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1.
J Chem Inf Model ; 62(20): 4916-4927, 2022 10 24.
Article in English | MEDLINE | ID: covidwho-2062143

ABSTRACT

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 outbreak that is affecting the entire planet. As the pandemic is still spreading worldwide, with multiple mutations of the virus, it is of interest and of help to employ computational methods for identifying potential inhibitors of the enzymes responsible for viral replication. Attractive antiviral nucleotide analogue RNA-dependent RNA polymerase (RdRp) chain terminator inhibitors are investigated with this purpose. This study, based on molecular dynamics (MD) simulations, addresses the important aspects of the incorporation of an endogenously synthesized nucleoside triphosphate, ddhCTP, in comparison with the natural nucleobase cytidine triphosphate (CTP) in RdRp. The ddhCTP species is the product of the viperin antiviral protein as part of the innate immune response. The absence of the ribose 3'-OH in ddhCTP could have important implications in its inhibitory mechanism of RdRp. We built an in silico model of the RNA strand embedded in RdRp using experimental methods, starting from the cryo-electron microscopy structure and exploiting the information obtained by spectrometry on the RNA sequence. We determined that the model was stable during the MD simulation time. The obtained results provide deeper insights into the incorporation of nucleoside triphosphates, whose molecular mechanism by the RdRp active site still remains elusive.


Subject(s)
COVID-19 , Cytidine Triphosphate , RNA-Dependent RNA Polymerase , SARS-CoV-2 , Humans , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Cryoelectron Microscopy , Cytidine Triphosphate/chemistry , Molecular Dynamics Simulation , Nucleosides , Nucleotides , Ribose , RNA, Viral , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism
2.
Viruses ; 14(7)2022 Jun 21.
Article in English | MEDLINE | ID: covidwho-1981455

ABSTRACT

Increasing evidence suggests that the polymerase acidic (PA) protein of influenza A viruses plays an important role in viral replication and pathogenicity. However, information regarding the interaction(s) of host factors with PA is scarce. By using a yeast two-hybrid screen, we identified a novel host factor, aryl hydrocarbon receptor nuclear translocator (ARNT), that interacts with the PA protein of the H5N1 virus. The interaction between PA and human ARNT was confirmed by co-immunoprecipitation and immunofluorescence microscopy. Moreover, overexpression of ARNT downregulated the polymerase activity and inhibited virus propagation, whereas knockdown of ARNT significantly increased the polymerase activity and virus replication. Mechanistically, overexpression of ARNT resulted in the accumulation of PA protein in the nucleus and inhibited both the replication and transcription of the viral genome. Interaction domain mapping revealed that the bHLH/PAS domain of ARNT mainly interacted with the C-terminal domain of PA. Together, our results demonstrate that ARNT inhibits the replication of the H5N1 virus and could be a target for the development of therapeutic strategies against H5N1 influenza viruses.


Subject(s)
Influenza A Virus, H5N1 Subtype , Influenza A virus , Influenza, Human , Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Humans , RNA-Dependent RNA Polymerase/metabolism , Virus Replication/genetics
3.
Nature ; 609(7928): 793-800, 2022 09.
Article in English | MEDLINE | ID: covidwho-1984402

ABSTRACT

The RNA genome of SARS-CoV-2 contains a 5' cap that facilitates the translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made in SARS-CoV-2 is not completely understood. Here we reconstitute the N7- and 2'-O-methylated SARS-CoV-2 RNA cap (7MeGpppA2'-O-Me) using virally encoded non-structural proteins (nsps). We show that the kinase-like nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain5 of nsp12 transfers the RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers the RNA to GDP, forming the core cap structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N terminus of nsp9 and the kinase-like active-site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.


