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1.
J Med Chem ; 65(8): 6231-6249, 2022 04 28.
Article in English | MEDLINE | ID: covidwho-1867997

ABSTRACT

Enzymes involved in RNA capping of SARS-CoV-2 are essential for the stability of viral RNA, translation of mRNAs, and virus evasion from innate immunity, making them attractive targets for antiviral agents. In this work, we focused on the design and synthesis of nucleoside-derived inhibitors against the SARS-CoV-2 nsp14 (N7-guanine)-methyltransferase (N7-MTase) that catalyzes the transfer of the methyl group from the S-adenosyl-l-methionine (SAM) cofactor to the N7-guanosine cap. Seven compounds out of 39 SAM analogues showed remarkable double-digit nanomolar inhibitory activity against the N7-MTase nsp14. Molecular docking supported the structure-activity relationships of these inhibitors and a bisubstrate-based mechanism of action. The three most potent inhibitors significantly stabilized nsp14 (ΔTm ≈ 11 °C), and the best inhibitor demonstrated high selectivity for nsp14 over human RNA N7-MTase.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/drug therapy , COVID-19/virology , Exoribonucleases/antagonists & inhibitors , Exoribonucleases/chemistry , Humans , Methyltransferases , Molecular Docking Simulation , RNA, Viral/genetics , S-Adenosylmethionine , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Sulfonamides/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry
2.
Cell Rep ; 39(11): 110954, 2022 Jun 14.
Article in English | MEDLINE | ID: covidwho-1866958

ABSTRACT

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.


Subject(s)
COVID-19 , Viral Nonstructural Proteins , Cell Line , Humans , RNA Stability , SARS-CoV-2 , Viral Nonstructural Proteins/metabolism
3.
Molecules ; 27(9)2022 Apr 23.
Article in English | MEDLINE | ID: covidwho-1855714

ABSTRACT

The COVID-19 pandemic is still affecting many people worldwide and causing a heavy burden to global health. To eliminate the disease, SARS-CoV-2, the virus responsible for the pandemic, can be targeted in several ways. One of them is to inhibit the 2'-O-methyltransferase (nsp16) enzyme that is crucial for effective translation of viral RNA and virus replication. For methylation of substrates, nsp16 utilizes S-adenosyl methionine (SAM). Binding of a small molecule in the protein site where SAM binds can disrupt the synthesis of viral proteins and, as a result, the replication of the virus. Here, we performed high-throughput docking into the SAM-binding site of nsp16 for almost 40 thousand structures, prepared for compounds from three libraries: Enamine Coronavirus Library, Enamine Nucleoside Mimetics Library, and Chemdiv Nucleoside Analogue Library. For the top scoring ligands, semi-empirical quantum-chemical calculations were performed, to better estimate protein-ligand binding enthalpy. Relying upon the calculated binding energies and predicted docking poses, we selected 21 compounds for experimental testing.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/drug therapy , Humans , Methyltransferases/chemistry , Molecular Docking Simulation , Pandemics , RNA, Viral/genetics , S-Adenosylmethionine , Viral Nonstructural Proteins/metabolism
4.
J Virol ; 96(11): e0046922, 2022 Jun 08.
Article in English | MEDLINE | ID: covidwho-1854236

ABSTRACT

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 (ß-CoVs), it may be a good target for vaccine development for other α-CoVs or ß-CoVs.


Subject(s)
Coronavirus Infections , Interferons , Porcine epidemic diarrhea virus , Swine Diseases , Viral Nonstructural Proteins , Animals , Animals, Newborn , Coronavirus Infections/immunology , Coronavirus Infections/veterinary , Diarrhea/veterinary , Diarrhea/virology , Female , Interferons/immunology , Mutation , Swine , Swine Diseases/immunology , Swine Diseases/virology , Viral Nonstructural Proteins/genetics
5.
Antiviral Res ; 203: 105344, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-1850638

