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1.
Int J Mol Sci ; 23(6)2022 Mar 08.
Article in English | MEDLINE | ID: covidwho-1732071

ABSTRACT

Nanobodies provide important advantages over traditional antibodies, including their smaller size and robust biochemical properties such as high thermal stability, high solubility, and the ability to be bioengineered into novel multivalent, multi-specific, and high-affinity molecules, making them a class of emerging powerful therapies against SARS-CoV-2. Recent research efforts on the design, protein engineering, and structure-functional characterization of nanobodies and their binding with SARS-CoV-2 S proteins reflected a growing realization that nanobody combinations can exploit distinct binding epitopes and leverage the intrinsic plasticity of the conformational landscape for the SARS-CoV-2 S protein to produce efficient neutralizing and mutation resistant characteristics. Structural and computational studies have also been instrumental in quantifying the structure, dynamics, and energetics of the SARS-CoV-2 spike protein binding with nanobodies. In this review, a comprehensive analysis of the current structural, biophysical, and computational biology investigations of SARS-CoV-2 S proteins and their complexes with distinct classes of nanobodies targeting different binding sites is presented. The analysis of computational studies is supplemented by an in-depth examination of mutational scanning simulations and identification of binding energy hotspots for distinct nanobody classes. The review is focused on the analysis of mechanisms underlying synergistic binding of multivalent nanobodies that can be superior to single nanobodies and conventional nanobody cocktails in combating escape mutations by effectively leveraging binding avidity and allosteric cooperativity. We discuss how structural insights and protein engineering approaches together with computational biology tools can aid in the rational design of synergistic combinations that exhibit superior binding and neutralization characteristics owing to avidity-mediated mechanisms.


Subject(s)
Binding Sites , Molecular Docking Simulation , Molecular Dynamics Simulation , Single-Domain Antibodies/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Amino Acids , Antibody Affinity , Epitopes/chemistry , Epitopes/metabolism , Humans , Multiprotein Complexes/chemistry , Mutagenesis , Protein Binding , Protein Engineering , Protein Interaction Domains and Motifs , Single-Domain Antibodies/genetics , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
2.
Biochem Biophys Res Commun ; 593: 52-56, 2022 02 19.
Article in English | MEDLINE | ID: covidwho-1633160

ABSTRACT

COVID-19, the respiratory infection caused by the novel coronavirus SARS-CoV-2, presents a clinical picture consistent with the dysregulation of many of the pathways mediated by the metalloprotease ADAM17. ADAM17 is a sheddase that plays a key role in the modulation of ACE2, the receptor which also functions as the point of attachment leading to cell entry by the virus. This work investigates the possibility that ADAM17 dysregulation and attachment of the SARS-CoV-2 virion to the ACE2 receptor are linked events, with the latter causing the former. Tetraspanins, the transmembrane proteins that function as scaffolds for the construction of viral entry platforms, are mooted as key components in this connection.


Subject(s)
ADAM17 Protein/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Receptors, Virus/metabolism , SARS-CoV-2/metabolism , Tetraspanin 29/metabolism , Virus Internalization , ADAM17 Protein/chemistry , Angiotensin-Converting Enzyme 2/chemistry , Binding Sites , COVID-19/epidemiology , COVID-19/transmission , COVID-19/virology , Humans , Models, Biological , Molecular Docking Simulation , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Pandemics , Protein Binding , Protein Domains , Receptors, Virus/chemistry , SARS-CoV-2/physiology , Tetraspanin 29/chemistry
3.
J Immunol ; 208(3): 753-761, 2022 02 01.
Article in English | MEDLINE | ID: covidwho-1614089

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has seriously threatened global public health. Severe COVID-19 has been reported to be associated with an impaired IFN response. However, the mechanisms of how SARS-CoV-2 antagonizes the host IFN response are poorly understood. In this study, we report that SARS-CoV-2 helicase NSP13 inhibits type I IFN production by directly targeting TANK-binding kinase 1 (TBK1) for degradation. Interestingly, inhibition of autophagy by genetic knockout of Beclin1 or pharmacological inhibition can rescue NSP13-mediated TBK1 degradation in HEK-293T cells. Subsequent studies revealed that NSP13 recruits TBK1 to p62, and the absence of p62 can also inhibit TBK1 degradation in HEK-293T and HeLa cells. Finally, TBK1 and p62 degradation and p62 aggregation were observed during SARS-CoV-2 infection in HeLa-ACE2 and Calu3 cells. Overall, our study shows that NSP13 inhibits type I IFN production by recruiting TBK1 to p62 for autophagic degradation, enabling it to evade the host innate immune response, which provides new insights into the transmission and pathogenesis of SARS-CoV-2 infection.