Subject(s)
RNA Caps , RNA, Viral , SARS-CoV-2 , Viral Proteins , Antiviral Agents , COVID-19/drug therapy , COVID-19/virology , Catalytic Domain , Guanosine Diphosphate/metabolism , Humans , Methyltransferases/metabolism , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/metabolism , Protein Domains , RNA Caps/chemistry , RNA Caps/genetics , RNA Caps/metabolism , RNA, Viral/chemistry , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism
4.
Viruses ; 13(8)2021 08 05.
Article in English | MEDLINE | ID: covidwho-1390162

ABSTRACT

Nucleotidylylation is a post-transcriptional modification important for replication in the picornavirus supergroup of RNA viruses, including members of the Caliciviridae, Coronaviridae, Picornaviridae and Potyviridae virus families. This modification occurs when the RNA-dependent RNA polymerase (RdRp) attaches one or more nucleotides to a target protein through a nucleotidyl-transferase reaction. The most characterized nucleotidylylation target is VPg (viral protein genome-linked), a protein linked to the 5' end of the genome in Caliciviridae, Picornaviridae and Potyviridae. The nucleotidylylation of VPg by RdRp is a critical step for the VPg protein to act as a primer for genome replication and, in Caliciviridae and Potyviridae, for the initiation of translation. In contrast, Coronaviridae do not express a VPg protein, but the nucleotidylylation of proteins involved in replication initiation is critical for genome replication. Furthermore, the RdRp proteins of the viruses that perform nucleotidylylation are themselves nucleotidylylated, and in the case of coronavirus, this has been shown to be essential for viral replication. This review focuses on nucleotidylylation within the picornavirus supergroup of viruses, including the proteins that are modified, what is known about the nucleotidylylation process and the roles that these modifications have in the viral life cycle.


Subject(s)
Nucleotides/metabolism , Positive-Strand RNA Viruses/metabolism , RNA-Dependent RNA Polymerase/metabolism , Viral Proteins/metabolism , Caliciviridae/genetics , Caliciviridae/metabolism , Coronaviridae/genetics , Coronaviridae/metabolism , Genome, Viral , Nidovirales/genetics , Nidovirales/metabolism , Picornaviridae/genetics , Picornaviridae/metabolism , Positive-Strand RNA Viruses/genetics , Potyviridae/genetics , Potyviridae/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Virus Replication
5.
Viruses ; 14(4)2022 03 25.
Article in English | MEDLINE | ID: covidwho-1834920

ABSTRACT

In only two years, the coronavirus disease 2019 (COVID-19) pandemic has had a devastating effect on public health all over the world and caused irreparable economic damage across all countries. Due to the limited therapeutic management of COVID-19 and the lack of tailor-made antiviral agents, finding new methods to combat this viral illness is now a priority. Herein, we report on a specific oligonucleotide-based RNA inhibitor targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It displayed remarkable spontaneous cellular uptake, >94% efficiency in reducing RNA-dependent RNA polymerase (RdRp) RNA levels in transfected lung cell lines, and >98% efficiency in reducing SARS-CoV-2 RNA levels in samples from patients hospitalized with COVID-19 following a single application.


Subject(s)
COVID-19 , Oligonucleotides , SARS-CoV-2 , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Humans , Oligonucleotides/pharmacology , Oligonucleotides/therapeutic use , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/genetics
6.
J Biol Chem ; 298(5): 101923, 2022 05.
Article in English | MEDLINE | ID: covidwho-1778265

ABSTRACT

Coronavirus (CoV) genomes consist of positive-sense single-stranded RNA and are among the largest viral RNAs known to date (∼30 kb). As a result, CoVs deploy sophisticated mechanisms to replicate these extraordinarily large genomes as well as to transcribe subgenomic messenger RNAs. Since 2003, with the emergence of three highly pathogenic CoVs (SARS-CoV, MERS-CoV, and SARS-CoV-2), significant progress has been made in the molecular characterization of the viral proteins and key mechanisms involved in CoV RNA genome replication. For example, to allow for the maintenance and integrity of their large RNA genomes, CoVs have acquired RNA proofreading 3'-5' exoribonuclease activity (in nonstructural protein nsp14). In order to replicate the large genome, the viral-RNA-dependent RNA polymerase (RdRp; in nsp12) is supplemented by a processivity factor (made of the viral complex nsp7/nsp8), making it the fastest known RdRp. Lastly, a viral structural protein, the nucleocapsid (N) protein, which is primarily involved in genome encapsidation, is required for efficient viral replication and transcription. Therefore, CoVs are a paradox among positive-strand RNA viruses in the sense that they use both a processivity factor and have proofreading activity reminiscent of DNA organisms in addition to structural proteins that mediate efficient RNA synthesis, commonly used by negative-strand RNA viruses. In this review, we present a historical perspective of these unsuspected discoveries and detail the current knowledge on the core replicative machinery deployed by CoVs.


