The SARS-CoV nsp12 Polymerase Active Site Is Tuned for Large-Genome Replication

Campagnola, Grace; Govindarajan, Vishnu; Pelletier, Annelise; Canard, Bruno; Peersen, Olve B.

doi:10.1128/jvi.00671-22

Cited by 14 publications

(14 citation statements)

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“…Recent work showed that a nsp8-nsp7 (nsp8L7) fusion protein can efficiently function as a nsp12 cofactor and allows the contributions of each nsp8 of a coronavirus core complex polymerase activity to be delineated [35]. We produced SARS-CoV-2 and PEDV nsp8L7 fusion proteins with six-residue linker regions ( Fig.…”

Section: Resultsmentioning

confidence: 99%

An alphacoronavirus polymerase structure reveals conserved co-factor functions

Anderson

Hoferle

Lee

et al. 2023

Preprint

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Coronaviruses are a diverse subfamily of viruses containing pathogens of humans and animals. This subfamily of viruses replicates their RNA genomes using a core polymerase complex composed of viral non-structural proteins: nsp7, nsp8 and nsp12. Most of our understanding of coronavirus molecular biology comes from the betacoronaviruses like SARS-CoV and SARS-CoV-2, the latter of which is the causative agent of COVID-19. In contrast, members of the alphacoronavirus genus are relatively understudied despite their importance in human and animal health. Here we have used cryo-electron microscopy to determine the structure of the alphacoronavirus porcine epidemic diarrhea virus (PEDV) core polymerase complex bound to RNA. Our structure shows an unexpected nsp8 stoichiometry in comparison to other published coronavirus polymerase structures. Biochemical analysis shows that the N-terminal extension of one nsp8 is not required for in vitro RNA synthesis for alpha and betacoronaviruses as previously hypothesized. Our work shows the importance of studying diverse coronaviruses to reveal aspects of coronavirus replication while also identifying areas of conservation to be targeted by antiviral drugs.

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Section: Resultsmentioning

confidence: 99%

An alphacoronavirus polymerase structure reveals conserved co-factor functions

Anderson

Hoferle

Lee

et al. 2023

Preprint

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“…Additionally, the core component of the RTC is the RNA synthase nsp12 [ 65 , 66 ], which stimulates its enzymatic activity with the help of cofactors nsp7 and nsp8 and together with nsp7 and nsp8 forms a core RdRp complex [ 54 ]. RdRp is a type of RNA synthetase that uses viral RNA as a template and four nucleoside triphosphates (NTPs) as substrates to synthesize RNA [ 67 , 68 , 69 , 70 , 71 ]. Currently, there are more than 30 crystal structures of SARS-CoV-2 RNA synthase in various functional states in the RCSB protein database [ 72 ].…”

Section: Drugs Targeting Rna-dependent Rna Polymerase Rdrpmentioning

confidence: 99%

Progress on COVID-19 Chemotherapeutics Discovery and Novel Technology

et al. 2022

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COVID-19 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel highly contagious and pathogenic coronavirus that emerged in late 2019. SARS-CoV-2 spreads primarily through virus-containing droplets and small particles of air pollution, which greatly increases the risk of inhaling these virus particles when people are in close proximity. COVID-19 is spreading across the world, and the COVID-19 pandemic poses a threat to human health and public safety. To date, there are no specific vaccines or effective drugs against SARS-CoV-2. In this review, we focus on the enzyme targets of the virus and host that may be critical for the discovery of chemical compounds and natural products as antiviral drugs, and describe the development of potential antiviral drugs in the preclinical and clinical stages. At the same time, we summarize novel emerging technologies applied to the research on new drug development and the pathological mechanisms of COVID-19.

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“…In order to copy equivalent levels of genetic information, and to rapidly produce full-length copies to avoid innate immune activation, the RTC must be considerably faster. Several studies have now shown the SARS-CoV RdRp (nsp12) and associated cofactors (nsp7 and nsp8) replicate approximately 3–5X faster than other viral polymerases ( Campagnola et al, 2022 ; Seifert et al, 2021 ; Shannon et al, 2020 ). However, replication-rate and fidelity are in direct competition, and faster polymerases are inevitably more error-prone ( Fitzsimmons et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

“…The guanine base of AT-9010 has more stacking power than the uracil of sofosbuvir, and likely helps to compensate for this loss in stability through interaction with residues in motif F of the fingers domain. This region is notably more open and flexible (in particular residue Arg555) in the CoV polymerase compared with other viral RdRps ( Campagnola et al, 2022 ). The missing interaction with the ribose of STP is therefore likely more detrimental, as the smaller uracil base is likely not well enough stabilized for efficient incorporation.…”

Section: Introductionmentioning

confidence: 99%

“…This serine is unique to large-genome nidovirus polymerases, with most other viral RdRps containing a glycine at this position (GDD triad). The side-chain of S759 usually forms a hydrogen bond with the ribose 2′-OH at the 3′-end of the product RNA, stabilizing it in place for catalysis and compensating for the increased flexibility of the fingers domain ( Campagnola et al, 2022 ). The S759A mutation was shown to decrease the preference for RDV-TP as a substrate ∼10-fold, predicted to be due to the loss of a potential (alternative) hydrogen bond formed between the RDV 1′-cyano group and the serine side-chain ( Stevens et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

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