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2.
Cell Mol Immunol ; 2022 Mar 10.
Article in English | MEDLINE | ID: covidwho-1735231

ABSTRACT

Neutrophil extracellular traps (NETs) can capture and kill viruses, such as influenza viruses, human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV), thus contributing to host defense. Contrary to our expectation, we show here that the histones released by NETosis enhance the infectivity of SARS-CoV-2, as found by using live SARS-CoV-2 and two pseudovirus systems as well as a mouse model. The histone H3 or H4 selectively binds to subunit 2 of the spike (S) protein, as shown by a biochemical binding assay, surface plasmon resonance and binding energy calculation as well as the construction of a mutant S protein by replacing four acidic amino acids. Sialic acid on the host cell surface is the key molecule to which histones bridge subunit 2 of the S protein. Moreover, histones enhance cell-cell fusion. Finally, treatment with an inhibitor of NETosis, histone H3 or H4, or sialic acid notably affected the levels of sgRNA copies and the number of apoptotic cells in a mouse model. These findings suggest that SARS-CoV-2 could hijack histones from neutrophil NETosis to promote its host cell attachment and entry process and may be important in exploring pathogenesis and possible strategies to develop new effective therapies for COVID-19.

4.
MedComm (2020) ; 2021 Dec 16.
Article in English | MEDLINE | ID: covidwho-1568243

ABSTRACT

Coronavirus disease 2019 (COVID-19) has brought about a great threat to global public health. Recently, a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant B.1.1.529 has been reported in South Africa and induced a rapid increase in COVID-19 cases. On November 24, 2021, B.1.1.529 named Omicron was designated as a variant under monitoring (VUM) by World Health Organization (WHO). Two days later, the Omicron variant was classified as a variant of concern (VOC). This variant harbors a high number of mutations, including 15 mutations in the receptor-binding domain (RBD) of spike. The Omicron variant also shares several mutations with the previous VOC Alpha, Beta, and Gamma variants, which immediately raised global concerns about viral transmissibility, pathogenicity, and immune evasion. Here we described the discovery and characteristics of the Omicron variant, compared the mutations of the spike in the five VOCs, and further raised possible strategies to prevent and overcome the prevalence of the Omicron variant.

6.
Signal Transduct Target Ther ; 6(1): 343, 2021 09 16.
Article in English | MEDLINE | ID: covidwho-1415924

ABSTRACT

SARS-CoV-2 recognizes, via its spike receptor-binding domain (S-RBD), human angiotensin-converting enzyme 2 (ACE2) to initiate infection. Ecto-domain protein of ACE2 can therefore function as a decoy. Here we show that mutations of S19W, T27W, and N330Y in ACE2 could individually enhance SARS-CoV-2 S-RBD binding. Y330 could be synergistically combined with either W19 or W27, whereas W19 and W27 are mutually unbeneficial. The structures of SARS-CoV-2 S-RBD bound to the ACE2 mutants reveal that the enhanced binding is mainly contributed by the van der Waals interactions mediated by the aromatic side-chains from W19, W27, and Y330. While Y330 and W19/W27 are distantly located and devoid of any steric interference, W19 and W27 are shown to orient their side-chains toward each other and to cause steric conflicts, explaining their incompatibility. Finally, using pseudotyped SARS-CoV-2 viruses, we demonstrate that these residue substitutions are associated with dramatically improved entry-inhibition efficacy toward both wild-type and antibody-resistant viruses. Taken together, our biochemical and structural data have delineated the basis for the elevated S-RBD binding associated with S19W, T27W, and N330Y mutations in ACE2, paving the way for potential application of these mutants in clinical treatment of COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , COVID-19 , Molecular Dynamics Simulation , Mutation, Missense , SARS-CoV-2/chemistry , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/chemistry , Antibodies, Viral/metabolism , Humans , SARS-CoV-2/genetics , SARS-CoV-2/metabolism
7.
Nucleic Acids Res ; 49(9): 5382-5392, 2021 05 21.
Article in English | MEDLINE | ID: covidwho-1387965

ABSTRACT

The emergence of SARS-CoV-2 infection has posed unprecedented threat to global public health. The virus-encoded non-structural protein 14 (nsp14) is a bi-functional enzyme consisting of an exoribonuclease (ExoN) domain and a methyltransferase (MTase) domain and plays a pivotal role in viral replication. Here, we report the structure of SARS-CoV-2 nsp14-ExoN domain bound to its co-factor nsp10 and show that, compared to the SARS-CoV nsp10/nsp14-full-length complex, SARS-CoV-2 nsp14-ExoN retains an integral exoribonuclease fold and preserves an active configuration in the catalytic center. Analysis of the nsp10/nsp14-ExoN interface reveals a footprint in nsp10 extensively overlapping with that observed in the nsp10/nsp16 structure. A marked difference in the co-factor when engaging nsp14 and nsp16 lies in helix-α1', which is further experimentally ascertained to be involved in nsp14-binding but not in nsp16-engagement. Finally, we also show that nsp10/nsp14-ExoN is enzymatically active despite the absence of nsp14-MTase domain. These data demonstrate that SARS-CoV-2 nsp10/nsp14-ExoN functions as an exoribonuclease with both structural and functional integrity.


