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1.
Elife ; 102021 12 07.
Article in English | MEDLINE | ID: covidwho-1555771

ABSTRACT

The emergence of SARS-CoV-2 variants threatens current vaccines and therapeutic antibodies and urgently demands powerful new therapeutics that can resist viral escape. We therefore generated a large nanobody repertoire to saturate the distinct and highly conserved available epitope space of SARS-CoV-2 spike, including the S1 receptor binding domain, N-terminal domain, and the S2 subunit, to identify new nanobody binding sites that may reflect novel mechanisms of viral neutralization. Structural mapping and functional assays show that indeed these highly stable monovalent nanobodies potently inhibit SARS-CoV-2 infection, display numerous neutralization mechanisms, are effective against emerging variants of concern, and are resistant to mutational escape. Rational combinations of these nanobodies that bind to distinct sites within and between spike subunits exhibit extraordinary synergy and suggest multiple tailored therapeutic and prophylactic strategies.


Subject(s)
COVID-19/immunology , SARS-CoV-2/immunology , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Binding Sites , Camelids, New World/immunology , Epitopes/genetics , Epitopes/immunology , HEK293 Cells , Humans , Male , Neutralization Tests , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
2.
Non-conventional in English | [Unspecified Source], Grey literature | ID: grc-750506

ABSTRACT

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryo-electron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template-product in complex with two molecules of the nsp13 helicase. The Nidovirus-order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12-thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapeutic development.

3.
Proc Natl Acad Sci U S A ; 118(19)2021 05 11.
Article in English | MEDLINE | ID: covidwho-1254144

ABSTRACT

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3' segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3' end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.


Subject(s)
COVID-19/virology , SARS-CoV-2/physiology , Virus Replication/genetics , Adenosine Monophosphate/pharmacology , Antiviral Agents/pharmacology , COVID-19/genetics , COVID-19/metabolism , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Cryoelectron Microscopy/methods , DNA Helicases/metabolism , Genome, Viral , Humans , Molecular Dynamics Simulation , RNA Helicases/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/metabolism , RNA-Dependent RNA Polymerase/physiology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics
4.
Structure ; 29(2): 186-195.e6, 2021 02 04.
Article in English | MEDLINE | ID: covidwho-939287

ABSTRACT

Recent advances in single-particle cryogenic electron microscopy (cryo-EM) have enabled the structural determination of numerous protein assemblies at high resolution, yielding unprecedented insights into their function. However, despite its extraordinary capabilities, cryo-EM remains time-consuming and resource-intensive. It is therefore beneficial to have a means for rapidly assessing and optimizing the quality of samples prior to lengthy cryo-EM analyses. To do this, we have developed a native mass spectrometry (nMS) platform that provides rapid feedback on sample quality and highly streamlined biochemical screening. Because nMS enables accurate mass analysis of protein complexes, it is well suited to routine evaluation of the composition, integrity, and homogeneity of samples prior to their plunge-freezing on EM grids. We demonstrate the utility of our nMS-based platform for facilitating cryo-EM studies using structural characterizations of exemplar bacterial transcription complexes as well as the replication-transcription assembly from the SARS-CoV-2 virus that is responsible for the COVID-19 pandemic.


Subject(s)
Cryoelectron Microscopy/methods , Mass Spectrometry/methods , Single Molecule Imaging/methods , Escherichia coli , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Methyltransferases/chemistry , Methyltransferases/metabolism , RNA Helicases/chemistry , RNA Helicases/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/ultrastructure , Transcription Factors/chemistry , Transcription Factors/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism
5.
Cell ; 182(6): 1560-1573.e13, 2020 09 17.
Article in English | MEDLINE | ID: covidwho-710427

ABSTRACT

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.


Subject(s)
Methyltransferases/chemistry , RNA Helicases/chemistry , RNA-Dependent RNA Polymerase/chemistry , Viral Nonstructural Proteins/chemistry , Virus Replication , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/metabolism , Betacoronavirus/genetics , Betacoronavirus/metabolism , Betacoronavirus/ultrastructure , Binding Sites , Coronavirus RNA-Dependent RNA Polymerase , Cryoelectron Microscopy , Holoenzymes/chemistry , Holoenzymes/metabolism , Magnesium/metabolism , Methyltransferases/metabolism , Protein Binding , RNA Helicases/metabolism , RNA, Viral/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2 , Viral Nonstructural Proteins/metabolism
6.
bioRxiv ; 2020 Jul 13.
Article in English | MEDLINE | ID: covidwho-663149

ABSTRACT

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryo-electron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template-product in complex with two molecules of the nsp13 helicase. The Nidovirus-order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12-thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapeutic development.

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