The Crystal Structure of Coxsackievirus B3 RNA-Dependent RNA Polymerase in Complex with Its Protein Primer VPg Confirms the Existence of a Second VPg Binding Site on Picornaviridae Polymerases

ABSTRACT The RNA-dependent RNA polymerase (RdRp) is a central piece in the replication machinery of RNA viruses. In picornaviruses this essential RdRp activity also uridylates the VPg peptide, which then serves as a primer for RNA synthesis. Previous genetic, binding, and biochemical data have identified a VPg binding site on poliovirus RdRp and have shown that is was implicated in VPg uridylation. More recent structural studies have identified a topologically distinct site on the closely related foot-and-mouth disease virus RdRp supposed to be the actual VPg-primer-binding site. Here, we report the crystal structure at 2.5-Å resolution of active coxsackievirus B3 RdRp (also named 3Dpol) in a complex with VPg and a pyrophosphate. The pyrophosphate is situated in the active-site cavity, occupying a putative binding site either for the coproduct of the reaction or an incoming NTP. VPg is bound at the base of the thumb subdomain, providing first structural evidence for the VPg binding site previously identified by genetic and biochemical methods. The binding mode of VPg to CVB3 3Dpol at this site excludes its uridylation by the carrier 3Dpol. We suggest that VPg at this position is either uridylated by another 3Dpol molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3Dpol/VPg complex structure is expected to contribute to the understanding of the multicomponent VPg-uridylation complex essential for the initiation of genome replication of picornaviruses.

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A crystal structure of alphavirus nonstructural protein 4 (nsP4) reveals an intrinsically dynamic RNA-dependent RNA polymerase

10.1101/2021.05.27.445971 ◽

2021 ◽

Author(s):

Yaw Bia Tan ◽

Laura Sandra Lello ◽

Xin Liu ◽

Yee-Song Law ◽

Congbao Kang ◽

...

Keyword(s):

Crystal Structure ◽

Rna Polymerase ◽

Nonstructural Protein ◽

3D Structure ◽

Rna Replication ◽

Viral Rna ◽

Rdrp Activity ◽

Rna Dependent Rna Polymerase ◽

Intrinsically Disordered ◽

Rdrp Domain

Alphaviruses such as Ross River virus (RRV), chikungunya virus and Venezuelan equine encephalitis virus are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report a crystal structure of the RdRp domain of nsP4 from RRV determined at a resolution of 2.6 Å. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses and picornaviruses. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods, we showed that in-solution nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were able to catalyze in vitro RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication.

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A single mutation attenuates both the transcription termination and RNA-dependent RNA polymerase activity of T7 RNA polymerase

10.1101/2021.01.11.426313 ◽

2021 ◽

Author(s):

Hui Wu ◽

Ting Wei ◽

Rui Cheng ◽

Fengtao Huang ◽

Xuelin Lu ◽

...

Keyword(s):

Rna Polymerase ◽

Transcription Termination ◽

Complex Structure ◽

T7 Rna Polymerase ◽

Prototype Model ◽

Single Mutation ◽

Rdrp Activity ◽

Rna Dependent Rna Polymerase ◽

Stem Loop

ABSTRACTTranscription termination is one of the least understood processes of gene expression. As the prototype model for transcription studies, the single-subunit T7 RNA polymerase (RNAP) was known to response to two types of termination signals, while the mechanism underlying such termination especially the specific elements of the polymerase involved in is still unclear, due to the lack of a termination complex structure. Here we applied phage-assisted continuous evolution to obtain variants of T7 RNAP that can bypass the typical class I T7 terminator with stem-loop structure. Through in vivo selection and in vitro characterization, we discovered a single mutation S43Y that significantly decreased the termination efficiency of T7 RNAP at all transcription terminators tested. Coincidently, the S43Y mutation almost eliminates the RNA-dependent RNA polymerase (RdRp) of T7 RNAP without affecting the major DNA-dependent RNA polymerase (DdRp) activity of the enzyme, indicating the relationship between transcription termination and RdRp activity, and suggesting a model in which the stem-loop terminator induces the RdRp activity which competes with the ongoing DdRp activity to cause transcription termination. The T7 RNAP S43Y mutant as an enzymatic reagent for in vitro transcription reduces the undesired termination in run-off RNA synthesis and produces RNA with higher terminal homogeneity.