Subject(s)
Autophagy , COVID-19/immunology , Coronavirus RNA-Dependent RNA Polymerase/physiology , Interferon Type I/biosynthesis , Methyltransferases/physiology , Protein Serine-Threonine Kinases/metabolism , RNA Helicases/physiology , SARS-CoV-2/physiology , Sequestosome-1 Protein/metabolism , Viral Nonstructural Proteins/physiology , Beclin-1/antagonists & inhibitors , Cell Line , Down-Regulation , Humans , Immune Evasion , Immunity, Innate , Immunoprecipitation , Interferon Type I/genetics , Multiprotein Complexes , Protein Aggregates , Protein Interaction Mapping
4.
FEBS J ; 288(17): 5130-5147, 2021 09.
Article in English | MEDLINE | ID: covidwho-1388264

ABSTRACT

SARS-CoV-2 virus has triggered a global pandemic with devastating consequences. The understanding of fundamental aspects of this virus is of extreme importance. In this work, we studied the viral ribonuclease nsp14, one of the most interferon antagonists from SARS-CoV-2. Nsp14 is a multifunctional protein with two distinct activities, an N-terminal 3'-to-5' exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), both critical for coronaviruses life cycle, indicating nsp14 as a prominent target for the development of antiviral drugs. In coronaviruses, nsp14 ExoN activity is stimulated through the interaction with the nsp10 protein. We have performed a biochemical characterization of nsp14-nsp10 complex from SARS-CoV-2. We confirm the 3'-5' exoribonuclease and MTase activities of nsp14 and the critical role of nsp10 in upregulating the nsp14 ExoN activity. Furthermore, we demonstrate that SARS-CoV-2 nsp14 N7-MTase activity is functionally independent of the ExoN activity and nsp10. A model from SARS-CoV-2 nsp14-nsp10 complex allowed mapping key nsp10 residues involved in this interaction. Our results show that a stable interaction between nsp10 and nsp14 is required for the nsp14-mediated ExoN activity of SARS-CoV-2. We studied the role of conserved DEDD catalytic residues of SARS-CoV-2 nsp14 ExoN. Our results show that motif I of ExoN domain is essential for the nsp14 function, contrasting to the functionality of these residues in other coronaviruses, which can have important implications regarding the specific pathogenesis of SARS-CoV-2. This work unraveled a basis for discovering inhibitors targeting specific amino acids in order to disrupt the assembly of this complex and interfere with coronaviruses replication.


Subject(s)
COVID-19/genetics , Exoribonucleases/genetics , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Viral Regulatory and Accessory Proteins/genetics , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/virology , Drug Design , Exoribonucleases/antagonists & inhibitors , Humans , Multiprotein Complexes/drug effects , Multiprotein Complexes/genetics , Protein Interaction Maps/genetics , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Virus Replication/genetics
5.
Nucleic Acids Res ; 48(3): 1392-1405, 2020 02 20.
Article in English | MEDLINE | ID: covidwho-1332861

ABSTRACT

The enterovirus 71 (EV71) 3Dpol is an RNA-dependent RNA polymerase (RdRP) that plays the central role in the viral genome replication, and is an important target in antiviral studies. Here, we report a crystal structure of EV71 3Dpol elongation complex (EC) at 1.8 Å resolution. The structure reveals that the 5'-end guanosine of the downstream RNA template interacts with a fingers domain pocket, with the base sandwiched by H44 and R277 side chains through hydrophobic stacking interactions, and these interactions are still maintained after one in-crystal translocation event induced by nucleotide incorporation, implying that the pocket could regulate the functional properties of the polymerase by interacting with RNA. When mutated, residue R277 showed an impact on virus proliferation in virological studies with residue H44 having a synergistic effect. In vitro biochemical data further suggest that mutations at these two sites affect RNA binding, EC stability, but not polymerase catalytic rate (kcat) and apparent NTP affinity (KM,NTP). We propose that, although rarely captured by crystallography, similar surface pocket interaction with nucleobase may commonly exist in nucleic acid motor enzymes to facilitate their processivity. Potential applications in antiviral drug and vaccine development are also discussed.


