https://www.ncbi.nlm.nih.gov/pubmed/31138817
(Kuvat)Figures)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538669/figure/Fig1/
Nat Commun. 2019 May 28;10(1):2342. doi: 10.1038/s41467-019-10280-3.
Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors.
Abstract
Recent
history is punctuated by the emergence of highly pathogenic
coronaviruses such as SARS- and MERS-CoV into human circulation. Upon
infecting host cells, coronaviruses assemble a multi-subunit
RNA-synthesis complex of viral non-structural proteins (nsp) responsible
for the replication and transcription of the viral genome.
Here, we present the 3.1 Å resolution structure of the SARS-CoV nsp12 polymerase bound to its essential co-factors, nsp7 and nsp8, using single particle cryo-electron microscopy.
nsp12 possesses an architecture common to all viral polymerases as well as a large N-terminal extension containing a kinase-like fold and is bound by two nsp8 co-factors.
This structure illuminates the assembly of the coronavirus core RNA-synthesis machinery, provides key insights into nsp12 polymerase catalysis and fidelity and acts as a template for the design of novel antiviral therapeutics.
Here, we present the 3.1 Å resolution structure of the SARS-CoV nsp12 polymerase bound to its essential co-factors, nsp7 and nsp8, using single particle cryo-electron microscopy.
nsp12 possesses an architecture common to all viral polymerases as well as a large N-terminal extension containing a kinase-like fold and is bound by two nsp8 co-factors.
This structure illuminates the assembly of the coronavirus core RNA-synthesis machinery, provides key insights into nsp12 polymerase catalysis and fidelity and acts as a template for the design of novel antiviral therapeutics.
- PMID:
- 31138817
- PMCID:
- PMC6538669
- DOI:
- 10.1038/s41467-019-10280-3
- [Indexed for MEDLINE]
Introduction
Coronaviruses
(CoV) represent a diverse family of positive-sense RNA viruses capable
of causing respiratory and enteric disease in human and animal hosts.
Though there are several human CoV responsible for a mild respiratory
disease1,
most notable are the highly pathogenic human CoVs: SARS-CoV and
MERS-CoV capable of causing a severe respiratory disease.
The zoonotic SARS-CoV emerged into human populations in 2002, spreading to 26 countries during its brief 9-month circulation in humans2. This epidemic resulted in >8000 infections with a ~10% case fatality rate and was eventually contained through public health measures, as there are no specific treatments approved for human CoV infections. With the exception of a smaller second SARS-CoV outbreak in 20043, SARS-CoV has been absent from human circulation since the initial outbreak ended. Despite the lack of recent human infections, SARS-CoV-like viruses continue to circulate in bat reservoirs4. Outbreaks of highly pathogenic human CoVs remain an emerging threat to global health security and are likely to continue to occur.
The threat of a novel emerging virus from the diverse CoV family necessitates antiviral strategies targeting conserved elements of the viral life cycle such as the viral machinery responsible for the replication and transcription of the positive-strand viral RNA genome.
The multi-subunit CoV RNA synthesis machinery is a complex of non-structural proteins (nsp) produced as cleavage products of the ORF1a and ORF1ab viral polyproteins5.
The nsp12 RNA-dependent RNA polymerase (RdRp) possesses some minimal activity on its own6, but the addition of the nsp7 and nsp8 co-factors greatly stimulates polymerase activity7. Though additional viral nsp subunits are likely necessary to carry out the full repertoire of replication and transcription activities, the nsp12-nsp7-nsp8 complex so far represents the minimal complex required for nucleotide polymerization. In addition, many other CoV nsps possess enzyme activities related to RNA modification8,9.
After the emergence of SARS-CoV, there was an intense effort to structurally characterize the replication complexes of CoVs. This resulted in high-resolution structure determination for many of the SARS-CoV nsps in isolation using X-ray crystallography and nuclear magnetic resonance10,11.
In addition, complexes of nsp7-nsp8, nsp10-nsp14 and nsp10-nsp16 were also determined12,13,14.