ABSTRACT

The emergence of several zoonotic viruses in the last twenty years, especially the pandemic outbreak of SARS-CoV-2, has exposed a dearth of antiviral drug therapies for viruses with pandemic potential. Developing a diverse drug portfolio will be critical to rapidly respond to novel coronaviruses (CoVs) and other viruses with pandemic potential. Here we focus on the SARS-CoV-2 conserved macrodomain (Mac1), a small domain of non-structural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that cleaves mono-ADP-ribose (MAR) from target proteins, protects the virus from the anti-viral effects of host ADP-ribosyltransferases, and is critical for the replication and pathogenesis of CoVs. In this study, a luminescent-based high-throughput assay was used to screen ∼38,000 small molecules for those that could inhibit Mac1-ADP-ribose binding. We identified 5 compounds amongst 3 chemotypes that inhibit SARS-CoV-2 Mac1-ADP-ribose binding in multiple assays with IC50 values less than 100 µM, inhibit ADP-ribosylhydrolase activity, and have evidence of direct Mac1 binding. These chemotypes are strong candidates for further derivatization into highly effective Mac1 inhibitors.


Subject(s)
COVID-19 , SARS-CoV-2 , Adenosine Diphosphate Ribose/metabolism , COVID-19/drug therapy , High-Throughput Screening Assays , Humans , Viral Nonstructural Proteins/chemistry
7.
J Cell Biol ; 221(6)2022 06 06.
Article in English | MEDLINE | ID: covidwho-1830915

ABSTRACT

ß-coronaviruses reshape host cell endomembranes to form double-membrane vesicles (DMVs) for genome replication and transcription. Ectopically expressed viral nonstructural proteins nsp3 and nsp4 interact to zipper and bend the ER for DMV biogenesis. Genome-wide screens revealed the autophagy proteins VMP1 and TMEM41B as important host factors for SARS-CoV-2 infection. Here, we demonstrated that DMV biogenesis, induced by virus infection or expression of nsp3/4, is impaired in the VMP1 KO or TMEM41B KO cells. In VMP1 KO cells, the nsp3/4 complex forms normally, but the zippered ER fails to close into DMVs. In TMEM41B KO cells, the nsp3-nsp4 interaction is reduced and DMV formation is suppressed. Thus, VMP1 and TMEM41B function at different steps during DMV formation. VMP1 was shown to regulate cross-membrane phosphatidylserine (PS) distribution. Inhibiting PS synthesis partially rescues the DMV defects in VMP1 KO cells, suggesting that PS participates in DMV formation. We provide molecular insights into the collaboration of host factors with viral proteins to remodel host organelles.


Subject(s)
COVID-19 , Membrane Proteins , SARS-CoV-2 , Viral Replication Compartments , Autophagy/genetics , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Organelles/metabolism , Phosphatidylserines , SARS-CoV-2/physiology , Viral Nonstructural Proteins/genetics , Virus Replication
8.
Molecules ; 27(9)2022 May 03.
Article in English | MEDLINE | ID: covidwho-1820344

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. While the development of vaccines and the emergence of antiviral therapeutics is promising, alternative strategies to combat COVID-19 (and potential future pandemics) remain an unmet need. Coronaviruses feature a unique mechanism that may present opportunities for therapeutic intervention: the RNA polymerase complex of coronaviruses is distinct in its ability to proofread and remove mismatched nucleotides during genome replication and transcription. The proofreading activity has been linked to the exonuclease (ExoN) activity of non-structural protein 14 (NSP14). Here, we review the role of NSP14, and other NSPs, in SARS-CoV-2 replication and describe the assays that have been developed to assess the ExoN function. We also review the nucleoside analogs and non-nucleoside inhibitors known to interfere with the proofreading activity of NSP14. Although not yet validated, the potential use of non-nucleoside proofreading inhibitors in combination with chain-terminating nucleosides may be a promising avenue for the development of anti-CoV agents.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Exoribonucleases/metabolism , Humans , Pandemics , RNA, Viral/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication
9.
Bioorg Chem ; 125: 105850, 2022 Aug.
Article in English | MEDLINE | ID: covidwho-1819433

