scholarly journals The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Carolina Q. Sacramento ◽  
Gabrielle R. de Melo ◽  
Caroline S. de Freitas ◽  
Natasha Rocha ◽  
Lucas Villas Bôas Hoelz ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Zika Virus ◽  
Neurological Disorders ◽  
Viral Genome ◽  
Antiviral Drug ◽  
Cellular Systems ◽  
Viral Rna ◽  
Amino Acid Residues ◽  
Direct Inhibition ◽  
Secondary Use

Abstract Zika virus (ZIKV) is a member of the Flaviviridae family, along with other agents of clinical significance such as dengue (DENV) and hepatitis C (HCV) viruses. Since ZIKV causes neurological disorders during fetal development and in adulthood, antiviral drugs are necessary. Sofosbuvir is clinically approved for use against HCV and targets the protein that is most conserved among the members of the Flaviviridae family, the viral RNA polymerase. Indeed, we found that sofosbuvir inhibits ZIKV RNA polymerase, targeting conserved amino acid residues. Sofosbuvir inhibited ZIKV replication in different cellular systems, such as hepatoma (Huh-7) cells, neuroblastoma (SH-Sy5y) cells, neural stem cells (NSC) and brain organoids. In addition to the direct inhibition of the viral RNA polymerase, we observed that sofosbuvir also induced an increase in A-to-G mutations in the viral genome. Together, our data highlight a potential secondary use of sofosbuvir, an anti-HCV drug, against ZIKV.

scholarly journals The clinically approved antiviral drug sofosbuvir impairs brazilian zika virus replication

2016 ◽  
Author(s):  
Caroline Q. Sacramento ◽  
Gabrielle R. de Melo ◽  
Natasha Rocha ◽  
Lucas Villas Bôas Hoelz ◽  
Milene Mesquita ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Zika Virus ◽  
Antiviral Drug ◽  
Time Frame ◽  
Polymerase Activity ◽  
Antiviral Drugs ◽  
South American ◽  
Dependent Manner ◽  
Amino Acid Residues ◽  
Uridine Nucleotide

SummaryZika virus (ZIKV) is a member of Flaviviridae family, as other agents of clinical significance, such as dengue (DENV) and hepatitis C (HCV) viruses. ZIKV spread from Africa to Pacific and South American territories, emerging as an etiological pathogen of neurological disorders, during fetal development and in adulthood. Therefore, antiviral drugs able to inhibit ZIKV replication are necessary. Broad spectrum antivirals, such as interferon, ribavirin and favipiravir, are harmful for pregnant animal models and women. The clinically approved uridine nucleotide analog anti-HCV drug, sofosbuvir, has not been affiliated to teratogenicity. Sofosbuvir target the most conserved protein over the members of the Flaviviridae family, the viral RNA polymerase. We thus studied ZIKV susceptibility to sofosbovir. We initially characterized a Brazilian ZIKV strain for use in experimental assays. Sofosbuvir inhibits the Brazilian ZIKV replication in a dose-dependent manner, both in BHK-21 cells and SH-Sy5y, by targeting ZIKV RNA polymerase activity, with the involvement of conserved amino acid residues over the members of Flaviviridae family. The identification of clinically approved antiviral drugs endowed with anti-ZIKV could reduce the time frame in pre-clinical development. Altogether, our data indicates that sofosbuvir chemical structure is endowed with anti-ZIKV activity.

scholarly journals Identifying Initiation and Elongation Inhibitors of Dengue Virus RNA Polymerase in a High-Throughput Lead-Finding Campaign

2014 ◽  
Vol 20 (1) ◽  
pp. 153-163 ◽  
Author(s):  
Thomas M. Smith ◽  
Siew Pheng Lim ◽  
Kimberley Yue ◽  
Scott A. Busby ◽  
Rishi Arora ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Rna Polymerase ◽  
High Throughput ◽  
Rna Synthesis ◽  
De Novo ◽  
Antiviral Drug ◽  
Viral Rna ◽  
Renilla Luciferase ◽  
Viral Pathogen ◽  
Open Conformation

