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      Structure of the RNA-dependent RNA polymerase from COVID-19 virus

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          Abstract

          A novel coronavirus (COVID-19 virus) outbreak has caused a global pandemic resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase (RdRp, also named nsp12) is the central component of coronaviral replication/transcription machinery and appears to be a primary target for the antiviral drug, remdesivir. We report the cryo-EM structure of COVID-19 virus full-length nsp12 in complex with cofactors nsp7 and nsp8 at 2.9-Å resolution. In addition to the conserved architecture of the polymerase core of the viral polymerase family, nsp12 possesses a newly identified β-hairpin domain at its N terminus. A comparative analysis model shows how remdesivir binds to this polymerase. The structure provides a basis for the design of new antiviral therapeutics targeting viral RdRp.

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          Most cited references 5

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          Nucleotide polymerase inhibitor sofosbuvir plus ribavirin for hepatitis C.

          The standard treatment for hepatitis C virus (HCV) infection is interferon, which is administered subcutaneously and can have troublesome side effects. We evaluated sofosbuvir, an oral nucleotide inhibitor of HCV polymerase, in interferon-sparing and interferon-free regimens for the treatment of HCV infection. We provided open-label treatment to eight groups of patients. A total of 40 previously untreated patients with HCV genotype 2 or 3 infection were randomly assigned to four groups; all four groups received sofosbuvir (at a dose of 400 mg once daily) plus ribavirin for 12 weeks. Three of these groups also received peginterferon alfa-2a for 4, 8, or 12 weeks. Two additional groups of previously untreated patients with HCV genotype 2 or 3 infection received sofosbuvir monotherapy for 12 weeks or sofosbuvir plus peginterferon alfa-2a and ribavirin for 8 weeks. Two groups of patients with HCV genotype 1 infection received sofosbuvir and ribavirin for 12 weeks: 10 patients with no response to prior treatment and 25 with no previous treatment. We report the rate of sustained virologic response 24 weeks after therapy. Of the 40 patients who underwent randomization, all 10 (100%) who received sofosbuvir plus ribavirin without interferon and all 30 (100%) who received sofosbuvir plus ribavirin for 12 weeks and interferon for 4, 8, or 12 weeks had a sustained virologic response at 24 weeks. For the other patients with HCV genotype 2 or 3 infection, all 10 (100%) who received sofosbuvir plus peginterferon alfa-2a and ribavirin for 8 weeks had a sustained virologic response at 24 weeks, as did 6 of 10 (60%) who received sofosbuvir monotherapy. Among patients with HCV genotype 1 infection, 21 of 25 previously untreated patients (84%) and 1 of 10 with no response to previous therapy (10%) had a sustained virologic response at 24 weeks. The most common adverse events were headache, fatigue, insomnia, nausea, rash, and anemia. Sofosbuvir plus ribavirin for 12 weeks may be effective in previously untreated patients with HCV genotype 1, 2, or 3 infection. (Funded by Pharmasset and Gilead Sciences; ClinicalTrials.gov number, NCT01260350.).
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            Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase.

            Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site. Copyright © 2015, American Association for the Advancement of Science.
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              Structural basis for active site closure by the poliovirus RNA-dependent RNA polymerase.

              Positive-strand RNA viruses include a large number of human and animal pathogens whose essential RNA-dependent RNA polymerases (RdRPs) share a structurally homologous core with an encircled active site. RdRPs are targets for antiviral drug development, but these efforts are hindered by limited structural information about the RdRP catalytic cycle. To further our understanding of RdRP function, we assembled, purified, and then crystallized poliovirus elongation complexes after multiple rounds of nucleotide incorporation. Here we present structures capturing the active polymerase and its nucleotide triphosphate complexes in four distinct states, leading us to propose a six-state catalytic cycle involving residues that are highly conserved among positive-strand RNA virus RdRPs. The structures indicate that RdRPs use a fully prepositioned templating base for nucleotide recognition and close their active sites for catalysis using a novel structural rearrangement in the palm domain. The data also suggest that translocation by RDRPs may not be directly linked to the conformational changes responsible for active site closure and reopening.
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                Author and article information

                Journal
                Science
                Science
                SCIENCE
                Science (New York, N.y.)
                American Association for the Advancement of Science
                0036-8075
                1095-9203
                10 April 2020
                Affiliations
                [1 ]Laboratory of Structural Biology, School of Life Sciences, and School of Medicine, Tsinghua University, Beijing, China.
                [2 ]Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
                [3 ]State Key Laboratory of Medicinal Chemical Biology, Frontiers Science Center for Cell Response, College of Life Sciences, and College of Pharmacy, Nankai University, Tianjin, China.
                [4 ]State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Science and Technology, Guangxi University, Nanning, China.
                [5 ]School of Life Sciences, Tianjin University, Tianjin, China.
                [6 ]School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, Australia.
                [7 ]National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, CAS, Beijing, China.
                Author notes
                [*]

                These authors contributed equally to this work.

                Article
                abb7498
                10.1126/science.abb7498
                7164392
                32277040
                Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

                This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).

                Funding
                Funded by: doi http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 81520108019
                Funded by: doi http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 813300237
                Funded by: doi http://dx.doi.org/10.13039/501100003399, Science and Technology Commission of Shanghai Municipality;
                Award ID: 20431900200
                Funded by: Strategic Priority Research Program of the Chinese Academy of Sciences;
                Award ID: XDB08020200
                Funded by: National Program on Key Research Project of China;
                Award ID: 2017YFC0840300
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