ABSTRACT

Nucleoside precursors and nucleoside analogs occupy an important place in the treatment of viral respiratory pathologies, especially during the current COVID-19 pandemic. From this perspective, the present study has been designed to explore and evaluate the synthesis and spectral characterisation of 5́-O-(lauroyl) thymidine analogs 2-6 with different aliphatic and aromatic groups through comprehensive in vitro antimicrobial screening, cytotoxicity assessment, physicochemical aspects, molecular docking and molecular dynamics analysis, along with pharmacokinetic prediction. A unimolar one-step lauroylation of thymidine under controlled conditions furnished the 5́-O-(lauroyl) thymidine and indicated the selectivity at C-5́ position and the development of thymidine based potential antimicrobial analogs, which were further converted into four newer 3́-O-(acyl)-5́-O-(lauroyl) thymidine analogs in reasonably good yields. The chemical structures of the newly synthesised analogs were ascertained by analysing their physicochemical, elemental, and spectroscopic data. In vitro antimicrobial tests against five bacteria and two fungi, along with the prediction of activity spectra for substances (PASS), indicated promising antibacterial functionality for these thymidine analogs compared to antifungal activity. In support of this observation, molecular docking experiments have been performed against the main protease of SARS-CoV-2, and significant binding affinities and non-bonding interactions were observed against the main protease (6LU7, 6Y84 and 7BQY), considering hydroxychloroquine (HCQ) as standard. Moreover, the 100 ns molecular dynamics simulation process was performed to monitor the behaviour of the complex structure formed by the main protease under in silico physiological conditions to examine its stability over time, and this revealed a stable conformation and binding pattern in a stimulating environment of thymidine analogs. Cytotoxicity determination confirmed that compounds were found less toxic. Pharmacokinetic predictions were investigated to evaluate their absorption, distribution, metabolism and toxic properties, and the combination of pharmacokinetic and drug-likeness predictions has shown promising results in silico. The POM analysis shows the presence of an antiviral (O1δ-, O2δ-) pharmacophore site. Overall, the current study should be of great help in the development of thymidine-based, novel, multiple drug-resistant antimicrobial and COVID-19 drugs.


Subject(s)
COVID-19 , SARS-CoV-2 , Anti-Bacterial Agents , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases , Cysteine Endopeptidases/metabolism , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Pandemics , Protease Inhibitors/chemistry , Thymidine/pharmacology , Viral Nonstructural Proteins/metabolism
10.
Comput Biol Med ; 146: 105572, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-1814281

ABSTRACT

BACKGROUND: The SARS-CoV-2 main protease (Mpro) is an attractive target in the COVID-19 drug development process. It catalyzes the polyprotein's translation from viral RNA and specifies a particular cleavage site. Due to the absence of identical cleavage specificity in human cell proteases, targeting Mpro with chemical compounds can obstruct the replication of the virus. METHODS: To explore the potential binding mechanisms of 1,2,3-triazole scaffolds in comparison to co-crystallized inhibitors 11a and 11b towards Mpro, we herein utilized molecular dynamics and enhanced sampling simulation studies. RESULTS AND CONCLUSION: All the 1,2,3-triazole scaffolds interacted with catalytic residues (Cys145 and His41) and binding pocket residues of Mpro involving Met165, Glu166, Ser144, Gln189, His163, and Met49. Furthermore, the adequate binding free energy and potential mean force of the topmost compound 3h was comparable to the experimental inhibitors 11a and 11b of Mpro. Overall, the current analysis could be beneficial in developing the SARS-CoV-2 Mpro potential inhibitors.


Subject(s)
COVID-19 , Molecular Dynamics Simulation , Benchmarking , COVID-19/drug therapy , Coronavirus 3C Proteases , Cysteine Endopeptidases/chemistry , Humans , Molecular Docking Simulation , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , SARS-CoV-2 , Triazoles , Viral Nonstructural Proteins/chemistry
11.
J Virol ; 96(9): e0040022, 2022 05 11.
Article in English | MEDLINE | ID: covidwho-1807320

ABSTRACT

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.


