes with PP1 for binding MDA5 and impedes the PP1-mediated dephosphorylation of MDA5 at S828, thereby blocking MDA5-mediated IFN production, revealing the complex mechanism utilized by PEDV nsp7 to efficiently escape host innate immunity.

SARS-CoV-2 RNA-dependent RNA polymerase as a target for high-throughput drug screening.

The ongoing COVID-19 pandemic caused by the SARS-CoV-2 has necessitated rapid development of drug screening tools. RNA-dependent RNA polymerase (RdRp) is a promising target due to its essential functions in replication and transcription of viral genome. To date, through minimal RNA synthesizing machinery established from cryo-electron microscopy structural data, there has been development of high-throughput screening assays for directly screening inhibitors that target the SARS-CoV-2 RdRp. Here, we analyze and present verified techniques that could be used to discover potential anti-RdRp agents or repurposing of approved drugs to target the SARS-CoV-2 RdRp. In addition, we highlight the characteristics and application value of cell-free or cell-based assays in drug discovery.

SARS-CoV-2 NSP7 inhibits type I and III IFN production by targeting the RIG-I/MDA5, TRIF, and STING signaling pathways.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a poor inducer of innate antiviral immunity, and the underlying mechanism still needs further investigation. Here, we reported that SARS-CoV-2 NSP7 inhibited the production of type I and III interferons (IFNs) by targeting the RIG-I/MDA5, Toll-like receptor (TLR3)-TRIF, and cGAS-STING signaling pathways. SARS-CoV-2 NSP7 suppressed the expression of IFNs and IFN-stimulated genes induced by poly (I:C) transfection and infection with Sendai virus or SARS-CoV-2 virus-like particles. NSP7 impaired type I and III IFN production activated by components of the cytosolic dsRNA-sensing pathway, including RIG-I, MDA5, and MAVS, but not TBK1, IKKε, and IRF3-5D, an active form of IRF3. In addition, NSP7 also suppressed TRIF- and STING-induced IFN responses. Mechanistically, NSP7 associated with RIG-I and MDA5 prevented the formation of the RIG-I/MDA5-MAVS signalosome and interacted with TRIF and STING to inhibit TRIF-TBK1 and STING-TBK1 complex formation, thus reducing the subsequent IRF3 phosphorylation and nuclear translocation that are essential for IFN induction. In addition, ectopic expression of NSP7 impeded innate immune activation and facilitated virus replication. Taken together, SARS-CoV-2 NSP7 dampens type I and III IFN responses via disruption of the signal transduction of the RIG-I/MDA5-MAVS, TLR3-TRIF, and cGAS-STING signaling pathways, thus providing novel insights into the interactions between SARS-CoV-2 and innate antiviral immunity.

Porcine Epidemic Diarrhea Virus nsp7 Inhibits Interferon-Induced JAK-STAT Signaling through Sequestering the Interaction between KPNA1 and STAT1.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus that causes high mortality in piglets. Interferon (IFN) responses are the primary defense mechanism against viral infection; however, viruses always evolve elaborate strategies to antagonize the antiviral action of IFN. Previous study showed that PEDV nonstructural protein 7 (nsp7), a component of the viral replicase polyprotein, can antagonize ploy(I:C)-induced type I IFN production. Here, we found that PEDV nsp7 also antagonized IFN-α-induced JAK-STAT signaling and the production of IFN-stimulated genes. PEDV nsp7 did not affect the protein and phosphorylation levels of JAK1, Tyk2, STAT1, and STAT2 or the formation of the interferon-stimulated gene factor 3 (ISGF3) complex. However, PEDV nsp7 prevented the nuclear translocation of STAT1 and STAT2. Mechanistically, PEDV nsp7 interacted with the DNA binding domain of STAT1/STAT2, which sequestered the interaction between karyopherin α1 (KPNA1) and STAT1, thereby blocking the nuclear transport of ISGF3. Collectively, these data reveal a new mechanism developed by PEDV to inhibit type I IFN signaling pathway. IMPORTANCE In recent years, an emerging porcine epidemic diarrhea virus (PEDV) variant has gained attention because of serious outbreaks of piglet diarrhea in China and the United States. Coronavirus nonstructural protein 7 (nsp7) has been proposed to act with nsp8 as part of an RNA primase to generate RNA primers for viral RNA synthesis. However, accumulating evidence indicates that coronavirus nsp7 can also antagonize type I IFN production. Our present study extends previous findings and demonstrates that PEDV nsp7 also antagonizes IFN-α-induced IFN signaling by competing with KPNA1 for binding to STAT1, thereby enriching the immune regulation function of coronavirus nsp7.