Dengue virus (DENV) is the most significant mosquito-borne viral pathogen in the world and is the cause of dengue fever. The DENV RNA-dependent RNA polymerase (RdRp) is conserved among the four viral serotypes and is an attractive target for antiviral drug development. During initiation of viral RNA synthesis, the polymerase switches from a “closed” to “open” conformation to accommodate the viral RNA template. Inhibitors that lock the “closed” or block the “open” conformation would prevent viral RNA synthesis. Herein, we describe a screening campaign that employed two biochemical assays to identify inhibitors of RdRp initiation and elongation. Using a DENV subgenomic RNA template that promotes RdRp de novo initiation, the first assay measures cytosine nucleotide analogue (Atto-CTP) incorporation. Liberated Atto fluorophore allows for quantification of RdRp activity via fluorescence. The second assay uses the same RNA template but is label free and directly detects RdRp-mediated liberation of pyrophosphates of native ribonucleotides via liquid chromatography–mass spectrometry. The ability of inhibitors to bind and stabilize a “closed” conformation of the DENV RdRp was further assessed in a differential scanning fluorimetry assay. Last, active compounds were evaluated in a renilla luciferase–based DENV replicon cell-based assay to monitor cellular efficacy. All assays described herein are medium to high throughput, are robust and reproducible, and allow identification of inhibitors of the open and closed forms of DENV RNA polymerase.

scholarly journals Identification of guanylyltransferase activity in the SARS-CoV-2 RNA polymerase

2021 ◽  
Author(s):  
Alexander P Walker ◽  
Haitian Fan ◽  
Jeremy R Keown ◽  
Jonathan M Grimes ◽  
Ervin Fodor
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Virus ◽  
Active Form ◽  
Antiviral Drug ◽  
Viral Rna ◽  
Protein Domain ◽  
Structural Protein ◽  
Viral Mrnas ◽  
Synthesis Mechanism

AbstractSARS-CoV-2 is a positive-sense RNA virus that is responsible for the ongoing Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors that are involved in RNA proofreading and 5’ capping of viral mRNAs. The formation of the 5’ cap-1 structure is known to require a guanylyltransferase (GTase) as well as 5’ triphosphatase and methyltransferase activities. However, the mechanism of SARS-CoV-2 mRNA capping remains poorly understood. Here we show that the SARS-CoV-2 RNA polymerase nsp12 functions as a GTase. We characterise this GTase activity and find that the nsp12 NiRAN (nidovirus RdRP-associated nucleotidyltransferase) domain is responsible for carrying out the addition of a GTP nucleotide to the 5’ end of viral RNA via a 5’ to 5’ triphosphate linkage. We also show that remdesivir triphosphate, the active form of the antiviral drug remdesivir, inhibits the SARS-CoV-2 GTase reaction as efficiently as RNA polymerase activity. These data improve understanding of coronavirus mRNA cap synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.ImportanceSARS-CoV-2 is a respiratory RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Coronaviruses encode an RNA polymerase which, in combination with other viral proteins, is responsible for synthesising capped viral mRNA. mRNA cap synthesis requires a guanylyltransferase enzyme; here we show that the SARS-CoV-2 guanylyltransferase is located in the viral RNA polymerase, and we identify the protein domain responsible for guanylyltransferase activity. Furthermore we demonstrate that remdesivir triphosphate, the active metabolite of remdesivir, inhibits both the guanylyltransferase and RNA polymerase functions of the SARS-CoV-2 RNA polymerase. These findings improve understanding of the coronavirus mRNA cap synthesis mechanism, in addition to highlighting a new target for the development of therapeutics to treat SARS-CoV-2 infection.

scholarly journals Non-nucleoside Inhibitors of Zika Virus RNA-Dependent RNA Polymerase