Subject(s)
Janus Kinase 1 , Porcine epidemic diarrhea virus , STAT1 Transcription Factor , Signal Transduction , Viral Nonstructural Proteins , alpha Karyopherins , Animals , Cell Line , Interferons/metabolism , Janus Kinase 1/metabolism , Porcine epidemic diarrhea virus/genetics , STAT1 Transcription Factor/metabolism , Swine , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , alpha Karyopherins/metabolism
12.
Sci Rep ; 12(1): 3860, 2022 03 09.
Article in English | MEDLINE | ID: covidwho-1799576

ABSTRACT

Non-structural protein 15 (Nsp15) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) forms a homo hexamer and functions as an endoribonuclease. Here, we propose that Nsp15 activity may be inhibited by preventing its hexamerization through drug binding. We first explored the stable conformation of the Nsp15 monomer as the global free energy minimum conformation in the free energy landscape using a combination of parallel cascade selection molecular dynamics (PaCS-MD) and the Markov state model (MSM), and found that the Nsp15 monomer forms a more open conformation with larger druggable pockets on the surface. Targeting the pockets with high druggability scores, we conducted ligand docking and identified compounds that tightly bind to the Nsp15 monomer. The top poses with Nsp15 were subjected to binding free energy calculations by dissociation PaCS-MD and MSM (dPaCS-MD/MSM), indicating the stability of the complexes. One of the identified pockets, which is distinctively bound by inosine analogues, may be an alternative binding site to stabilize viral RNA binding and/or an alternative catalytic site. We constructed a stable RNA structure model bound to both UTP and alternative binding sites, providing a reasonable proposed model of the Nsp15/RNA complex.


Subject(s)
Endoribonucleases/metabolism , RNA, Viral/chemistry , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Binding Sites , COVID-19/pathology , COVID-19/virology , Endoribonucleases/antagonists & inhibitors , Humans , Markov Chains , Molecular Docking Simulation , Molecular Dynamics Simulation , Nucleic Acid Conformation , Protein Multimerization , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Static Electricity , Viral Nonstructural Proteins/antagonists & inhibitors
13.
FEBS Lett ; 596(9): 1203-1213, 2022 May.
Article in English | MEDLINE | ID: covidwho-1798052

ABSTRACT

Nonstructural protein 1 (Nsp1) of SARS-CoV-2 inhibits host cell translation through an interaction between its C-terminal domain and the 40S ribosome. The N-terminal domain (NTD) of Nsp1 is a target of recurring deletions, some of which are associated with altered COVID-19 disease progression. Here, we characterize the efficiency of translational inhibition by clinically observed Nsp1 deletion variants. We show that a frequent deletion of residues 79-89 severely reduces the ability of Nsp1 to inhibit translation while not abrogating Nsp1 binding to the 40S. Notably, while the SARS-CoV-2 5' untranslated region enhances translation of mRNA, it does not protect from Nsp1-mediated inhibition. Finally, thermal stability measurements and structure predictions reveal a correlation between stability of the NTD and the efficiency of translation inhibition.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/genetics , Humans , Protein Biosynthesis , Ribosomes/genetics , Ribosomes/metabolism , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism
14.
Molecules ; 27(7)2022 Mar 31.
Article in English | MEDLINE | ID: covidwho-1792596