SARS-CoV-2 Proteins Bind to Hemoglobin and Its Metabolites.

(1) Background: coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to hematological dysfunctions, but there are little experimental data that explain this. Spike (S) and Nucleoprotein (N) proteins have been putatively associated with these dysfunctions. In this work, we analyzed the recruitment of hemoglobin (Hb) and other metabolites (hemin and protoporphyrin IX-PpIX) by SARS-Cov2 proteins using different approaches. (2) Methods: shotgun proteomics (LC-MS/MS) after affinity column adsorption identified hemin-binding SARS-CoV-2 proteins. The parallel synthesis of the peptides technique was used to study the interaction of the receptor bind domain (RBD) and N-terminal domain (NTD) of the S protein with Hb and in silico analysis to identify the binding motifs of the N protein. The plaque assay was used to investigate the inhibitory effect of Hb and the metabolites hemin and PpIX on virus adsorption and replication in Vero cells. (3) Results: the proteomic analysis by LC-MS/MS identified the S, N, M, Nsp3, and Nsp7 as putative hemin-binding proteins. Six short sequences in the RBD and 11 in the NTD of the spike were identified by microarray of peptides to interact with Hb and tree motifs in the N protein by in silico analysis to bind with heme. An inhibitory effect in vitro of Hb, hemin, and PpIX at different levels was observed. Strikingly, free Hb at 1mM suppressed viral replication (99%), and its interaction with SARS-CoV-2 was localized into the RBD region of the spike protein. (4) Conclusions: in this study, we identified that (at least) five proteins (S, N, M, Nsp3, and Nsp7) of SARS-CoV-2 recruit Hb/metabolites. The motifs of the RDB of SARS-CoV-2 spike, which binds Hb, and the sites of the heme bind-N protein were disclosed. In addition, these compounds and PpIX block the virus's adsorption and replication. Furthermore, we also identified heme-binding motifs and interaction with hemin in N protein and other structural (S and M) and non-structural (Nsp3 and Nsp7) proteins.

Mutations in SARS-CoV-2 nsp7 and nsp8 proteins and their predicted impact on replication/transcription complex structure.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) has been identified to be a mutation hot spot, with the P323L mutation being commonly observed in viral genomes isolated from North America. RdRp forms a complex with nonstructural proteins nsp7 and nsp8 to form the minimal replication/transcription machinery required for genome replication. As mutations in RdRp may affect formation of the RdRp-nsp7-nsp8 supercomplex, we analyzed viral genomes to identify mutations in nsp7 and nsp8 protein sequences. Based on in silico analysis of predicted structures of the supercomplex comprising of native and mutated proteins, we demonstrate that specific mutations in nsp7 and nsp8 proteins may have a role in stabilization of the replication/transcription complex.

Genome sequencing of SARS-CoV-2 in a cohort of Egyptian patients revealed mutation hotspots that are related to clinical outcomes.

BACKGROUND: Severe acute respiratory syndrome-2 (SARS-CoV-2) exhibits a broad spectrum of clinical manifestations. Despite the fact that SARS-CoV-2 has slower evolutionary rate than other coronaviruses, different mutational hotspots have been identified along the SARS-CoV-2 genome. METHODS: We performed whole-genome high throughput sequencing on isolates from 50 Egyptian patients to see if the variation in clinical symptoms was related to mutations in the SARS-CoV-2 genome. Then, we investigated the relationship between the observed mutations and the clinical characteristics of the patients. RESULTS: Among the 36 most common mutations, we found two frameshift deletions linked to an increased risk of shortness of breath, a V6 deletion in the spike glycoprotein's signal peptide region linked to an increased risk of fever, longer fever duration and nasal congestion, and L3606-nsp6 deletion linked to a higher prevalence of cough and conjunctival congestion. S5398L nsp13-helicase was linked to an increased risk of fever duration and progression. The most common mutations (241, 3037, 14,408, and 23,403) were not linked to clinical variability. However, the E3909G-nsp7 variant was more common in children (2-13 years old) and was associated with a shorter duration of symptoms. The duration of fever was significantly reduced with E1363D-nsp3 and E3073A-nsp4. CONCLUSIONS: The most common mutations, D614G/spike-glycoprotein and P4715L/RNA-dependent-RNA-polymerase, were linked to transmissibility regardless of symptom variability. E3909G-nsp7 could explain why children recover so quickly. Nsp6-L3606fs, spike-glycoprotein-V6fs, and nsp13-S5398L variants may be linked to clinical symptom worsening. These variations related to host-virus interactions might open new therapeutic avenues for symptom relief and disease containment.