Subject(s)
Biocatalysis , Exoribonucleases/chemistry , Exoribonucleases/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Exoribonucleases/genetics , Guanine , Methyltransferases/chemistry , Methyltransferases/deficiency , Methyltransferases/genetics , Methyltransferases/metabolism , Models, Molecular , Protein Domains/genetics , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Viral Regulatory and Accessory Proteins/genetics
9.
Science ; 371(6536): 1374-1378, 2021 03 26.
Article in English | MEDLINE | ID: covidwho-1255508

ABSTRACT

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continually poses serious threats to global public health. The main protease (Mpro) of SARS-CoV-2 plays a central role in viral replication. We designed and synthesized 32 new bicycloproline-containing Mpro inhibitors derived from either boceprevir or telaprevir, both of which are approved antivirals. All compounds inhibited SARS-CoV-2 Mpro activity in vitro, with 50% inhibitory concentration values ranging from 7.6 to 748.5 nM. The cocrystal structure of Mpro in complex with MI-23, one of the most potent compounds, revealed its interaction mode. Two compounds (MI-09 and MI-30) showed excellent antiviral activity in cell-based assays. In a transgenic mouse model of SARS-CoV-2 infection, oral or intraperitoneal treatment with MI-09 or MI-30 significantly reduced lung viral loads and lung lesions. Both also displayed good pharmacokinetic properties and safety in rats.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/pharmacology , Animals , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , COVID-19/pathology , COVID-19/virology , Cell Line , Cell Survival/drug effects , Chemokine CXCL10/metabolism , Disease Models, Animal , Drug Design , Humans , Interferon-beta/metabolism , Lung/immunology , Lung/pathology , Lung/virology , Mice , Mice, Transgenic , Oligopeptides , Proline/analogs & derivatives , Protease Inhibitors/chemistry , Protease Inhibitors/therapeutic use , Protease Inhibitors/toxicity , Rats , Rats, Sprague-Dawley , Viral Load/drug effects , Virus Replication
10.
MedComm (2020) ; 2021 May 17.
Article in English | MEDLINE | ID: covidwho-1222647

ABSTRACT

The emerging variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in pandemic call for the urgent development of universal corona virus disease 2019 (COVID-19) vaccines which could be effective for both wild-type SARS-CoV-2 and mutant strains. In the current study, we formulated protein subunit vaccines with AS03 adjuvant and recombinant proteins of S1 subunit of SARS-CoV-2 (S1-WT) and S1 variant (K417N, E484K, N501Y, and D614G) subunit (S1-Mut), and immunized transgenic mice that express human angiotensin-converting enzyme 2 (hACE2). The S1 protein-specific antibody production and the neutralization capability for SARS-CoV-2 and B.1.351 variant were measured after immunization in mice. The results revealed that the S1-Mut antigens were more effective in inhibiting the receptor-binding domain and ACE2 binding in B.1.351 variant than in wild-type SARS-CoV-2. Furthermore, the development of a bivalent vaccine exhibited the ideal neutralization properties against wild-type and B.1.351 variant, as well as other variants. Our findings may provide a rationale for the development of a bivalent recombinant vaccine targeting the S1 protein that can induce the neutralizing antibodies against both SARS-CoV-2 variants and wild-type of the virus and may be of importance to explore the potential clinical use of bivalent recombinant vaccine in the future.

11.
Nucleic Acids Res ; 49(9): 5382-5392, 2021 05 21.
Article in English | MEDLINE | ID: covidwho-1217861

ABSTRACT

The emergence of SARS-CoV-2 infection has posed unprecedented threat to global public health. The virus-encoded non-structural protein 14 (nsp14) is a bi-functional enzyme consisting of an exoribonuclease (ExoN) domain and a methyltransferase (MTase) domain and plays a pivotal role in viral replication. Here, we report the structure of SARS-CoV-2 nsp14-ExoN domain bound to its co-factor nsp10 and show that, compared to the SARS-CoV nsp10/nsp14-full-length complex, SARS-CoV-2 nsp14-ExoN retains an integral exoribonuclease fold and preserves an active configuration in the catalytic center. Analysis of the nsp10/nsp14-ExoN interface reveals a footprint in nsp10 extensively overlapping with that observed in the nsp10/nsp16 structure. A marked difference in the co-factor when engaging nsp14 and nsp16 lies in helix-α1', which is further experimentally ascertained to be involved in nsp14-binding but not in nsp16-engagement. Finally, we also show that nsp10/nsp14-ExoN is enzymatically active despite the absence of nsp14-MTase domain. These data demonstrate that SARS-CoV-2 nsp10/nsp14-ExoN functions as an exoribonuclease with both structural and functional integrity.