ABSTRACT

As a continuation of our earlier work against SARS-CoV-2, seven FDA-approved drugs were designated as the best SARS-CoV-2 nsp16-nsp10 2'-o-methyltransferase (2'OMTase) inhibitors through 3009 compounds. The in silico inhibitory potential of the examined compounds against SARS-CoV-2 nsp16-nsp10 2'-o-methyltransferase (PDB ID: (6W4H) was conducted through a multi-step screening approach. At the beginning, molecular fingerprints experiment with SAM (S-Adenosylmethionine), the co-crystallized ligand of the targeted enzyme, unveiled the resemblance of 147 drugs. Then, a structural similarity experiment recommended 26 compounds. Therefore, the 26 compounds were docked against 2'OMTase to reveal the potential inhibitory effect of seven promising compounds (Protirelin, (1187), Calcium folinate (1913), Raltegravir (1995), Regadenoson (2176), Ertapenem (2396), Methylergometrine (2532), and Thiamine pyrophosphate hydrochloride (2612)). Out of the docked ligands, Ertapenem (2396) showed an ideal binding mode like that of the co-crystallized ligand (SAM). It occupied all sub-pockets of the active site and bound the crucial amino acids. Accordingly, some MD simulation experiments (RMSD, RMSF, Rg, SASA, and H-bonding) have been conducted for the 2'OMTase-Ertapenem complex over 100 ns. The performed MD experiments verified the correct binding mode of Ertapenem against 2'OMTase exhibiting low energy and optimal dynamics. Finally, MM-PBSA studies indicated that Ertapenem bonded advantageously to the targeted protein with a free energy value of -43 KJ/mol. Furthermore, the binding free energy analysis revealed the essential amino acids of 2'OMTase that served positively to the binding. The achieved results bring hope to find a treatment for COVID-19 via in vitro and in vivo studies for the pointed compounds.


Subject(s)
Methyltransferases , SARS-CoV-2 , Viral Nonstructural Proteins , Viral Regulatory and Accessory Proteins , Ertapenem/pharmacology , Ligands , Methyltransferases/antagonists & inhibitors , Molecular Docking Simulation , Molecular Dynamics Simulation , S-Adenosylmethionine/chemistry , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/antagonists & inhibitors
15.
J Virol ; 96(6): e0205921, 2022 03 23.
Article in English | MEDLINE | ID: covidwho-1788916

ABSTRACT

The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious global pathogen prevalent in all types of poultry flocks. IBV is responsible for economic losses and welfare issues in domestic poultry, resulting in a significant risk to food security. IBV vaccines are currently generated by serial passage of virulent IBV field isolates through embryonated hens' eggs. The different patterns of genomic variation accumulated during this process means that the exact mechanism of attenuation is unknown and presents a risk of reversion to virulence. Additionally, the passaging process adapts the virus to replicate in chicken embryos, increasing embryo lethality. Vaccines produced in this manner are therefore unsuitable for in ovo application. We have developed a reverse genetics system, based on the pathogenic IBV strain M41, to identify genes which can be targeted for rational attenuation. During the development of this reverse genetics system, we identified four amino acids, located in nonstructural proteins (nsps) 10, 14, 15, and 16, which resulted in attenuation both in vivo and in ovo. Further investigation highlighted a role of amino acid changes, Pro85Leu in nsp 10 and Val393Leu in nsp 14, in the attenuated in vivo phenotype observed. This study provides evidence that mutations in nsps offer a promising mechanism for the development of rationally attenuated live vaccines against IBV, which have the potential for in ovo application. IMPORTANCE The Gammacoronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute, highly contagious, economically important disease of poultry. Vaccination is achieved using a mixture of live attenuated vaccines for young chicks and inactivated vaccines as boosters for laying hens. Live attenuated vaccines are generated through serial passage in embryonated hens' eggs, an empirical process which achieves attenuation but retains immunogenicity. However, these vaccines have a risk of reversion to virulence, and they are lethal to the embryo. In this study, we identified amino acids in the replicase gene which attenuated IBV strain M41, both in vivo and in ovo. Stability assays indicate that the attenuating amino acids are stable and unlikely to revert. The data in this study provide evidence that specific modifications in the replicase gene offer a promising direction for IBV live attenuated vaccine development, with the potential for in ovo application.


