Primary Antioxidant Power and Mpro SARS-CoV-2 Non-Covalent Inhibition Capabilities of Miquelianin.

The antioxidant power of quercetin-3-O-glucuronide (miquelianin) has been studied, at the density functional level of theory, in both lipid-like and aqueous environments. In the aqueous phase, the computed pKa equilibria allowed the identification of the neutral and charged species present in solution that can react with the ⋅OOH radical. The Hydrogen Atom Transfer (HAT), Single Electron Transfer (SET) and Radical Adduct Formation (RAF) mechanisms were considered, and the individual, total and fraction corrected rate constants were obtained. Potential non-covalent inhibition of Mpro from SARS-CoV-2 by miquelianin has been also evaluated.

Oral Simnotrelvir for Adult Patients with Mild-to-Moderate Covid-19.

BACKGROUND: Simnotrelvir is an oral 3-chymotrypsin-like protease inhibitor that has been found to have in vitro activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and potential efficacy in a phase 1B trial. METHODS: In this phase 2-3, double-blind, randomized, placebo-controlled trial, we assigned patients who had mild-to-moderate coronavirus disease 2019 (Covid-19) and onset of symptoms within the past 3 days in a 1:1 ratio to receive 750 mg of simnotrelvir plus 100 mg of ritonavir or placebo twice daily for 5 days. The primary efficacy end point was the time to sustained resolution of symptoms, defined as the absence of 11 Covid-19-related symptoms for 2 consecutive days. Safety and changes in viral load were also assessed. RESULTS: A total of 1208 patients were enrolled at 35 sites in China; 603 were assigned to receive simnotrelvir and 605 to receive placebo. Among patients in the modified intention-to-treat population who received the first dose of trial drug or placebo within 72 hours after symptom onset, the time to sustained resolution of Covid-19 symptoms was significantly shorter in the simnotrelvir group than in the placebo group (180.1 hours [95% confidence interval {CI}, 162.1 to 201.6] vs. 216.0 hours [95% CI, 203.4 to 228.1]; median difference, -35.8 hours [95% CI, -60.1 to -12.4]; P = 0.006 by Peto-Prentice test). On day 5, the decrease in viral load from baseline was greater in the simnotrelvir group than in the placebo group (mean difference [±SE], -1.51±0.14 log10 copies per milliliter; 95% CI, -1.79 to -1.24). The incidence of adverse events during treatment was higher in the simnotrelvir group than in the placebo group (29.0% vs. 21.6%). Most adverse events were mild or moderate. CONCLUSIONS: Early administration of simnotrelvir plus ritonavir shortened the time to the resolution of symptoms among adult patients with Covid-19, without evident safety concerns. (Funded by Jiangsu Simcere Pharmaceutical; ClinicalTrials.gov number, NCT05506176.).

Coronavirus M Protein Trafficking in Epithelial Cells Utilizes a Myosin Vb Splice Variant and Rab10.

The membrane (M) glycoprotein of coronaviruses (CoVs) serves as the nidus for virion assembly. Using a yeast two-hybrid screen, we identified the interaction of the cytosolic tail of Murine Hepatitis Virus (MHV-CoV) M protein with Myosin Vb (MYO5B), specifically with the alternative splice variant of cellular MYO5B including exon D (MYO5B+D), which mediates interaction with Rab10. When co-expressed in human lung epithelial A549 and canine kidney epithelial MDCK cells, MYO5B+D co-localized with the MHV-CoV M protein, as well as with the M proteins from Porcine Epidemic Diarrhea Virus (PEDV-CoV), Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome 2 (SARS-CoV-2). Co-expressed M proteins and MYO5B+D co-localized with endogenous Rab10 and Rab11a. We identified point mutations in MHV-CoV M that blocked the interaction with MYO5B+D in yeast 2-hybrid assays. One of these point mutations (E121K) was previously shown to block MHV-CoV virion assembly and its interaction with MYO5B+D. The E to K mutation at homologous positions in PEDV-CoV, MERS-CoV and SARS-CoV-2 M proteins also blocked colocalization with MYO5B+D. The knockdown of Rab10 blocked the co-localization of M proteins with MYO5B+D and was rescued by re-expression of CFP-Rab10. Our results suggest that CoV M proteins traffic through Rab10-containing systems, in association with MYO5B+D.