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Higher-order structures of the foot-and-mouth disease virus RNA-dependent RNA polymerase required for dynamic inter-molecular interactions involved in viral genome replication

10.1101/2020.12.17.423260 ◽

2020 ◽

Author(s):

Eleni-Anna Loundras ◽

James Streetley ◽

Morgan R. Herod ◽

Rebecca F. Thompson ◽

Mark Harris ◽

...

Keyword(s):

Rna Polymerase ◽

Disease Virus ◽

Molecular Interactions ◽

Foot And Mouth Disease ◽

Higher Order ◽

Mouth Disease ◽

Genome Replication ◽

Rna Dependent Rna Polymerase ◽

Mouth Disease Virus ◽

Foot And Mouth

AbstractReplication of many positive-sense RNA viruses occurs within intracellular membrane-associated compartments. These are believed to provide a favourable environment for replication to occur, concentrating essential viral structural and non-structural components, as well as protecting these components from host-cell pathogen recognition and innate immune responses. However, the details of the molecular interactions and dynamics within these structures is very limited. One of the key components of the replication machinery is the RNA-dependent RNA polymerase, RdRp. This enzyme has been shown to form higher-order fibrils in vitro. Here, using the RdRp from foot-and-mouth disease virus (termed 3Dpol), we report fibril structures, solved at ~7-9 Å resolution by cryo-EM, revealing multiple conformations of a flexible assembly. Fitting high-resolution coordinates led to the definition of potential intermolecular interactions. We employed mutagenesis using a sub-genomic replicon system to probe the importance of these interactions for replication. We use these data to propose models for the role of higher order 3Dpol complexes as a dynamic scaffold within which RNA replication can occur.

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Mechanism of SARS-CoV-2 polymerase stalling by remdesivir

Nature Communications ◽

10.1038/s41467-020-20542-0 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Goran Kokic ◽

Hauke S. Hillen ◽

Dimitry Tegunov ◽

Christian Dienemann ◽

Florian Seitz ◽

...

Keyword(s):

Electron Microscopy ◽

Rna Polymerase ◽

Binding Site ◽

Molecular Mechanism ◽

Active Center ◽

Rna Synthesis ◽

Active Form ◽

Nucleoside Triphosphate ◽

Rna Dependent Rna Polymerase ◽

Cryo Electron Microscopy

AbstractRemdesivir 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 3ʹ-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 3ʹ-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 3ʹ-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

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Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp12/7/8 RNA-dependent RNA Polymerase

10.1101/2021.04.07.438807 ◽

2021 ◽

Author(s):

Agustina P. Bertolin ◽

Florian Weissmann ◽

Jingkun Zeng ◽

Viktor Posse ◽

Jennifer C. Milligan ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Virus ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Etiologic Agent ◽

Host Cells ◽

Rdrp Activity ◽

Chemical Library ◽

Rna Dependent Rna Polymerase

SummaryThe coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologs in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer (FRET)-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified 3 novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.

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De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus

Journal of Virology ◽

10.1128/jvi.74.2.851-863.2000 ◽

2000 ◽

Vol 74 (2) ◽

pp. 851-863 ◽

Cited By ~ 217

Author(s):

Guangxiang Luo ◽

Robert K. Hamatake ◽

Danielle M. Mathis ◽

Jason Racela ◽

Karen L. Rigat ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Synthesis ◽

De Novo ◽

Oligonucleotide Primer ◽

Transferase Activity ◽

Rdrp Activity ◽

Rna Dependent Rna Polymerase ◽

Hcv Ns5b

ABSTRACT Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn2+ than in the presence of Mg2+. When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a “copy-back” mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3′ end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (≥50 μM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.