Subject(s)
Biocatalysis , Exoribonucleases/chemistry , Exoribonucleases/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Exoribonucleases/genetics , Guanine , Methyltransferases/chemistry , Methyltransferases/deficiency , Methyltransferases/genetics , Methyltransferases/metabolism , Models, Molecular , Protein Domains/genetics , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Viral Regulatory and Accessory Proteins/genetics
13.
Cell ; 181(4): 894-904.e9, 2020 05 14.
Article in English | MEDLINE | ID: covidwho-45975

ABSTRACT

The recent emergence of a novel coronavirus (SARS-CoV-2) in China has caused significant public health concerns. Recently, ACE2 was reported as an entry receptor for SARS-CoV-2. In this study, we present the crystal structure of the C-terminal domain of SARS-CoV-2 (SARS-CoV-2-CTD) spike (S) protein in complex with human ACE2 (hACE2), which reveals a hACE2-binding mode similar overall to that observed for SARS-CoV. However, atomic details at the binding interface demonstrate that key residue substitutions in SARS-CoV-2-CTD slightly strengthen the interaction and lead to higher affinity for receptor binding than SARS-RBD. Additionally, a panel of murine monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) against SARS-CoV-S1/receptor-binding domain (RBD) were unable to interact with the SARS-CoV-2 S protein, indicating notable differences in antigenicity between SARS-CoV and SARS-CoV-2. These findings shed light on the viral pathogenesis and provide important structural information regarding development of therapeutic countermeasures against the emerging virus.


Subject(s)
Betacoronavirus/chemistry , Peptidyl-Dipeptidase A/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Betacoronavirus/physiology , Epitopes , Humans , Models, Molecular , Peptidyl-Dipeptidase A/metabolism , Phylogeny , Protein Domains , SARS Virus/chemistry , SARS Virus/physiology , SARS-CoV-2 , Sequence Alignment , Spike Glycoprotein, Coronavirus/metabolism
14.
J Virol ; 94(5)2020 02 14.
Article in English | MEDLINE | ID: covidwho-2071

ABSTRACT

Continued reports of Middle East respiratory syndrome coronavirus (MERS-CoV) infecting humans have occurred since the identification of this virus in 2012. MERS-CoV is prone to cause endemic disease in the Middle East, with several dozen spillover infections to other continents. It is hypothesized that MERS-CoV originated from bat coronaviruses and that dromedary camels are its natural reservoir. Although gene segments identical to MERS-CoV were sequenced from certain species of bats and one species experimentally shed the virus, it is still unknown whether other bats can transmit the virus. Here, at the molecular level, we found that all purified bat CD26s (bCD26s) from a diverse range of species interact with the receptor binding domain (RBD) of MERS-CoV, with equilibrium dissociation constant values ranging from several to hundreds at the micromolar level. Moreover, all bCD26s expressed in this study mediated the entry of pseudotyped MERS-CoV to receptor-expressing cells, indicating the broad potential engagement of bCD26s as MERS-CoV receptors. Further structural analysis indicated that in the bat receptor, compared to the human receptor, substitutions of key residues and their adjacent amino acids leads to decreased binding affinity to the MERS-RBD. These results add more evidence to the existing belief that bats are the original source of MERS-CoV and suggest that bCD26s in many species can mediate the entry of the virus, which has significant implications for the surveillance and control of MERS-CoV infection.IMPORTANCE In this study, we found that bat CD26s (bCD26s) from different species exhibit large diversities, especially in the region responsible for binding to the receptor binding domain (RBD) of Middle East respiratory syndrome coronavirus (MERS-CoV). However, they maintain the interaction with MERS-RBD at varied affinities and support the entry of pseudotyped MERS-CoV. These bat receptors polymorphisms seem to confer evolutionary pressure for the adaptation of CD26-binding virus, such as the ancestor of MERS-CoV, and led to the generation of diversified CD26-engaging CoV strains. Thus, our data add more evidence to support that bats are the reservoir of MERS-CoV and similar viruses, as well as further emphasize the necessity to survey MERS-CoV and other CoVs among bats.


Subject(s)
Dipeptidyl Peptidase 4 , Middle East Respiratory Syndrome Coronavirus , Virus Attachment , Animals , Cell Line , Chiroptera , Dipeptidyl Peptidase 4/chemistry , Dipeptidyl Peptidase 4/genetics , Dipeptidyl Peptidase 4/metabolism , Humans , Middle East Respiratory Syndrome Coronavirus/chemistry , Middle East Respiratory Syndrome Coronavirus/metabolism , Protein Domains , Species Specificity
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