Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex

Chen, James; Malone, Brandon; Llewellyn, Eliza; Grasso, Michael; Shelton, Patrick M. M.; Olinares, Paul Dominic B.; Maruthi, Kashyap; Eng, Edward T.; Vatandaslar, Hasan; Chait, Brian T.; Kapoor, Tarun M.; Darst, Seth A.; Campbell, Elizabeth A.

doi:10.1101/2020.07.08.194084

Cited by 34 publications

(50 citation statements)

References 97 publications

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“…1d). These shifts indicate stabilization via ligand binding, and are consistent with recent structural data showing ADP:AlF3 binding within the nsp13 active site 5 (Fig. 1d).…”

Section: Biochemical Characterization Of Sars-cov-2 Nsp13supporting

confidence: 91%

“…The efficiency of nsp13 unwindase activity is low compared to other helicases, but may be enhanced by either cooperativity or by interaction with the viral RdRp [8][9][10] . Consistently, recent structural data has shown that SARS-CoV-2 nsp13 can form a complex with RdRP 5 . Nonetheless, the potential mechanisms underlying nsp13 activation have not been explored.…”

supporting

confidence: 81%

“…These mechanisms are not mutually exclusive, and a continuum between them likely exists 32 . Nsp13 is non-hexameric 5 , and based on the shape of the force-velocity curve (Fig. 4d), we propose that it more closely matches the definition of a passive helicase.…”

Section: Discussionmentioning

confidence: 54%

“…4e). While the recent structural data does not directly inform on how nsp13 and the RdRp together work on the same RNA substrate 5 , existing kinetic data shows that RdRP enhances nsp13 helicases activity 8 . Moreover, there are numerous instances of polymerases activating helicases in other viral systems, including Zika 24 , T7 12,21,25 , and T4 [26][27][28] .…”

Section: Discussionmentioning

confidence: 99%

“…However, the positively-charged His6-tag may also artificially bias polynucleotide binding. Therefore, for our analyses we used recombinant affinity tag-free, full-length SARS-CoV-2 nsp13 (hereafter, nsp13), which we have reported recently 5 . Briefly, the protein was subjected to Ni-NTA affinity, cation exchange (CaptoS HP) and size exclusion (S200 increase) chromatography.…”

Section: Biochemical Characterization Of Sars-cov-2 Nsp13mentioning

confidence: 99%

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Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

Mickolajczyk

Shelton

Grasso

et al. 2020

Preprint

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The superfamily-1 helicase non-structural protein 13 (nsp13) is required for SARS-CoV-2 replication, making it an important antiviral therapeutic target. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the dsRNA, suggesting a passive unwinding mechanism. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be potently activated by picoNewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, drawing stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.

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“…1d). These shifts indicate stabilization via ligand binding, and are consistent with recent structural data showing ADP:AlF3 binding within the nsp13 active site 5 (Fig. 1d).…”

Section: Biochemical Characterization Of Sars-cov-2 Nsp13supporting

confidence: 91%

supporting

confidence: 81%

Section: Discussionmentioning

confidence: 54%

Section: Discussionmentioning

confidence: 99%

Section: Biochemical Characterization Of Sars-cov-2 Nsp13mentioning

confidence: 99%

See 3 more Smart Citations

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

Mickolajczyk

Shelton

Grasso

et al. 2020

Preprint

Self Cite

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Dissecting the Drug Development Strategies Against SARS-CoV-2 Through Diverse Computational Modeling Techniques

Adhikari

Amin

Jha

2020

Methods in Pharmacology and Toxicology

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The world of the twenty-first century has not experienced such a lockdown situation before, which leads to a complete shutdown of economy not until the novel coronavirus disease 2019 (COVID-19) came caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It becomes an unacceptable global threat to human lives. The COVID-19 has been known only worldwide in the last few months, but it is spreading in a speed of light from Wuhan, China, to the rest of the world. About 2 crore of people have been infected and more than 7 lakh of people have died worldwide till now due to the deadly SARS-CoV-2 infection. There are still no drugs exclusively available in the market for the treatment of SARS-CoV-2 infection, though supportive treatment of hydroxychloroquine, ribavirin, favipiravir, and remdesivir have shown clinical evidences to treat COVID-19. Therefore, it is utmost importance for the medicinal chemists to design and discover novel drugs urgently to rescue or to protect the humanity worldwide from this deadly virus. However, lack of experimental evidences and understanding the behavior of the SARS-CoV-2 within this short period may hinder the process of drug discovery. Still a ray of hope resides in the structural features of SARS-CoV-2 and related coronaviruses (SARS-CoV and MERS-CoV) as these are homologous. Therefore, depending on the established viral target proteins (spike protein, ACE-2, 3CL pro , PL pro , RdRp, helicase, as well as other viral proteins), novel chemical entities may be designed. In this context, several computational modeling approaches (generally structure-based modeling techniques) may be utilized which are cost-effective and less time-consuming. Different structure-based modeling techniques, namely, homology modeling, robust molecular docking, molecular dynamics simulation, and structure-based pharmacophore mapping followed by in silico virtual screening, may be effective and fruitful approaches to design compounds against SARS-CoV-2. In this chapter, various structure-based drug design and discovery strategies from target identification that could be optimized against SARS-CoV-2 have been discussed in detail. Additionally, ongoing and previously reported computational modeling techniques performed by different groups of researchers on various SARS-CoV-2 target proteins have been highlighted elaborately. In addition to the identification techniques of drugs, this chapter also discloses their binding mode of action along with the pharmacokinetics and toxicity criteria computed by modeling techniques. This chapter, therefore, may be a stepping-stone for the researchers to open up a new horizon in the discovery of novel anti-coronavirus drugs in the future.

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RNA-dependent RNA polymerase: Structure, mechanism, and drug discovery for COVID-19

Jiang

Yin

2021

Biochemical and Biophysical Research Communications

131

109

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Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly become a global pandemic. Although great efforts have been made to develop effective therapeutic interventions, only the nucleotide analog remdesivir was approved for emergency use against COVID-19. Remdesivir targets the RNA-dependent RNA polymerase (RdRp), an essential enzyme for viral RNA replication and a promising drug target for COVID-19. Recently, several structures of RdRp in complex with substrate RNA and remdesivir were reported, providing insights into the mechanisms of RNA recognition by RdRp. These structures also reveal the mechanism of RdRp inhibition by nucleotide inhibitors and offer a molecular template for the development of RdRp-targeting drugs. This review discusses the recognition mechanism of RNA and nucleotide inhibitor by RdRp, and its implication in drug discovery.

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Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex

Cited by 34 publications

References 97 publications

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

Dissecting the Drug Development Strategies Against SARS-CoV-2 Through Diverse Computational Modeling Techniques

RNA-dependent RNA polymerase: Structure, mechanism, and drug discovery for COVID-19

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