Subject(s)
Enterovirus A, Human/ultrastructure , Multiprotein Complexes/ultrastructure , Protein Conformation , RNA-Dependent RNA Polymerase/ultrastructure , Antiviral Agents/chemistry , Binding Sites , Crystallography, X-Ray , Enterovirus A, Human/chemistry , Enterovirus A, Human/genetics , Genome, Viral , Humans , Models, Molecular , Multiprotein Complexes/chemistry , Nucleotides/chemistry , RNA, Viral/chemistry , RNA, Viral/ultrastructure , RNA-Dependent RNA Polymerase/chemistry , Virus Replication/genetics
6.
Nucleic Acids Res ; 49(10): 5956-5966, 2021 06 04.
Article in English | MEDLINE | ID: covidwho-1231040

ABSTRACT

Replication of the ∼30 kb-long coronavirus genome is mediated by a complex of non-structural proteins (NSP), in which NSP7 and NSP8 play a critical role in regulating the RNA-dependent RNA polymerase (RdRP) activity of NSP12. The assembly of NSP7, NSP8 and NSP12 proteins is highly dynamic in solution, yet the underlying mechanism remains elusive. We report the crystal structure of the complex between NSP7 and NSP8 of SARS-CoV-2, revealing a 2:2 heterotetrameric form. Formation of the NSP7-NSP8 complex is mediated by two distinct oligomer interfaces, with interface I responsible for heterodimeric NSP7-NSP8 assembly, and interface II mediating the heterotetrameric interaction between the two NSP7-NSP8 dimers. Structure-guided mutagenesis, combined with biochemical and enzymatic assays, further reveals a structural coupling between the two oligomer interfaces, as well as the importance of these interfaces for the RdRP activity of the NSP7-NSP8-NSP12 complex. Finally, we identify an NSP7 mutation that differentially affects the stability of the NSP7-NSP8 and NSP7-NSP8-NSP12 complexes leading to a selective impairment of the RdRP activity. Together, this study provides deep insights into the structure and mechanism for the dynamic assembly of NSP7 and NSP8 in regulating the replication of the SARS-CoV-2 genome, with important implications for antiviral drug development.


Subject(s)
COVID-19 , Coronavirus RNA-Dependent RNA Polymerase/chemistry , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/chemistry , Chromatography, Gel , Coronavirus RNA-Dependent RNA Polymerase/biosynthesis , Coronavirus RNA-Dependent RNA Polymerase/genetics , Crystallography, X-Ray , Dimerization , Models, Molecular , Multiprotein Complexes , Mutagenesis , Mutation , Protein Conformation , Protein Domains , Protein Interaction Mapping , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Structure-Activity Relationship , Viral Nonstructural Proteins/genetics , Virus Replication
7.
Nat Commun ; 12(1): 2843, 2021 05 14.
Article in English | MEDLINE | ID: covidwho-1228252

