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1.
Nature ; 614(7949): 781-787, 2023 02.
Article in English | MEDLINE | ID: covidwho-2221840

ABSTRACT

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart adenosine triphosphate3,4. Our results explain the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap6.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase , Cryoelectron Microscopy , SARS-CoV-2 , Humans , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Coronavirus RNA-Dependent RNA Polymerase/ultrastructure , COVID-19/virology , Nucleosides/metabolism , Nucleosides/pharmacology , RNA, Viral/biosynthesis , RNA, Viral/chemistry , RNA, Viral/metabolism , SARS-CoV-2/enzymology , Substrate Specificity , Guanosine Triphosphate/metabolism , RNA Caps
2.
J Biol Chem ; 299(3): 102954, 2023 03.
Article in English | MEDLINE | ID: covidwho-2210672

ABSTRACT

COVID-19, caused by the coronavirus SARS-CoV-2, represents a serious worldwide health issue, with continually emerging new variants challenging current therapeutics. One promising alternate therapeutic avenue is represented by nanobodies, small single-chain antibodies derived from camelids with numerous advantageous properties and the potential to neutralize the virus. For identification and characterization of a broad spectrum of anti-SARS-CoV-2 Spike nanobodies, we further optimized a yeast display method, leveraging a previously published mass spectrometry-based method, using B-cell complementary DNA from the same immunized animals as a source of VHH sequences. Yeast display captured many of the sequences identified by the previous approach, as well as many additional sequences that proved to encode a large new repertoire of nanobodies with high affinities and neutralization activities against different SARS-CoV-2 variants. We evaluated DNA shuffling applied to the three complementarity-determining regions of antiviral nanobodies. The results suggested a surprising degree of modularity to complementarity-determining region function. Importantly, the yeast display approach applied to nanobody libraries from immunized animals allows parallel interrogation of a vast number of nanobodies. For example, we employed a modified yeast display to carry out massively parallel epitope binning. The current yeast display approach proved comparable in efficiency and specificity to the mass spectrometry-based approach, while requiring none of the infrastructure and expertise required for that approach, making these highly complementary approaches that together appear to comprehensively explore the paratope space. The larger repertoires produced maximize the likelihood of discovering broadly specific reagents and those that powerfully synergize in mixtures.


Subject(s)
Antibodies, Neutralizing , SARS-CoV-2 , Single-Domain Antibodies , Animals , Antibodies, Neutralizing/genetics , Antibodies, Viral/genetics , Complementarity Determining Regions , Saccharomyces cerevisiae/genetics , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Single-Domain Antibodies/genetics , Spike Glycoprotein, Coronavirus/immunology
3.
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
4.
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
5.
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
6.
J Cell Biol ; 219(10)2020 10 05.
Article in English | MEDLINE | ID: covidwho-713813

ABSTRACT

With the rapid global spread of SARS-CoV-2, we have become acutely aware of the inadequacies of our ability to respond to viral epidemics. Although disrupting the viral life cycle is critical for limiting viral spread and disease, it has proven challenging to develop targeted and selective therapeutics. Synthetic lethality offers a promising but largely unexploited strategy against infectious viral disease; as viruses infect cells, they abnormally alter the cell state, unwittingly exposing new vulnerabilities in the infected cell. Therefore, we propose that effective therapies can be developed to selectively target the virally reconfigured host cell networks that accompany altered cellular states to cripple the host cell that has been converted into a virus factory, thus disrupting the viral life cycle.


Subject(s)
Antiviral Agents/pharmacology , Host Microbial Interactions/drug effects , Virus Diseases/drug therapy , Virus Replication/drug effects , Drug Discovery , Humans , Immunologic Factors/pharmacology , Metabolic Networks and Pathways/drug effects , Protein Interaction Maps , Proteolysis , RNA Viruses/drug effects , RNA Viruses/physiology , Virus Diseases/genetics
7.
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
8.
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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