Subject(s)
Genome, Viral , Positive-Strand RNA Viruses , SARS-CoV-2 , COVID-19/virology , Genome, Viral/genetics , Humans , Mutation , Positive-Strand RNA Viruses/genetics , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/genetics
7.
Commun Biol ; 5(1): 154, 2022 02 22.
Article in English | MEDLINE | ID: covidwho-1699831

ABSTRACT

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Exonucleases/antagonists & inhibitors , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Amino Acid Sequence , Anilides/pharmacology , Animals , Base Sequence , Benzimidazoles/pharmacology , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Drug Synergism , Exonucleases/genetics , Exonucleases/metabolism , Humans , Proline/pharmacology , Pyrrolidines/pharmacology , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Valine/pharmacology , Vero Cells , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects , Virus Replication/genetics
8.
Nat Commun ; 13(1): 621, 2022 02 02.
Article in English | MEDLINE | ID: covidwho-1671551

ABSTRACT

The guanosine analog AT-527 represents a promising candidate against Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2). AT-527 recently entered phase III clinical trials for the treatment of COVID-19. Once in cells, AT-527 is converted into its triphosphate form, AT-9010, that presumably targets the viral RNA-dependent RNA polymerase (RdRp, nsp12), for incorporation into viral RNA. Here we report a 2.98 Å cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-nsp82-RNA complex, showing AT-9010 bound at three sites of nsp12. In the RdRp active-site, one AT-9010 is incorporated at the 3' end of the RNA product strand. Its modified ribose group (2'-fluoro, 2'-methyl) prevents correct alignment of the incoming NTP, in this case a second AT-9010, causing immediate termination of RNA synthesis. The third AT-9010 is bound to the N-terminal domain of nsp12 - known as the NiRAN. In contrast to native NTPs, AT-9010 is in a flipped orientation in the active-site, with its guanine base unexpectedly occupying a previously unnoticed cavity. AT-9010 outcompetes all native nucleotides for NiRAN binding, inhibiting its nucleotidyltransferase activity. The dual mechanism of action of AT-527 at both RdRp and NiRAN active sites represents a promising research avenue against COVID-19.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Guanosine Monophosphate/analogs & derivatives , Phosphoramides/chemistry , Phosphoramides/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/enzymology , Viral Proteins/antagonists & inhibitors , Viral Proteins/metabolism , COVID-19/virology , Cryoelectron Microscopy , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Guanosine Monophosphate/chemistry , Guanosine Monophosphate/pharmacology , Humans , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Viral Proteins/genetics
9.
Science ; 375(6577): 161-167, 2022 Jan 14.
Article in English | MEDLINE | ID: covidwho-1648160

ABSTRACT

The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and older adults. We describe 4'-fluorouridine (4'-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with high selectivity index in cells and human airway epithelia organoids. Polymerase inhibition within in vitro RNA-dependent RNA polymerase assays established for RSV and SARS-CoV-2 revealed transcriptional stalling after incorporation. Once-daily oral treatment was highly efficacious at 5 milligrams per kilogram (mg/kg) in RSV-infected mice or 20 mg/kg in ferrets infected with different SARS-CoV-2 variants of concern, initiated 24 or 12 hours after infection, respectively. These properties define 4'-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2, and related RNA virus infections.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Respiratory Syncytial Virus Infections/drug therapy , Respiratory Syncytial Virus, Human/drug effects , SARS-CoV-2/drug effects , Uracil Nucleotides/pharmacology , Administration, Oral , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/metabolism , COVID-19/virology , Cell Line , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Disease Models, Animal , Female , Ferrets , Humans , Mice , Mice, Inbred BALB C , Microbial Sensitivity Tests , Mononegavirales/drug effects , Mononegavirales/physiology , RNA-Dependent RNA Polymerase/metabolism , Respiratory Mucosa/virology , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/genetics , Respiratory Syncytial Virus, Human/physiology , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Transcription, Genetic , Uracil Nucleotides/administration & dosage , Uracil Nucleotides/metabolism , Virus Replication/drug effects
10.
Molecules ; 27(3)2022 Jan 26.
Article in English | MEDLINE | ID: covidwho-1648677