Prevalence and genetic variation of the M, N, and S2 genes of feline coronavirus in Shandong Province, China.

Feline coronavirus (FCoV) is the causative agent of feline infectious peritonitis and diarrhoea in kittens worldwide. In this study, a total of 73 feline diarrhoeal faecal samples were collected from animal hospitals and pet markets in ShanDong province from 2017 to 2019. FCoV was detected in 58.23% (46/73) of the samples, using the RT-PCR method. The results showed that the detection rate of FCoV in healthy cats and sick cats was 41.7% (10/24) and 81.6% (40/49), respectively. Full gene amplification and sequencing of the N, M, and S2 genes of FCoV isolates were performed. An amino acid mutation (M1058L) in the S2 gene was found that can be used as a marker for distinguishing feline enteric coronavirus (FECV) from feline infectious peritonitis virus (FIPV). This study provides new epidemiological information about FCoV that will aid in the prevention of FCoV in China.

The TGEV Membrane Protein Interacts with HSC70 To Direct Virus Internalization through Clathrin-Mediated Endocytosis.

Coronavirus membrane protein is a major component of the viral envelope and plays a central role in the viral life cycle. Studies of the coronavirus membrane protein (M) have mainly focused on its role in viral assembly and budding, but whether M protein is involved in the initial stage of viral replication remains unclear. In this study, eight proteins in transmissible gastroenteritis virus (TGEV)-infected cells coimmunoprecipitated with monoclonal antibodies (MAb) against M protein in PK-15 cells, heat shock cognate protein 70 (HSC70), and clathrin were identified by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF MS). Further studies demonstrated that HSC70 and TGEV M colocalized on the cell surface in early stages of TGEV infection; specifically, HSC70 bound M protein through its substrate-binding domain (SBD) and preincubation of TGEV with anti-M serum to block the interaction of M and HSC70 reduced the internalization of TGEV, thus demonstrating that the M-HSC70 interaction mediates the internalization of TGEV. Remarkably, the process of internalization was dependent on clathrin-mediated endocytosis (CME) in PK-15 cells. Furthermore, inhibition of the ATPase activity of HSC70 reduced the efficiency of CME. Collectively, our results indicated that HSC70 is a newly identified host factor involved in TGEV infection. Taken together, our findings clearly illustrate a novel role for TGEV M protein in the viral life cycle and present a unique strategy used by HSC70 to promote TGEV infection in which the interaction with M protein directs viral internalization. These studies provide new insights into the life cycle of coronaviruses. IMPORTANCE TGEV is the causative agent of porcine diarrhea, a viral disease that economically affects the pig industry in many countries. However, the molecular mechanisms underlying viral replication remain incompletely understood. Here, we provide evidence of a previously undescribed role of M protein in viral replication during early stages. We also identified HSC70 as a new host factor affecting TGEV infection. We demonstrate that the interaction between M and HSC70 directs TGEV internalization in a manner dependent on CME, thus revealing a novel mechanism for TGEV replication. We believe that this study may change our understanding of the first steps of infection of cells with coronavirus. This study should facilitate the development of anti-TGEV therapeutic agents by targeting the host factors and may provide a new strategy for the control of porcine diarrhea.

Characterization of the Interaction Between SARS-CoV-2 Membrane Protein (M) and Proliferating Cell Nuclear Antigen (PCNA) as a Potential Therapeutic Target.

SARS-CoV-2 is an emerging virus from the Coronaviridae family and is responsible for the ongoing COVID-19 pandemic. In this work, we explored the previously reported SARS-CoV-2 structural membrane protein (M) interaction with human Proliferating Cell Nuclear Antigen (PCNA). The M protein is responsible for maintaining virion shape, and PCNA is a marker of DNA damage which is essential for DNA replication and repair. We validated the M-PCNA interaction through immunoprecipitation, immunofluorescence co-localization, and PLA (Proximity Ligation Assay). In cells infected with SARS-CoV-2 or transfected with M protein, using immunofluorescence and cell fractioning, we documented a reallocation of PCNA from the nucleus to the cytoplasm and the increase of PCNA and γH2AX (another DNA damage marker) expression. We also observed an increase in PCNA and γH2AX expression in the lung of a COVID-19 patient by immunohistochemistry. In addition, the inhibition of PCNA translocation by PCNA I1 and Verdinexor led to a reduction of plaque formation in an in vitro assay. We, therefore, propose that the transport of PCNA to the cytoplasm and its association with M could be a virus strategy to manipulate cell functions and may be considered a target for COVID-19 therapy.