ABSTRACT

Although the accessory proteins are considered non-essential for coronavirus replication, accumulating evidences demonstrate they are critical to virus-host interaction and pathogenesis. Orf9b is a unique accessory protein of SARS-CoV-2 and SARS-CoV. It is implicated in immune evasion by targeting mitochondria, where it associates with the versatile adapter TOM70. Here, we determined the crystal structure of SARS-CoV-2 orf9b in complex with the cytosolic segment of human TOM70 to 2.2 Å. A central portion of orf9b occupies the deep pocket in the TOM70 C-terminal domain (CTD) and adopts a helical conformation strikingly different from the ß-sheet-rich structure of the orf9b homodimer. Interactions between orf9b and TOM70 CTD are primarily hydrophobic and distinct from the electrostatic interaction between the heat shock protein 90 (Hsp90) EEVD motif and the TOM70 N-terminal domain (NTD). Using isothermal titration calorimetry (ITC), we demonstrated that the orf9b dimer does not bind TOM70, but a synthetic peptide harboring a segment of orf9b (denoted C-peptide) binds TOM70 with nanomolar KD. While the interaction between C-peptide and TOM70 CTD is an endothermic process, the interaction between Hsp90 EEVD and TOM70 NTD is exothermic, which underscores the distinct binding mechanisms at NTD and CTD pockets. Strikingly, the binding affinity of Hsp90 EEVD motif to TOM70 NTD is reduced by ~29-fold when orf9b occupies the pocket of TOM70 CTD, supporting the hypothesis that orf9b allosterically inhibits the Hsp90/TOM70 interaction. Our findings shed light on the mechanism underlying SARS-CoV-2 orf9b mediated suppression of interferon responses.


Subject(s)
Coronavirus Nucleocapsid Proteins/chemistry , Mitochondrial Membrane Transport Proteins/chemistry , Multiprotein Complexes/chemistry , Recombinant Proteins/chemistry , Binding Sites/genetics , COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Cryoelectron Microscopy , Crystallography, X-Ray , Escherichia coli/genetics , Host Microbial Interactions , Humans , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism , Models, Molecular , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding , Protein Domains , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , SARS-CoV-2/physiology
8.
Proc Natl Acad Sci U S A ; 118(21)2021 05 25.
Article in English | MEDLINE | ID: covidwho-1223143

ABSTRACT

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2'-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.


Subject(s)
RNA Caps/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , SARS-CoV-2/chemistry , Crystallography , Methylation , Methyltransferases/chemistry , Methyltransferases/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , RNA Cap Analogs/chemistry , RNA Cap Analogs/metabolism , RNA Caps/chemistry , RNA, Messenger/chemistry , RNA, Viral/chemistry , S-Adenosylhomocysteine/chemistry , S-Adenosylhomocysteine/metabolism , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Synchrotrons , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism
9.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Article in English | MEDLINE | ID: covidwho-1172591

ABSTRACT

In order to understand the transmission and virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to understand the functions of each of the gene products encoded in the viral genome. One feature of the SARS-CoV-2 genome that is not present in related, common coronaviruses is ORF10, a putative 38-amino acid protein-coding gene. Proteomic studies found that ORF10 binds to an E3 ubiquitin ligase containing Cullin-2, Rbx1, Elongin B, Elongin C, and ZYG11B (CRL2ZYG11B). Since CRL2ZYG11B mediates protein degradation, one possible role for ORF10 is to "hijack" CRL2ZYG11B in order to target cellular, antiviral proteins for ubiquitylation and subsequent proteasomal degradation. Here, we investigated whether ORF10 hijacks CRL2ZYG11B or functions in other ways, for example, as an inhibitor or substrate of CRL2ZYG11B While we confirm the ORF10-ZYG11B interaction and show that the N terminus of ORF10 is critical for it, we find no evidence that ORF10 is functioning to inhibit or hijack CRL2ZYG11B Furthermore, ZYG11B and its paralog ZER1 are dispensable for SARS-CoV-2 infection in cultured cells. We conclude that the interaction between ORF10 and CRL2ZYG11B is not relevant for SARS-CoV-2 infection in vitro.


Subject(s)
COVID-19/metabolism , Cell Cycle Proteins/metabolism , Cullin Proteins/metabolism , Multiprotein Complexes/metabolism , Open Reading Frames , SARS-CoV-2/metabolism , Viral Proteins/metabolism , COVID-19/genetics , Cell Cycle Proteins/genetics , Cullin Proteins/genetics , HEK293 Cells , Humans , Multiprotein Complexes/genetics , SARS-CoV-2/genetics , Viral Proteins/genetics
10.
Proc Natl Acad Sci U S A ; 117(48): 30687-30698, 2020 12 01.
Article in English | MEDLINE | ID: covidwho-922313