ABSTRACT

The human population is still facing appalling conditions due to several outbreaks of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) virus. The absence of specific drugs, appropriate vaccines for mutants, and knowledge of potential therapeutic agents makes this situation more difficult. Several 1, 2, 4-triazolo [1, 5-a] pyrimidine (TP)-derivative compounds were comprehensively studied for antiviral activities against RNA polymerase of HIV, HCV, and influenza viruses, and showed immense pharmacological interest. Therefore, TP-derivative compounds can be repurposed against the RNA-dependent RNA polymerase (RdRp) protein of SARS-CoV-2. In this study, a meta-analysis was performed to ensure the genomic variability and stability of the SARS-CoV-2 RdRp protein. The molecular docking of natural and synthetic TP compounds to RdRp and molecular dynamic (MD) simulations were performed to analyse the dynamic behaviour of TP compounds at the active site of the RdRp protein. TP compounds were also docked against other non-structural proteins (NSP1, NSP2, NSP3, NSP5, NSP8, NSP13, and NSP15) of SARS-CoV-2. Furthermore, the inhibition potential of TP compounds was compared with Remdesivir and Favipiravir drugs as a positive control. Additionally, TP compounds were analysed for inhibitory activity against SARS-CoV RdRp protein. This study demonstrates that TP analogues (monomethylated triazolopyrimidine and essramycin) represent potential lead molecules for designing an effective inhibitor to control viral replication. Furthermore, in vitro and in vivo studies will strengthen the use of these inhibitors as suitable drug candidates against SARS-CoV-2.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase/drug effects , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Pyrimidines/pharmacology , Triazoles/pharmacology , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Amides/pharmacology , COVID-19/drug therapy , COVID-19/metabolism , Catalytic Domain/drug effects , Computational Biology/methods , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Pyrazines/pharmacology , Pyrimidines/chemistry , RNA, Viral/drug effects , RNA-Dependent RNA Polymerase/drug effects , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Triazoles/chemistry , Virus Replication/drug effects
11.
J Biol Chem ; 298(2): 101529, 2022 02.
Article in English | MEDLINE | ID: covidwho-1587355

ABSTRACT

Remdesivir (RDV) is a direct-acting antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2. RDV exhibits broad-spectrum antiviral activity against positive-sense RNA viruses, for example, severe acute respiratory syndrome coronavirus and hepatitis C virus, and nonsegmented negative-sense RNA viruses, for example, Nipah virus, whereas segmented negative-sense RNA viruses such as influenza virus or Crimean-Congo hemorrhagic fever virus are not sensitive to the drug. The reasons for this apparent efficacy pattern are unknown. Here, we expressed and purified representative RNA-dependent RNA polymerases and studied three biochemical parameters that have been associated with the inhibitory effects of RDV-triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV-TP, (ii) the effect of the incorporated RDV-monophosphate (MP) on primer extension, and (iii) the effect of RDV-MP in the template during incorporation of the complementary UTP. We found a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Inhibition in primer extension reactions was heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP was seen with all polymerases. Molecular modeling suggests a steric conflict between the 1'-cyano group of the inhibitor and residues of the structurally conserved RNA-dependent RNA polymerase motif F. We conclude that future efforts in the development of nucleotide analogs with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1'-modification.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Models, Molecular , RNA Viruses/enzymology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Alanine/chemistry , Alanine/pharmacology , Antiviral Agents/pharmacology , Hepacivirus/drug effects , Hepacivirus/enzymology , Negative-Sense RNA Viruses/drug effects , Negative-Sense RNA Viruses/enzymology , Nipah Virus/drug effects , Nipah Virus/enzymology , Positive-Strand RNA Viruses/drug effects , Positive-Strand RNA Viruses/enzymology , RNA Viruses/drug effects , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Virus Replication/drug effects
12.
Biomed Pharmacother ; 146: 112517, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1561313