SARS-CoV-2 Membrane Protein: From Genomic Data to Structural New Insights.

Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) is composed of four structural proteins and several accessory non-structural proteins. SARS-CoV-2's most abundant structural protein, Membrane (M) protein, has a pivotal role both during viral infection cycle and host interferon antagonism. This is a highly conserved viral protein, thus an interesting and suitable target for drug discovery. In this paper, we explain the structural nature of M protein homodimer. To do so, we developed and applied a detailed and robust in silico workflow to predict M protein dimeric structure, membrane orientation, and interface characterization. Single Nucleotide Polymorphisms (SNPs) in M protein were retrieved from over 1.2 M SARS-CoV-2 genomes and proteins from the Global Initiative on Sharing All Influenza Data (GISAID) database, 91 of which were located at the predicted dimer interface. Among those, we identified SNPs in Variants of Concern (VOC) and Variants of Interest (VOI). Binding free energy differences were evaluated for dimer interfacial SNPs to infer mutant protein stabilities. A few high-prevalent mutated residues were found to be especially relevant in VOC and VOI. This realization may be a game-changer to structure-driven formulation of new therapeutics for SARS-CoV-2.

DNA Vaccines Expressing the Envelope and Membrane Proteins Provide Partial Protection Against SARS-CoV-2 in Mice.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a public health emergency of international concern, and an effective vaccine is urgently needed to control the pandemic. Envelope (E) and membrane (M) proteins are highly conserved structural proteins among SARS-CoV-2 and SARS-CoV and have been proposed as potential targets for the development of cross-protective vaccines. Here, synthetic DNA vaccines encoding SARS-CoV-2 E/M proteins (called p-SARS-CoV-2-E/M) were developed, and mice were immunised with three doses via intramuscular injection and electroporation. Significant cellular immune responses were elicited, whereas no robust humoral immunity was detected. In addition, novel H-2d-restricted T-cell epitopes were identified. Notably, although no drop in lung tissue virus titre was detected in DNA-vaccinated mice post-challenge with SARS-CoV-2, immunisation with either p-SARS-CoV-2-E or p-SARS-CoV-2-M provided minor protection and co-immunisation with p-SARS-CoV-2-E+M increased protection. Therefore, E/M proteins should be considered as vaccine candidates as they may be valuable in the optimisation of vaccination strategies against COVID-19.

A Thermostable Oral SARS-CoV-2 Vaccine Induces Mucosal and Protective Immunity.

An ideal protective vaccine against SARS-CoV-2 should not only be effective in preventing disease, but also in preventing virus transmission. It should also be well accepted by the population and have a simple logistic chain. To fulfill these criteria, we developed a thermostable, orally administered vaccine that can induce a robust mucosal neutralizing immune response. We used our platform based on retrovirus-derived enveloped virus-like particles (eVLPs) harnessed with variable surface proteins (VSPs) from the intestinal parasite Giardia lamblia, affording them resistance to degradation and the triggering of robust mucosal cellular and antibody immune responses after oral administration. We made eVLPs expressing various forms of the SARS-CoV-2 Spike protein (S), with or without membrane protein (M) expression. We found that prime-boost administration of VSP-decorated eVLPs expressing a pre-fusion stabilized form of S and M triggers robust mucosal responses against SARS-CoV-2 in mice and hamsters, which translate into complete protection from a viral challenge. Moreover, they dramatically boosted the IgA mucosal response of intramuscularly injected vaccines. We conclude that our thermostable orally administered eVLP vaccine could be a valuable addition to the current arsenal against SARS-CoV-2, in a stand-alone prime-boost vaccination strategy or as a boost for existing vaccines.