SARS-CoV-2 and SARS-CoV: Virtual screening of potential inhibitors targeting RNA-dependent RNA polymerase activity (NSP12).

Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the harm caused by coronaviruses to the world cannot be underestimated. Recently, a novel coronavirus (severe acute respiratory syndrome coronavirus-2 [SARS-CoV-2]) initially found to trigger human severe respiratory illness in Wuhan City of China in 2019, has infected more than six million people worldwide by 21 June 2020, and which has been recognized as a public health emergency of international concern as well. And the virus has spread to more than 200 countries around the world. However, the effective drug has not yet been officially licensed or approved to treat SARS-Cov-2 and SARS-Cov infection. NSP12-NSP7-NSP8 complex of SARS-CoV-2 or SARS-CoV, essential for viral replication and transcription, is generally regarded as a potential target to fight against the virus. According to the NSP12-NSP7-NSP8 complex (PDB ID: 7BW4) structure of SARS-CoV-2 and the NSP12-NSP7-NSP8 complex (PDB ID: 6NUR) structure of SARS-CoV, NSP12-NSP7 interface model, and NSP12-NSP8 interface model were established for virtual screening in the present study. Eight compounds (Nilotinib, Saquinavir, Tipranavir, Lonafarnib, Tegobuvir, Olysio, Filibuvir, and Cepharanthine) were selected for binding free energy calculations based on virtual screening and docking scores. All eight compounds can combine well with NSP12-NSP7-NSP8 in the crystal structure, providing drug candidates for the treatment and prevention of coronavirus disease 2019 and SARS.

Nonstructural protein 7 and 8 complexes of SARS-CoV-2.

The pandemic outbreak of coronavirus disease 2019 (COVID-19) across the world has led to millions of infection cases and caused a global public health crisis. Current research suggests that SARS-CoV-2 is a highly contagious coronavirus that spreads rapidly through communities. To understand the mechanisms of viral replication, it is imperative to investigate coronavirus viral replicase, a huge protein complex comprising up to 16 viral nonstructural and associated host proteins, which is the most promising antiviral target for inhibiting viral genome replication and transcription. Recently, several components of the viral replicase complex in SARS-CoV-2 have been solved to provide a basis for the design of new antiviral therapeutics. Here, we report the crystal structure of the SARS-CoV-2 nsp7+8 tetramer, which comprises two copies of each protein representing nsp7's full-length and the C-terminus of nsp8 owing to N-terminus proteolysis during the process of crystallization. We also identified a long helical extension and highly flexible N-terminal domain of nsp8, which is preferred for interacting with single-stranded nucleic acids.

Development of a Simple In Vitro Assay To Identify and Evaluate Nucleotide Analogs against SARS-CoV-2 RNA-Dependent RNA Polymerase.

Nucleotide analogs targeting viral RNA polymerase have been proved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In this study, we developed a robust in vitro nonradioactive primer extension assay to quantitatively evaluate the efficiency of incorporation of nucleotide analogs by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analogs over those of natural nucleotides were measured to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RdRp. In agreement with the data published in the literature, we found that the incorporation efficiency of remdesivir-TP is higher than that of ATP and incorporation of remdesivir-TP caused delayed chain termination, which can be overcome by higher concentrations of the next nucleotide to be incorporated. Our data also showed that the delayed chain termination pattern caused by remdesivir-TP incorporation is different for different template sequences. Multiple incorporations of remdesivir-TP caused chain termination under our assay conditions. Incorporation of sofosbuvir-TP is very low, suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2'-C-methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2'-C-methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful for evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp and for studying the mechanism of action of selected nucleotide analogs.

Expanding our understanding of the role polyprotein conformation plays in the coronavirus life cycle.

Coronavirus are the causative agents in many globally concerning respiratory disease outbreaks such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and coronavirus disease-2019 (COVID-19). It is therefore important that we improve our understanding of how the molecular components of the virus facilitate the viral life cycle. These details will allow for the design of effective interventions. Krichel and coauthors in their article in the Biochemical Journal provide molecular details of how the viral polyprotein (nsp7-10) produced from the positive single stranded RNA genome, is cleaved to form proteins that are part of the replication/transcription complex. The authors highlight the impact the polyprotein conformation has on the cleavage efficiency of the main protease (Mpro) and hence the order of release of non-structural proteins 7-10 (nsp7-10) of the SARS-CoV. Cleavage order is important in controlling viral processes and seems to have relevance in terms of the protein-protein complexes formed. The authors made use of mass spectrometry to advance our understanding of the mechanism by which coronaviruses control nsp 7, 8, 9 and 10 production in the virus life cycle.