ABSTRACT

Rapid changes in the viral genome allow viruses to evade threats posed by the host immune response or antiviral drugs, and can lead to viral persistence in the host cells. RNA-dependent RNA polymerase (RdRp) is an essential enzyme in RNA viruses, which is involved in RNA synthesis through the formation of phosphodiester bonds. Therefore, in RNA viral infections such as SARS-CoV-2, RdRp could be a crucial therapeutic target. The present review discusses the promising application of RdRp inhibitors, previously approved or currently being tested in human clinical trials, in the treatment of RNA virus infections. Nucleoside inhibitors (NIs) bind to the active site of RdRp, while nonnucleoside inhibitors (NNIs) bind to allosteric sites. Given the absence of highly effective drugs for the treatment of COVID-19, the discovery of an efficient treatment for this pandemic is an urgent concern for researchers around the world. We review the evidence for molnupiravir (MK-4482, EIDD-2801), an antiviral drug originally designed for Alphavirus infections, as a potential preventive and therapeutic agent for the management of COVID-19. At the beginning of this pandemic, molnupiravir was in preclinical development for seasonal influenza. When COVID-19 spread dramatically, the timeline for development was accelerated to focus on the treatment of this pandemic. Real time consultation with regulators took place to expedite this program. We summarize the therapeutic potential of RdRp inhibitors, and highlight molnupiravir as a new small molecule drug for COVID-19 treatment.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/enzymology , Cytidine/analogs & derivatives , Hydroxylamines/therapeutic use , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Animals , Antiviral Agents/pharmacology , Clinical Trials as Topic/methods , Cytidine/pharmacology , Cytidine/therapeutic use , Humans , Hydroxylamines/pharmacology , RNA-Dependent RNA Polymerase/metabolism
13.
Sci Rep ; 11(1): 23465, 2021 12 06.
Article in English | MEDLINE | ID: covidwho-1556248

ABSTRACT

Human coronavirus NL63 (HCoV-NL63) mainly affects young children and immunocompromised patients, causing morbidity and mortality in a subset of patients. Since no specific treatment is available, this study aims to explore the anti-SARS-CoV-2 agents including favipiravir and remdesivir for treating HCoV-NL63 infection. We first successfully modelled the 3D structure of HCoV-NL63 RNA-dependent RNA polymerase (RdRp) based on the experimentally solved SARS-CoV-2 RdRp structure. Molecular docking indicated that favipiravir has similar binding affinities to SARS-CoV-2 and HCoV-NL63 RdRp with LibDock scores of 75 and 74, respectively. The LibDock scores of remdesivir to SARS-CoV-2 and HCoV-NL63 were 135 and 151, suggesting that remdesivir may have a higher affinity to HCoV-NL63 compared to SARS-CoV-2 RdRp. In cell culture models infected with HCoV-NL63, both favipiravir and remdesivir significantly inhibited viral replication and production of infectious viruses. Overall, remdesivir compared to favipiravir is more potent in inhibiting HCoV-NL63 in cell culture. Importantly, there is no evidence of resistance development upon long-term exposure to remdesivir. Furthermore, combining favipiravir or remdesivir with the clinically used antiviral cytokine interferon-alpha resulted in synergistic effects. These findings provided a proof-of-concept that anti-SARS-CoV-2 drugs, in particular remdesivir, have the potential to be repurposed for treating HCoV-NL63 infection.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Amides/chemistry , Antiviral Agents/chemistry , Coronavirus NL63, Human/enzymology , Pyrazines/chemistry , RNA-Dependent RNA Polymerase/chemistry , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Adenosine Monophosphate/pharmacology , Alanine/chemistry , Alanine/metabolism , Alanine/pharmacology , Amides/metabolism , Amides/pharmacology , Animals , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Binding Sites , Cell Culture Techniques , Cell Line , Coronavirus NL63, Human/physiology , Haplorhini , Humans , Molecular Docking Simulation , Pyrazines/metabolism , Pyrazines/pharmacology , RNA-Dependent RNA Polymerase/metabolism , Virus Replication/drug effects
14.
Infect Genet Evol ; 96: 105155, 2021 12.
Article in English | MEDLINE | ID: covidwho-1525880

ABSTRACT

The present study aimed to predict the binding potential of carbon nanotube and nano fullerene towards multiple targets of SARS-CoV-2. Based on the virulent functions, the spike glycoprotein, RNA-dependent RNA polymerase, main protease, papain-like protease, and RNA binding domain of the nucleocapsid proteins of SARS-CoV-2 were prioritized as the molecular targets and their three-dimensional (3D) structures were retrieved from the Protein Data Bank. The 3D structures of carbon nanotubes and nano-fullerene were computationally modeled, and the binding potential of these nanoparticles to the selected molecular targets was predicted by molecular docking and molecular dynamic (MD) simulations. The drug-likeness and pharmacokinetic features of the lead molecules were computationally predicted. The current study suggested that carbon fullerene and nanotube demonstrated significant binding towards the prioritized multi-targets of SARS-CoV-2. Interestingly, carbon nanotube showed better interaction with these targets when compared to carbon fullerene. MD simulation studies clearly showed that the interaction of nanoparticles and selected targets possessed stability and conformational changes. This study revealed that carbon nanotubes and fullerene are probably used as effectual binders to multiple targets of SARS-CoV-2, and the study offers insights into the experimental validation and highlights the relevance of utilizing carbon nanomaterials as a therapeutic remedy against COVID-19.


