Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
1.
Commun Biol ; 4(1): 999, 2021 08 24.
Article in English | MEDLINE | ID: covidwho-1371605

ABSTRACT

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Previous structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the enzyme and resolve its structure at 5.5 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 each and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs.


Subject(s)
SARS-CoV-2/enzymology , Dimerization , Humans , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism
2.
Nat Struct Mol Biol ; 28(9): 740-746, 2021 09.
Article in English | MEDLINE | ID: covidwho-1354110

ABSTRACT

Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, ß-D-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp-RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.


Subject(s)
COVID-19/prevention & control , Cytidine/analogs & derivatives , Hydroxylamines/metabolism , Mutagenesis/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Base Sequence , COVID-19/drug therapy , COVID-19/virology , Cytidine/chemistry , Cytidine/metabolism , Cytidine/pharmacology , Humans , Hydroxylamines/chemistry , Hydroxylamines/pharmacology , Models, Molecular , Molecular Structure , Mutagenesis/drug effects , Mutation/drug effects , Mutation/genetics , Nucleic Acid Conformation , Protein Binding/drug effects , Protein Conformation , RNA, Viral/chemistry , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Virus Replication/drug effects , Virus Replication/genetics
3.
EMBO J ; 40(19): e107985, 2021 10 01.
Article in English | MEDLINE | ID: covidwho-1323478

ABSTRACT

Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein, we isolated 45 infection-blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS-CoV-2 at 17-50 pM concentration (0.2-0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X-ray and cryo-EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune-escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low-picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such "fold-promoting" nanobodies may allow for simplified production of vaccines and their adaptation to viral escape-mutations.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Mutation/immunology , SARS-CoV-2/immunology , Single-Domain Antibodies/immunology , Animals , COVID-19/virology , Camelids, New World/immunology , Camelids, New World/virology , Cell Line , Escherichia coli/virology , Female , Humans , Spike Glycoprotein, Coronavirus/immunology
4.
Biospektrum (Heidelb) ; 27(1): 49-53, 2021.
Article in German | MEDLINE | ID: covidwho-1092773

ABSTRACT

Coronaviruses use an RNA-dependent RNA polymerase to replicate and transcribe their RNA genome. The structure of the SARS-CoV-2 polymerase was determined by cryo-electron microscopy within a short time in spring 2020. The structure explains how the viral enzyme synthesizes RNA and how it replicates the exceptionally large genome in a processive manner. The most recent structure-function studies further reveal the mechanism of polymerase inhibition by remdesivir, an approved drug for the treatment of COVID-19.

5.
Nat Commun ; 12(1): 279, 2021 01 12.
Article in English | MEDLINE | ID: covidwho-1026823

ABSTRACT

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3'-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3'-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3'-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , RNA-Dependent RNA Polymerase/drug effects , SARS-CoV-2/drug effects , Antiviral Agents/pharmacology , Aptamers, Nucleotide , COVID-19/drug therapy , Coronavirus RNA-Dependent RNA Polymerase/drug effects , Nucleotides , RNA, Viral , RNA-Dependent RNA Polymerase/genetics , SARS-CoV-2/enzymology , Virus Replication/drug effects
6.
Nature ; 584(7819): 154-156, 2020 08.
Article in English | MEDLINE | ID: covidwho-326051

ABSTRACT

The new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes1-3. Here we present a cryo-electron microscopy structure of the SARS-CoV-2 RdRp in an active form that mimics the replicating enzyme. The structure comprises the viral proteins non-structural protein 12 (nsp12), nsp8 and nsp7, and more than two turns of RNA template-product duplex. The active-site cleft of nsp12 binds to the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the second turn of RNA. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged 'sliding poles'. These sliding poles can account for the known processivity of RdRp that is required for replicating the long genome of coronaviruses3. Our results enable a detailed analysis of the inhibitory mechanisms that underlie the antiviral activity of substances such as remdesivir, a drug for the treatment of coronavirus disease 2019 (COVID-19)4.


Subject(s)
Betacoronavirus/enzymology , Cryoelectron Microscopy , RNA, Viral/biosynthesis , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Betacoronavirus/drug effects , Betacoronavirus/genetics , Betacoronavirus/ultrastructure , Coronavirus RNA-Dependent RNA Polymerase , Models, Molecular , Protein Conformation , RNA, Viral/chemistry , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/ultrastructure , SARS-CoV-2 , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/ultrastructure
SELECTION OF CITATIONS
SEARCH DETAIL