Neutralizing-antibody-independent SARS-CoV-2 control correlated with intranasal-vaccine-induced CD8+ T cell responses.

Effective vaccines are essential for the control of the coronavirus disease 2019 (COVID-19) pandemic. Currently developed vaccines inducing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-antigen-specific neutralizing antibodies (NAbs) are effective, but the appearance of NAb-resistant S variant viruses is of great concern. A vaccine inducing S-independent or NAb-independent SARS-CoV-2 control may contribute to containment of these variants. Here, we investigate the efficacy of an intranasal vaccine expressing viral non-S antigens against intranasal SARS-CoV-2 challenge in cynomolgus macaques. Seven vaccinated macaques exhibit significantly reduced viral load in nasopharyngeal swabs on day 2 post-challenge compared with nine unvaccinated controls. The viral control in the absence of SARS-CoV-2-specific NAbs is significantly correlated with vaccine-induced, viral-antigen-specific CD8+ T cell responses. Our results indicate that CD8+ T cell induction by intranasal vaccination can result in NAb-independent control of SARS-CoV-2 infection, highlighting a potential of vaccine-induced CD8+ T cell responses to contribute to COVID-19 containment.

Predicted 3D model of the M protein of Porcine Epidemic Diarrhea Virus and analysis of its immunogenic potential.

The membrane protein M of the Porcine Epidemic Diarrhea Virus (PEDV) is the most abundant component of the viral envelope. The M protein plays a central role in the morphogenesis and assembly of the virus through protein interactions of the M-M, M-Spike (S) and M-nucleocapsid (N) type. The M protein is known to induce protective antibodies in pigs and to participate in the antagonistic response of the cellular antiviral system coordinated by the type I and type III interferon pathways. The 3D structure of the PEDV M protein is still unknown. The present work exposes a predicted 3D model of the M protein generated using the Robetta protocol. The M protein model is organized into a transmembrane and a globular region. The obtained 3D model of the PEDV M protein was compared with 3D models of the SARS-CoV-2 M protein created using neural networks and with initial machine learning-based models created using trRosetta. The 3D model of the present study predicted four linear B-cell epitopes (RSVNASSGTG and KHGDYSAVSNPSALT peptides are noteworthy), six discontinuous B-cell epitopes, forty weak binding and fourteen strong binding T-cell epitopes in the CV777 M protein. A high degree of conservation of the epitopes predicted in the PEDV M protein was observed among different PEDV strains isolated in different countries. The data suggest that the M protein could be a potential candidate for the development of new treatments or strategies that activate protective cellular mechanisms against viral diseases.

Immune Response in Moderate to Critical Breakthrough COVID-19 Infection After mRNA Vaccination.

SARS-CoV-2 variants of concern (VOCs) can trigger severe endemic waves and vaccine breakthrough infections (VBI). We analyzed the cellular and humoral immune response in 8 patients infected with the alpha variant, resulting in moderate to fatal COVID-19 disease manifestation, after double mRNA-based anti-SARS-CoV-2 vaccination. In contrast to the uninfected vaccinated control cohort, the diseased individuals had no detectable high-avidity spike (S)-reactive CD4+ and CD8+ T cells against the alpha variant and wild type (WT) at disease onset, whereas a robust CD4+ T-cell response against the N- and M-proteins was generated. Furthermore, a delayed alpha S-reactive high-avidity CD4+ T-cell response was mounted during disease progression. Compared to the vaccinated control donors, these patients also had lower neutralizing antibody titers against the alpha variant at disease onset. The delayed development of alpha S-specific cellular and humoral immunity upon VBI indicates reduced immunogenicity against the S-protein of the alpha VOC, while there was a higher and earlier N- and M-reactive T-cell response. Our findings do not undermine the current vaccination strategies but underline a potential need for the inclusion of VBI patients in alternative vaccination strategies and additional antigenic targets in next-generation SARS-CoV-2 vaccines.

Evaluation of Xa inhibitors as potential inhibitors of the SARS-CoV-2 Mpro protease.