One notable gap in our structural knowledge of the CoV RNA synthesis complex was a structure of the nsp12 RNA polymerase. This structural gap included information regarding the unique N-terminal extension of nidovirus polymerases, which has been proposed to contain a nucleotidyltransferase activity (nidovirus RdRp-associated nucleotidyltransferase (NiRAN))9. It had also been unclear what the role of nsp8 and the nsp7-nsp8 complex within the viral RNA synthesis complex play, with proposed functions ranging from acting as processivity factors14 to possessing RNA primase activity15,16.
Here we used cryo-electron microscopy (cryo-EM) to determine the 3.1 Å resolution structure of SARS-CoV nsp12 bound to the nsp7 and nsp8 co-factors. This 160 kDa complex defines the largest portion of the viral RNA synthesis complex yet to be structurally characterized at high resolution.
In addition to a structurally conserved polymerase domain, this complex provides the first views of a nidovirus-unique nsp12 N-terminal extension that possesses a kinase-like fold and demonstrates an unexpected binding stoichiometry of nsp7 and nsp8 co-factors.
The zoonotic SARS-CoV emerged into human populations in 2002, spreading to 26 countries during its brief 9-month circulation in humans2. This epidemic resulted in >8000 infections with a ~10% case fatality rate and was eventually contained through public health measures, as there are no specific treatments approved for human CoV infections. With the exception of a smaller second SARS-CoV outbreak in 20043, SARS-CoV has been absent from human circulation since the initial outbreak ended. Despite the lack of recent human infections, SARS-CoV-like viruses continue to circulate in bat reservoirs4. Outbreaks of highly pathogenic human CoVs remain an emerging threat to global health security and are likely to continue to occur.
The threat of a novel emerging virus from the diverse CoV family necessitates antiviral strategies targeting conserved elements of the viral life cycle such as the viral machinery responsible for the replication and transcription of the positive-strand viral RNA genome.
The multi-subunit CoV RNA synthesis machinery is a complex of non-structural proteins (nsp) produced as cleavage products of the ORF1a and ORF1ab viral polyproteins5.
The nsp12 RNA-dependent RNA polymerase (RdRp) possesses some minimal activity on its own6, but the addition of the nsp7 and nsp8 co-factors greatly stimulates polymerase activity7. Though additional viral nsp subunits are likely necessary to carry out the full repertoire of replication and transcription activities, the nsp12-nsp7-nsp8 complex so far represents the minimal complex required for nucleotide polymerization. In addition, many other CoV nsps possess enzyme activities related to RNA modification8,9.
After the emergence of SARS-CoV, there was an intense effort to structurally characterize the replication complexes of CoVs. This resulted in high-resolution structure determination for many of the SARS-CoV nsps in isolation using X-ray crystallography and nuclear magnetic resonance10,11.
In addition, complexes of nsp7-nsp8, nsp10-nsp14 and nsp10-nsp16 were also determined12,13,14.
One notable gap in our structural knowledge of the CoV RNA synthesis complex was a structure of the nsp12 RNA polymerase. This structural gap included information regarding the unique N-terminal extension of nidovirus polymerases, which has been proposed to contain a nucleotidyltransferase activity (nidovirus RdRp-associated nucleotidyltransferase (NiRAN))9. It had also been unclear what the role of nsp8 and the nsp7-nsp8 complex within the viral RNA synthesis complex play, with proposed functions ranging from acting as processivity factors14 to possessing RNA primase activity15,16.
Here we used cryo-electron microscopy (cryo-EM) to determine the 3.1 Å resolution structure of SARS-CoV nsp12 bound to the nsp7 and nsp8 co-factors. This 160 kDa complex defines the largest portion of the viral RNA synthesis complex yet to be structurally characterized at high resolution.
In addition to a structurally conserved polymerase domain, this complex provides the first views of a nidovirus-unique nsp12 N-terminal extension that possesses a kinase-like fold and demonstrates an unexpected binding stoichiometry of nsp7 and nsp8 co-factors.
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