Subject(s)
Amino Acids , Coronavirus Infections , Infectious bronchitis virus , Poultry Diseases , Viral Nonstructural Proteins , Viral Vaccines , Amino Acids/chemistry , Amino Acids/genetics , Animals , Chick Embryo , Chickens , Coronavirus Infections/prevention & control , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Female , Infectious bronchitis virus/genetics , Poultry Diseases/prevention & control , Poultry Diseases/virology , Vaccines, Attenuated/genetics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Vaccines/genetics
16.
J Mol Biol ; 434(10): 167583, 2022 May 30.
Article in English | MEDLINE | ID: covidwho-1778319

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 infection has impacted the world economy and healthcare infrastructure. Key reagents with high specificity to SARS-CoV-2 proteins are currently lacking, which limits our ability to understand the pathophysiology of SARS-CoV-2 infections. To address this need, we initiated a series of studies to generate and develop highly specific antibodies against proteins from SARS-CoV-2 using an antibody engineering platform. These efforts resulted in 18 monoclonal antibodies against nine SARS-CoV-2 proteins. Here we report the characterization of several antibodies, including those that recognize Nsp1, Nsp8, Nsp12, and Orf3b viral proteins. Our validation studies included evaluation for use of antibodies in ELISA, western blots, and immunofluorescence assays (IFA). We expect that availability of these antibodies will enhance our ability to further characterize host-viral interactions, including specific roles played by viral proteins during infection, to acquire a better understanding of the pathophysiology of SARS-CoV-2 infections.


Subject(s)
Antibodies, Monoclonal , Antibodies, Viral , COVID-19 , SARS-CoV-2 , Viral Proteins , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Viral/genetics , Antibodies, Viral/immunology , COVID-19/metabolism , Cell Surface Display Techniques , Coronavirus RNA-Dependent RNA Polymerase/analysis , Enzyme-Linked Immunosorbent Assay , Humans , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/analysis , Viral Proteins/analysis
17.
J Biol Chem ; 298(5): 101923, 2022 05.
Article in English | MEDLINE | ID: covidwho-1778265

ABSTRACT

Coronavirus (CoV) genomes consist of positive-sense single-stranded RNA and are among the largest viral RNAs known to date (∼30 kb). As a result, CoVs deploy sophisticated mechanisms to replicate these extraordinarily large genomes as well as to transcribe subgenomic messenger RNAs. Since 2003, with the emergence of three highly pathogenic CoVs (SARS-CoV, MERS-CoV, and SARS-CoV-2), significant progress has been made in the molecular characterization of the viral proteins and key mechanisms involved in CoV RNA genome replication. For example, to allow for the maintenance and integrity of their large RNA genomes, CoVs have acquired RNA proofreading 3'-5' exoribonuclease activity (in nonstructural protein nsp14). In order to replicate the large genome, the viral-RNA-dependent RNA polymerase (RdRp; in nsp12) is supplemented by a processivity factor (made of the viral complex nsp7/nsp8), making it the fastest known RdRp. Lastly, a viral structural protein, the nucleocapsid (N) protein, which is primarily involved in genome encapsidation, is required for efficient viral replication and transcription. Therefore, CoVs are a paradox among positive-strand RNA viruses in the sense that they use both a processivity factor and have proofreading activity reminiscent of DNA organisms in addition to structural proteins that mediate efficient RNA synthesis, commonly used by negative-strand RNA viruses. In this review, we present a historical perspective of these unsuspected discoveries and detail the current knowledge on the core replicative machinery deployed by CoVs.


Subject(s)
Genome, Viral , Positive-Strand RNA Viruses , SARS-CoV-2 , COVID-19/virology , Genome, Viral/genetics , Humans , Mutation , Positive-Strand RNA Viruses/genetics , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/genetics
18.
Eur J Med Chem ; 228: 114030, 2022 Jan 15.
Article in English | MEDLINE | ID: covidwho-1768048