Subject(s)
Fullerenes/metabolism , Nanotubes, Carbon , SARS-CoV-2/metabolism , Viral Proteins/chemistry , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/metabolism , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Fullerenes/chemistry , Fullerenes/pharmacokinetics , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Nanotubes, Carbon/chemistry , Phosphoproteins/chemistry , Phosphoproteins/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Viral Proteins/metabolism
15.
J Biol Chem ; 297(6): 101399, 2021 12.
Article in English | MEDLINE | ID: covidwho-1509947

ABSTRACT

The nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 is a critical viral protein that suppresses host gene expression by blocking the assembly of the ribosome on host mRNAs. To understand the mechanism of inhibition of host gene expression, we sought to identify cellular proteins that interact with nsp1. Using proximity-dependent biotinylation followed by proteomic analyses of biotinylated proteins, here we captured multiple dynamic interactions of nsp1 with host cell proteins. In addition to ribosomal proteins, we identified several pre-mRNA processing proteins that interact with nsp1, including splicing factors and transcription termination proteins, as well as exosome, and stress granule (SG)-associated proteins. We found that the interactions with transcription termination factors are primarily governed by the C-terminal region of nsp1 and are disrupted by the mutation of amino acids K164 and H165 that are essential for its host shutoff function. We further show that nsp1 interacts with Ras GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) and colocalizes with G3BP1 in SGs under sodium arsenite-induced stress. Finally, we observe that the presence of nsp1 disrupts the maturation of SGs over a long period. Isolation of SG core at different times shows a gradual loss of G3BP1 in the presence of nsp1.


Subject(s)
COVID-19/metabolism , RNA-Dependent RNA Polymerase/metabolism , SARS Virus/metabolism , SARS-CoV-2/metabolism , Severe Acute Respiratory Syndrome/metabolism , Viral Nonstructural Proteins/metabolism , Biotinylation , COVID-19/virology , HEK293 Cells , Host-Pathogen Interactions , Humans , Proteomics , Ribosomal Proteins/metabolism , SARS Virus/physiology , SARS-CoV-2/physiology , Severe Acute Respiratory Syndrome/virology , /metabolism
16.
Mol Cell ; 81(21): 4467-4480.e7, 2021 11 04.
Article in English | MEDLINE | ID: covidwho-1473419

ABSTRACT

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.


Subject(s)
Pyrazines/chemistry , RNA Viruses/genetics , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/genetics , Recombination, Genetic , Ribonucleotides/chemistry , Animals , Antiviral Agents , Catalysis , Cells, Cultured , Genetic Techniques , Genome , Genome, Viral , Homologous Recombination , Humans , Kinetics , Mice , Mice, Transgenic , Molecular Dynamics Simulation , Mutagenesis , Nucleotides/genetics , Protein Conformation , RNA/chemistry , RNA-Dependent RNA Polymerase/metabolism , RNA-Seq , Transgenes , Virulence
17.
Int J Mol Sci ; 22(20)2021 Oct 15.
Article in English | MEDLINE | ID: covidwho-1470891

ABSTRACT

SARS-CoV-2, or severe acute respiratory syndrome coronavirus 2, represents a new pathogen from the family of Coronaviridae that caused a global pandemic of COVID-19 disease. In the absence of effective antiviral drugs, research of novel therapeutic targets such as SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) becomes essential. This viral protein is without a human counterpart and thus represents a unique prospective drug target. However, in vitro biological evaluation testing on RdRp remains difficult and is not widely available. Therefore, we prepared a database of commercial small-molecule compounds and performed an in silico high-throughput virtual screening on the active site of the SARS-CoV-2 RdRp using ensemble docking. We identified a novel thioether-amide or guanidine-linker class of potential RdRp inhibitors and calculated favorable binding free energies of representative hits by molecular dynamics simulations coupled with Linear Interaction Energy calculations. This innovative procedure maximized the respective phase-space sampling and yielded non-covalent inhibitors representing small optimizable molecules that are synthetically readily accessible, commercially available as well as suitable for further biological evaluation and mode of action studies.