Based on previous large-scale in silico screening several factor Xa inhibitors were proposed to potentially inhibit SARS-CoV-2 Mpro. In addition to their known anticoagulants activity this potential inhibition could have an additional therapeutic effect on patients with COVID-19 disease. In this study we examined the binding of the Apixaban, Betrixaban and Rivaroxaban to the SARS-CoV-2 Mpro with the use of the MicroScale Thermophoresis technique. Our results indicate that the experimentally measured binding affinity is weak and the therapeutic effect due to the SARS-CoV-2 Mpro inhibition is rather negligible.

SARS-CoV-2 membrane protein causes the mitochondrial apoptosis and pulmonary edema via targeting BOK.

Deaths caused by coronavirus disease 2019 (COVID-19) are largely due to the lungs edema resulting from the disruption of the lung alveolo-capillary barrier, induced by SARS-CoV-2-triggered pulmonary cell apoptosis. However, the molecular mechanism underlying the proapoptotic role of SARS-CoV-2 is still unclear. Here, we revealed that SARS-CoV-2 membrane (M) protein could induce lung epithelial cells mitochondrial apoptosis. Notably, M protein stabilized B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) via inhibiting its ubiquitination and promoted BOK mitochondria translocation. The endodomain of M protein was required for its interaction with BOK. Knockout of BOK by CRISPR/Cas9 increased cellular resistance to M protein-induced apoptosis. BOK was rescued in the BOK-knockout cells, which led to apoptosis induced by M protein. M protein induced BOK to trigger apoptosis in the absence of BAX and BAK. Furthermore, the BH2 domain of BOK was required for interaction with M protein and proapoptosis. In vivo M protein recombinant lentivirus infection induced caspase-associated apoptosis and increased alveolar-capillary permeability in the mouse lungs. BOK knockdown improved the lung edema due to lentivirus-M protein infection. Overall, M protein activated the BOK-dependent apoptotic pathway and thus exacerbated SARS-CoV-2 associated lung injury in vivo. These findings proposed a proapoptotic role for M protein in SARS-CoV-2 pathogenesis, which may provide potential targets for COVID-19 treatments.

Construction of SARS-CoV-2 virus-like particles in plant.

The pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a public health emergency, and research on the development of various types of vaccines is rapidly progressing at an unprecedented development speed internationally. Some vaccines have already been approved for emergency use and are being supplied to people around the world, but there are still many ongoing efforts to create new vaccines. Virus-like particles (VLPs) enable the construction of promising platforms in the field of vaccine development. Here, we demonstrate that non-infectious SARS-CoV-2 VLPs can be successfully assembled by co-expressing three important viral proteins membrane (M), envelop (E) and nucleocapsid (N) in plants. Plant-derived VLPs were purified by sedimentation through a sucrose cushion. The shape and size of plant-derived VLPs are similar to native SARS-CoV-2 VLPs without spike. Although the assembled VLPs do not have S protein spikes, they could be developed as formulations that can improve the immunogenicity of vaccines including S antigens, and further could be used as platforms that can carry S antigens of concern for various mutations.

Evaluating cepharanthine analogues as natural drugs against SARS-CoV-2.

Cepharanthine (CEP) is a natural biscoclaurine alkaloid of plant origin and was recently demonstrated to have anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) activity. In this study, we evaluated whether natural analogues of CEP may act as potential anti-coronavirus disease 2019 drugs. A total of 24 compounds resembling CEP were extracted from the KNApSAcK database, and their binding affinities to target proteins, including the spike protein and main protease of SARS-CoV-2, NPC1 and TPC2 in humans, were predicted via molecular docking simulations. Selected analogues were further evaluated by a cell-based SARS-CoV-2 infection assay. In addition, the efficacies of CEP and its analogue tetrandrine were assessed. A comparison of the docking conformations of these compounds suggested that the diphenyl ester moiety of the molecules was a putative pharmacophore of the CEP analogues.

A new insight into sex-specific non-coding RNAs and networks in response to SARS-CoV-2.