ABSTRACT

The SARS-CoV-2 pandemic has made it clear that we have a desperate need for antivirals. We present work that the mammalian SKI complex is a broad-spectrum, host-directed, antiviral drug target. Yeast suppressor screening was utilized to find a functional genetic interaction between proteins from influenza A virus (IAV) and Middle East respiratory syndrome coronavirus (MERS-CoV) with eukaryotic proteins that may be potential host factors involved in replication. This screening identified the SKI complex as a potential host factor for both viruses. In mammalian systems siRNA-mediated knockdown of SKI genes inhibited replication of IAV and MERS-CoV. In silico modeling and database screening identified a binding pocket on the SKI complex and compounds predicted to bind. Experimental assays of those compounds identified three chemical structures that were antiviral against IAV and MERS-CoV along with the filoviruses Ebola and Marburg and two further coronaviruses, SARS-CoV and SARS-CoV-2. The mechanism of antiviral activity is through inhibition of viral RNA production. This work defines the mammalian SKI complex as a broad-spectrum antiviral drug target and identifies lead compounds for further development.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus/drug effects , Filoviridae/drug effects , Host-Pathogen Interactions/drug effects , Multiprotein Complexes/metabolism , Orthomyxoviridae/drug effects , Cell Line , Genes, Suppressor , Models, Molecular , Molecular Targeted Therapy , Protein Binding , RNA, Small Interfering/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Saccharomyces cerevisiae/genetics , Viral Proteins/metabolism , Virus Replication/drug effects
11.
Cell Rep ; 31(11): 107774, 2020 06 16.
Article in English | MEDLINE | ID: covidwho-594914

ABSTRACT

The ongoing global pandemic of coronavirus disease 2019 (COVID-19) has caused a huge number of human deaths. Currently, there are no specific drugs or vaccines available for this virus (SARS-CoV-2). The viral polymerase is a promising antiviral target. Here, we describe the near-atomic-resolution structure of the SARS-CoV-2 polymerase complex consisting of the nsp12 catalytic subunit and nsp7-nsp8 cofactors. This structure highly resembles the counterpart of SARS-CoV with conserved motifs for all viral RNA-dependent RNA polymerases and suggests a mechanism of activation by cofactors. Biochemical studies reveal reduced activity of the core polymerase complex and lower thermostability of individual subunits of SARS-CoV-2 compared with SARS-CoV. These findings provide important insights into RNA synthesis by coronavirus polymerase and indicate adaptation of SARS-CoV-2 toward humans with a relatively lower body temperature than the natural bat hosts.


Subject(s)
Betacoronavirus/enzymology , Cryoelectron Microscopy , RNA-Dependent RNA Polymerase/chemistry , Viral Nonstructural Proteins/chemistry , Amino Acid Substitution , Coronavirus RNA-Dependent RNA Polymerase , Escherichia coli/genetics , Evolution, Molecular , Models, Molecular , Multiprotein Complexes/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS Virus/enzymology , SARS-CoV-2 , Viral Nonstructural Proteins/metabolism
12.
J Struct Biol ; 211(2): 107548, 2020 08 01.
Article in English | MEDLINE | ID: covidwho-593332

ABSTRACT

We report the crystal structure of the SARS-CoV-2 putative primase composed of the nsp7 and nsp8 proteins. We observed a dimer of dimers (2:2 nsp7-nsp8) in the crystallographic asymmetric unit. The structure revealed a fold with a helical core of the heterotetramer formed by both nsp7 and nsp8 that is flanked with two symmetry-related nsp8 ß-sheet subdomains. It was also revealed that two hydrophobic interfaces one of approx. 1340 Å2 connects the nsp7 to nsp8 and a second one of approx. 950 Å2 connects the dimers and form the observed heterotetramer. Interestingly, analysis of the surface electrostatic potential revealed a putative RNA binding site that is formed only within the heterotetramer.