Subject(s)
Antiviral Agents/chemistry , Enzyme Inhibitors/chemistry , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/enzymology , Viral Proteins/antagonists & inhibitors , Amides/chemistry , Antiviral Agents/metabolism , Antiviral Agents/therapeutic use , Binding Sites , COVID-19/drug therapy , COVID-19/virology , Catalytic Domain , Databases, Chemical , Drug Design , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/therapeutic use , Guanidine/chemistry , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/isolation & purification , Structure-Activity Relationship , Sulfides/chemistry , Thermodynamics , Viral Proteins/metabolism
18.
Int J Mol Sci ; 22(20)2021 Oct 13.
Article in English | MEDLINE | ID: covidwho-1470887

ABSTRACT

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has infected >235 million people and killed over 4.8 million individuals worldwide. Although vaccines have been developed for prophylactic management, there are no clinically proven antivirals to treat the viral infection. Continuous efforts are being made all over the world to develop effective drugs but these are being delayed by periodic outbreak of mutated SARS-CoV-2 and a lack of knowledge of molecular mechanisms underlying viral pathogenesis and post-infection complications. In this regard, the involvement of Annexin A2 (AnxA2), a lipid-raft related phospholipid-binding protein, in SARS-CoV-2 attachment, internalization, and replication has been discussed. In addition to the evidence from published literature, we have performed in silico docking of viral spike glycoprotein and RNA-dependent RNA polymerase with human AnxA2 to find the molecular interactions. Overall, this review provides the molecular insights into a potential role of AnxA2 in the SARS-CoV-2 pathogenesis and post-infection complications, especially thrombosis, cytokine storm, and insulin resistance.


Subject(s)
Annexin A2/metabolism , COVID-19/pathology , Annexin A2/chemistry , COVID-19/virology , Cytokine Release Syndrome/metabolism , Cytokine Release Syndrome/pathology , Humans , Molecular Docking Simulation , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Thrombosis/metabolism , Thrombosis/pathology , Virus Internalization
19.
Virology ; 564: 33-38, 2021 12.
Article in English | MEDLINE | ID: covidwho-1447220

ABSTRACT

Endemic seasonal coronaviruses cause morbidity and mortality in a subset of patients, but no specific treatment is available. Molnupiravir is a promising pipeline antiviral drug for treating SARS-CoV-2 infection potentially by targeting RNA-dependent RNA polymerase (RdRp). This study aims to evaluate the potential of repurposing molnupiravir for treating seasonal human coronavirus (HCoV) infections. Molecular docking revealed that the active form of molnupiravir, ß-D-N4-hydroxycytidine (NHC), has similar binding affinity to RdRp of SARS-CoV-2 and seasonal HCoV-NL63, HCoV-OC43 and HCoV-229E. In cell culture models, treatment of molnupiravir effectively inhibited viral replication and production of infectious viruses of the three seasonal coronaviruses. A time-of-drug-addition experiment indicates the specificity of molnupiravir in inhibiting viral components. Furthermore, combining molnupiravir with the protease inhibitor GC376 resulted in enhanced antiviral activity. Our findings highlight that the great potential of repurposing molnupiravir for treating seasonal coronavirus infected patients.


Subject(s)
Coronavirus 229E, Human/genetics , Coronavirus Infections/drug therapy , Coronavirus NL63, Human/genetics , Coronavirus OC43, Human/genetics , Cytidine/analogs & derivatives , Hydroxylamines/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Common Cold/drug therapy , Coronavirus 229E, Human/drug effects , Coronavirus 229E, Human/physiology , Coronavirus NL63, Human/drug effects , Coronavirus NL63, Human/physiology , Coronavirus OC43, Human/drug effects , Coronavirus OC43, Human/physiology , Cytidine/pharmacology , Humans , Molecular Docking Simulation , Protein Binding/drug effects , Pyrrolidines/pharmacology , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Seasons , Sulfonic Acids/pharmacology , Virus Replication/drug effects , Virus Replication/genetics
20.
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
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