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Structure of Foot-and-Mouth Disease Virus RNA-dependent RNA Polymerase and Its Complex with a Template-Primer RNA

Journal of Biological Chemistry ◽

10.1074/jbc.m405465200 ◽

2004 ◽

Vol 279 (45) ◽

pp. 47212-47221 ◽

Cited By ~ 170

Author(s):

Cristina Ferrer-Orta ◽

Armando Arias ◽

Rosa Perez-Luque ◽

Cristina Escarmís ◽

Esteban Domingo ◽

...

Keyword(s):

Rna Polymerase ◽

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Rna Dependent Rna Polymerase ◽

Virus Rna ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Primer Rna

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Characterization of Soluble Hepatitis C Virus RNA-Dependent RNA Polymerase Expressed in Escherichia coli

Journal of Virology ◽

10.1128/jvi.73.2.1649-1654.1999 ◽

1999 ◽

Vol 73 (2) ◽

pp. 1649-1654 ◽

Cited By ~ 172

Author(s):

Eric Ferrari ◽

Jacquelyn Wright-Minogue ◽

Jane W. S. Fang ◽

Bahige M. Baroudy ◽

Johnson Y. N. Lau ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Nonstructural Protein ◽

Full Length ◽

Dependent Manner ◽

Rdrp Activity ◽

Rna Dependent Rna Polymerase ◽

Hydrophobic Domain ◽

Hcv Ns5b

ABSTRACT Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BΔCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.

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(F)uridylylated Peptides Linked to VPg1 of Foot-and- Mouth Disease Virus (FMDV): Design, Synthesis and X-Ray Crystallography of the Complexes with FMDV RNA-Dependent RNA Polymerase

Molecules ◽

10.3390/molecules24132360 ◽

2019 ◽

Vol 24 (13) ◽

pp. 2360 ◽

Cited By ~ 2

Author(s):

Sonia de Castro ◽

Cristina Ferrer-Orta ◽

Alberto Mills ◽

Gloria Fernández-Cureses ◽

Federico Gago ◽

...

Keyword(s):

Rna Polymerase ◽

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Viral Rna ◽

Hydroxyl Group ◽

Rna Dependent Rna Polymerase ◽

X Ray ◽

Mouth Disease Virus ◽

Foot And Mouth

Foot-and-mouth disease virus (FMDV) is an RNA virus belonging to the Picornaviridae family that contains three small viral proteins (VPgs), named VPg1, VPg2 and VPg3, linked to the 5′-end of the viral genome. These VPg proteins act as primers for RNA replication, which is initiated by the consecutive binding of two UMP molecules to the hydroxyl group of Tyr3 in VPg. This process, termed uridylylation, is catalyzed by the viral RNA-dependent RNA polymerase named 3Dpol. 5-Fluorouridine triphosphate (FUTP) is a potent competitive inhibitor of VPg uridylylation. Peptide analysis showed FUMP covalently linked to the Tyr3 of VPg. This fluorouridylylation prevents further incorporation of the second UMP residue. The molecular basis of how the incorporated FUMP blocks the incorporation of the second UMP is still unknown. To investigate the mechanism of inhibition of VPg uridylylation by FUMP, we have prepared a simplified 15-mer model of VPg1 containing FUMP and studied its x-ray crystal structure in complex with 3Dpol. Unfortunately, the fluorouridylylated VPg1 was disordered and not visible in the electron density maps; however, the structure of 3Dpol in the presence of VPg1-FUMP showed an 8 Å movement of the β9-α11 loop of the polymerase towards the active site cavity relative to the complex of 3Dpol with VPg1-UMP. The conformational rearrangement of this loop preceding the 3Dpol B motif seems to block the access of the template nucleotide to the catalytic cavity. This result may be useful in the design of new antivirals against not only FMDV but also other picornaviruses, since all members of this family require the uridylylation of their VPg proteins to initiate the viral RNA synthesis.

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