SARS-CoV-2 is the RNA virus responsible for COVID-19, the prognosis of which has been found to be slightly worse in men. The present study aimed to analyze the expression of different mRNAs and their regulatory molecules (miRNAs and lncRNAs) to consider the potential existence of sex-specific expression patterns and COVID-19 susceptibility using bioinformatics analysis. The binding sites of all human mature miRNA sequences on the SARS-CoV-2 genome nucleotide sequence were predicted by the miRanda tool. Sequencing data was excavated using the Galaxy web server from GSE157103, and the output of feature counts was analyzed using DEseq2 packages to obtain differentially expressed genes (DEGs). Gene set enrichment analysis (GSEA) and DEG annotation analyses were performed using the ToppGene and Metascape tools. Using the RNA Interactome Database, we predicted interactions between differentially expressed lncRNAs and differentially expressed mRNAs. Finally, their networks were constructed with top miRNAs. We identified 11 miRNAs with three to five binding sites on the SARS-COVID-2 genome reference. MiR-29c-3p, miR-21-3p, and miR-6838-5p occupied four binding sites, and miR-29a-3p had five binding sites on the SARS-CoV-2 genome. Moreover, miR-29a-3p, and miR-29c-3p were the top miRNAs targeting DEGs. The expression levels of miRNAs (125, 181b, 130a, 29a, b, c, 212, 181a, 133a) changed in males with COVID-19, in whom they regulated ACE2 expression and affected the immune response by affecting phagosomes, complement activation, and cell-matrix adhesion. Our results indicated that XIST lncRNA was up-regulated, and TTTY14, TTTY10, and ZFY-AS1 lncRN as were down-regulated in both ICU and non-ICU men with COVID-19. Dysregulation of noncoding-RNAs has critical effects on the pathophysiology of men with COVID-19, which is why they may be used as biomarkers and therapeutic agents. Overall, our results indicated that the miR-29 family target regulation patterns and might become promising biomarkers for severity and survival outcome in men with COVID-19.

Structure and dynamics of membrane protein in SARS-CoV-2.

SARS-CoV-2 membrane (M) protein performs a variety of critical functions in virus infection cycle. However, the expression and purification of membrane protein structure is difficult despite tremendous progress. In this study, the 3 D structure is modeled followed by intensive validation and molecular dynamics simulation. The lack of suitable homologous templates (>30% sequence identities) leads us to construct the membrane protein models using template-free modeling (de novo or ab initio) approach with Robetta and trRosetta servers. Comparing with other model structures, it is evident that trRosetta (TM-score: 0.64; TM region RMSD: 2 Å) can provide the best model than Robetta (TM-score: 0.61; TM region RMSD: 3.3 Å) and I-TASSER (TM-score: 0.45; TM region RMSD: 6.5 Å). 100 ns molecular dynamics simulations are performed on the model structures by incorporating membrane environment. Moreover, secondary structure elements and principal component analysis (PCA) have also been performed on MD simulation data. Finally, trRosetta model is utilized for interpretation and visualization of interacting residues during protein-protein interactions. The common interacting residues including Phe103, Arg107, Met109, Trp110, Arg131, and Glu135 in the C-terminal domain of M protein are identified in membrane-spike and membrane-nucleocapsid protein complexes. The active site residues are also predicted for potential drug and peptide binding. Overall, this study might be helpful to design drugs and peptides against the modeled membrane protein of SARS-CoV-2 to accelerate further investigation. Communicated by Ramaswamy H. Sarma.

Protease Inhibitors as Promising Weapons against COVID-19: Focus on Repurposing of Drugs used to Treat HIV and HCV Infections.

As a part of the efforts to quickly develop pharmaceutical treatments for COVID-19 through repurposing existing drugs, some researchers around the world have combined the recently released crystal structure of SARS-CoV-2 Mpro in complex with a covalently bonded inhibitor with virtual screening procedures employing molecular docking approaches. In this context, protease inhibitors (PIs) clinically available and currently used to treat infectious diseases, particularly viral ones, are relevant sources of promising drug candidates to inhibit the SARS-CoV-2 Mpro, a key viral enzyme involved in crucial events during its life cycle. In the present perspective, we summarized the published studies showing the promising use of HIV and HCV PIs as potential repurposing drugs against the SARS-CoV-2 Mpro.