RESUMEN
The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA-dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication-transcription complex or RTC) associated with two copies of nsp13 (nsp132-RTC). One copy of nsp13 interacts with the template-RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backward on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here we use cryogenic-electron microscopy and molecular dynamics simulations to analyze the nsp132-RTC, revealing four distinct conformational states of the helicases. The results indicate a mechanism for the nsp132-RTC to turn backtracking on and off, using an allosteric mechanism to switch between RNA synthesis or backtracking in response to stimuli at the RdRp active site.
Asunto(s)
COVID-19 , SARS-CoV-2 , Microscopía por Crioelectrón , Humanos , ARN Helicasas/química , Proteínas no Estructurales Virales/química , Replicación ViralRESUMEN
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.
RESUMEN
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.
Asunto(s)
COVID-19/virología , SARS-CoV-2/fisiología , Replicación Viral/genética , Adenosina Monofosfato/farmacología , Antivirales/farmacología , COVID-19/genética , COVID-19/metabolismo , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , Microscopía por Crioelectrón/métodos , ADN Helicasas/metabolismo , Genoma Viral , Humanos , Simulación de Dinámica Molecular , ARN Helicasas/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/fisiología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
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.
Asunto(s)
Microscopía por Crioelectrón/métodos , Espectrometría de Masas/métodos , Imagen Individual de Molécula/métodos , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Metiltransferasas/química , Metiltransferasas/metabolismo , ARN Helicasas/química , ARN Helicasas/metabolismo , SARS-CoV-2/enzimología , SARS-CoV-2/ultraestructura , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismoRESUMEN
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.
Asunto(s)
Metiltransferasas/química , ARN Helicasas/química , ARN Polimerasa Dependiente del ARN/química , Proteínas no Estructurales Virales/química , Replicación Viral , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Betacoronavirus/genética , Betacoronavirus/metabolismo , Betacoronavirus/ultraestructura , Sitios de Unión , ARN Polimerasa Dependiente de ARN de Coronavirus , Microscopía por Crioelectrón , Holoenzimas/química , Holoenzimas/metabolismo , Magnesio/metabolismo , Metiltransferasas/metabolismo , Unión Proteica , ARN Helicasas/metabolismo , ARN Viral/química , ARN Polimerasa Dependiente del ARN/metabolismo , SARS-CoV-2 , Proteínas no Estructurales Virales/metabolismoRESUMEN
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.