Subject(s)
Betacoronavirus/chemistry , DNA Primase/chemistry , Viral Nonstructural Proteins/chemistry , Binding Sites , Coronavirus RNA-Dependent RNA Polymerase , Crystallography, X-Ray , DNA Primase/metabolism , Models, Molecular , Multiprotein Complexes , Protein Conformation , Protein Multimerization , RNA/metabolism , SARS-CoV-2 , Viral Nonstructural Proteins/metabolism
13.
Science ; 368(6498): 1499-1504, 2020 06 26.
Article in English | MEDLINE | ID: covidwho-154668

ABSTRACT

The pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global crisis. Replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp) enzyme, a target of the antiviral drug remdesivir. Here we report the cryo-electron microscopy structure of the SARS-CoV-2 RdRp, both in the apo form at 2.8-angstrom resolution and in complex with a 50-base template-primer RNA and remdesivir at 2.5-angstrom resolution. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp, where remdesivir is covalently incorporated into the primer strand at the first replicated base pair, and terminates chain elongation. Our structures provide insights into the mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/chemistry , Betacoronavirus/enzymology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Adenosine Monophosphate/pharmacology , Alanine/chemistry , Alanine/metabolism , Alanine/pharmacology , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Betacoronavirus/physiology , Catalytic Domain , Coronavirus RNA-Dependent RNA Polymerase , Cryoelectron Microscopy , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Models, Molecular , Multiprotein Complexes/chemistry , Protein Conformation , RNA, Viral/chemistry , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2 , Viral Nonstructural Proteins/metabolism , Virus Replication
14.
Science ; 368(6492): 779-782, 2020 05 15.
Article in English | MEDLINE | ID: covidwho-47347

ABSTRACT

A novel coronavirus [severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)] outbreak has caused a global coronavirus disease 2019 (COVID-19) pandemic, resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase [(RdRp), also named nsp12] is the central component of coronaviral replication and transcription machinery, and it appears to be a primary target for the antiviral drug remdesivir. We report the cryo-electron microscopy structure of COVID-19 virus full-length nsp12 in complex with cofactors nsp7 and nsp8 at 2.9-angstrom resolution. In addition to the conserved architecture of the polymerase core of the viral polymerase family, nsp12 possesses a newly identified ß-hairpin domain at its N terminus. A comparative analysis model shows how remdesivir binds to this polymerase. The structure provides a basis for the design of new antiviral therapeutics that target viral RdRp.


Subject(s)
Betacoronavirus/enzymology , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/ultrastructure , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/ultrastructure , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/metabolism , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/metabolism , Alanine/pharmacology , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Catalytic Domain , Coronavirus RNA-Dependent RNA Polymerase , Cryoelectron Microscopy , Drug Design , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Protein Conformation, beta-Strand , Protein Domains , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2 , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism
15.
J Med Virol ; 92(9): 1649-1656, 2020 09.
Article in English | MEDLINE | ID: covidwho-27178

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the recent COVID-19 public health crisis. Bat is the widely believed original host of SARS-CoV-2. However, its intermediate host before transmitting to humans is not clear. Some studies proposed pangolin, snake, or turtle as the intermediate hosts. Angiotensin-converting enzyme 2 (ACE2) is the receptor for SARS-CoV-2, which determines the potential host range for SARS-CoV-2. On the basis of structural information of the complex of human ACE2 and SARS-CoV-2 receptor-binding domain (RBD), we analyzed the affinity to S protein of the 20 key residues in ACE2 from mammal, bird, turtle, and snake. Several ACE2 proteins from Primates, Bovidae, Cricetidae, and Cetacea maintained the majority of key residues in ACE2 for associating with SARS-CoV-2 RBD. The simulated structures indicated that ACE2 proteins from Bovidae and Cricetidae were able to associate with SARS-CoV-2 RBD. We found that nearly half of the key residues in turtle, snake, and bird were changed. The simulated structures showed several key contacts with SARS-CoV-2 RBD in turtle and snake ACE2 were abolished. This study demonstrated that neither snake nor turtle was the intermediate hosts for SARS-CoV-2, which further reinforced the concept that the reptiles are resistant against infection of coronavirus. This study suggested that Bovidae and Cricetidae should be included in the screening of intermediate hosts for SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , COVID-19/virology , Receptors, Virus/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/chemistry , Animals , Arvicolinae , Cattle , Humans , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Protein Binding , Receptors, Virus/chemistry , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Structure-Activity Relationship , Viral Tropism
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