ABSTRACT

The epidemic coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has now spread worldwide and efficacious therapeutics are urgently needed. 3-Chymotrypsin-like cysteine protease (3CLpro) is an indispensable protein in viral replication and represents an attractive drug target for fighting COVID-19. Herein, we report the discovery of 9,10-dihydrophenanthrene derivatives as non-peptidomimetic and non-covalent inhibitors of the SARS-CoV-2 3CLpro. The structure-activity relationships of 9,10-dihydrophenanthrenes as SARS-CoV-2 3CLpro inhibitors have carefully been investigated and discussed in this study. Among all tested 9,10-dihydrophenanthrene derivatives, C1 and C2 display the most potent SARS-CoV-2 3CLpro inhibition activity, with IC50 values of 1.55 ± 0.21 µM and 1.81 ± 0.17 µM, respectively. Further enzyme kinetics assays show that these two compounds dose-dependently inhibit SARS-CoV-2 3CLprovia a mixed-inhibition manner. Molecular docking simulations reveal the binding modes of C1 in the dimer interface and substrate-binding pocket of the target. In addition, C1 shows outstanding metabolic stability in the gastrointestinal tract, human plasma, and human liver microsome, suggesting that this agent has the potential to be developed as an orally administrated SARS-CoV-2 3CLpro inhibitor.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Drug Discovery/methods , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Gastrointestinal Tract/metabolism , Humans , Kinetics , Microsomes, Liver/metabolism , Molecular Docking Simulation , Protein Binding , Structure-Activity Relationship , Viral Nonstructural Proteins/antagonists & inhibitors
19.
Elife ; 112022 Mar 24.
Article in English | MEDLINE | ID: covidwho-1761115

ABSTRACT

The SARS-CoV-2 non-structural protein 1 (Nsp1) contains an N-terminal domain and C-terminal helices connected by a short linker region. The C-terminal helices of Nsp1 (Nsp1-C-ter) from SARS-CoV-2 bind in the mRNA entry channel of the 40S ribosomal subunit and blocks mRNA entry, thereby shutting down host protein synthesis. Nsp1 suppresses host immune function and is vital for viral replication. Hence, Nsp1 appears to be an attractive target for therapeutics. In this study, we have in silico screened Food and Drug Administration (FDA)-approved drugs against Nsp1-C-ter. Among the top hits obtained, montelukast sodium hydrate binds to Nsp1 with a binding affinity (KD) of 10.8 ± 0.2 µM in vitro. It forms a stable complex with Nsp1-C-ter in simulation runs with -95.8 ± 13.3 kJ/mol binding energy. Montelukast sodium hydrate also rescues the inhibitory effect of Nsp1 in host protein synthesis, as demonstrated by the expression of firefly luciferase reporter gene in cells. Importantly, it shows antiviral activity against SARS-CoV-2 with reduced viral replication in HEK cells expressing ACE2 and Vero-E6 cells. We, therefore, propose montelukast sodium hydrate can be used as a lead molecule to design potent inhibitors to help combat SARS-CoV-2 infection.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Delivery Systems , Humans , Pharmaceutical Preparations , RNA, Messenger/metabolism , Viral Nonstructural Proteins/metabolism
20.
Biomolecules ; 12(4)2022 03 22.
Article in English | MEDLINE | ID: covidwho-1753432

ABSTRACT

The merging of distinct computational approaches has become a powerful strategy for discovering new biologically active compounds. By using molecular modeling, significant efforts have recently resulted in the development of new molecules, demonstrating high efficiency in reducing the replication of severe acute respiratory coronavirus 2 (SARS-CoV-2), the agent responsible for the COVID-19 pandemic. We have focused our interest on non-structural protein Nsp13 (NTPase/helicase), as a crucial protein, embedded in the replication-transcription complex (RTC), that controls the virus life cycle. To assist in the identification of the most druggable surfaces of Nsps13, we applied a combination of four computational tools: FTMap, SiteMap, Fpocket and LigandScout. These software packages explored the binding sites for different three-dimensional structures of RTC complexes (PDB codes: 6XEZ, 7CXM, 7CXN), thus, detecting several hot spots, that were clustered to obtain ensemble consensus sites, through a combination of four different approaches. The comparison of data provided new insights about putative druggable sites that might be employed for further docking simulations on druggable surfaces of Nsps13, in a scenario of repurposing drugs.


Subject(s)
Antiviral Agents , RNA Helicases , SARS-CoV-2 , Viral Nonstructural Proteins , Antiviral Agents/chemistry , Binding Sites , COVID-19 , Humans , Pandemics , RNA Helicases/antagonists & inhibitors , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors
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