2020 ◽  
Vol 94 (21) ◽  
Author(s):  
Aicha Gharbi-Ayachi ◽  
Sridhar Santhanakrishnan ◽  
Yee Hwa Wong ◽  
Kitti W. K. Chan ◽  
Siok Thing Tan ◽  
...  
Keyword(s):  
High Resolution ◽  
Dengue Virus ◽  
Rna Polymerase ◽  
Zika Virus ◽  
Nonstructural Protein ◽  
Specific Binding ◽  
Antiviral Drug ◽  
Polymerase Activity ◽  
Health Concern ◽  
Rna Dependent Rna Polymerase

ABSTRACT Zika virus (ZIKV) remains a potentially significant public health concern because it can cause teratogenic effects, such as microcephaly in newborns and neurological disease, like Guillain-Barré syndrome. Together with efforts to develop a vaccine, the discovery of antiviral molecules is important to control ZIKV infections and to prevent its most severe symptoms. Here, we report the development of small nonnucleoside inhibitors (NNIs) of ZIKV RNA-dependent RNA polymerase (RdRp) activity. These NNIs target an allosteric pocket (N pocket) located next to a putative hinge region between the thumb and the palm subdomains that was originally described for dengue virus (DENV) RdRp. We first tested the activity of DENV RdRp N-pocket inhibitors against ZIKV RdRp, introduced chemical modifications into these molecules, and assessed their potency using both enzymatic and cell-based assays. The most potent compound had a 50% inhibitory concentration value of 7.3 μM and inhibited ZIKV replication in a cell-based assay with a 50% effective concentration value of 24.3 μM. Importantly, we report four high-resolution crystal structures detailing how these NNIs insert into the N pocket of ZIKV RdRp. Our observations point to subtle differences in the size, shape, chemical environment, and hydration of the N pocket from ZIKV RdRp from those of the N pocket from DENV RdRp that are crucial for the design of improved antiviral inhibitors with activity against ZIKV. IMPORTANCE Zika virus belongs to the Flavivirus genus, which comprises several important human pathogens. There is currently neither an approved vaccine nor antiviral drugs available to prevent infection by ZIKV. The nonstructural protein 5 (NS5) polymerase, which is responsible for replicating the viral RNA genome, represents one of the most promising targets for antiviral drug development. Starting from compounds recently developed against dengue virus NS5, we designed and synthesized inhibitors targeting Zika virus NS5. We show that these novel compounds inhibit viral replication by targeting the polymerase activity. High-resolution X-ray crystallographic structures of protein-inhibitor complexes demonstrated specific binding to an allosteric site within the polymerase, called the N pocket. This work paves the way for the future structure-based design of potent compounds specifically targeting ZIKV RNA polymerase activity.

scholarly journals Mutation of an Influenza Virus Polymerase 3′ RNA Promoter Binding Site Inhibits Transcription Elongation

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Alexander P. Walker ◽  
Jane Sharps ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Binding Site ◽  
Binding Sites ◽  
Viral Genome ◽  
Influenza Viruses ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Genome Transcription

ABSTRACT Influenza viruses encode a viral RNA-dependent RNA polymerase (FluPol), which is responsible for transcribing and replicating the negative-sense viral RNA (vRNA) genome. FluPol transcribes vRNA using a host-capped mRNA primer and replicates it by synthesizing a positive-sense cRNA intermediate, which is copied back into vRNA. To carry out these functions, FluPol interacts with vRNA and cRNA using conserved promoter elements at the 5′ and 3′ termini. Recent structural studies have identified a new surface binding site for the 3′ vRNA and cRNA promoters on FluPol, referred to as the mode B site. However, the role of this binding site in FluPol function is unknown. In this study, we used a combination of cell-based and biochemical assays to show that the mode B site is important for both viral genome transcription and replication in influenza A virus. Furthermore, we show that the mode B site is not needed for initiating transcription in vitro but is required to synthesize a full-length product. This is consistent with a model in which the 3′ terminus of the vRNA template binds in the mode B site during elongation. Our data provide the first functional insights into the role of the mode B site on FluPol, which advances our understanding of FluPol function and influenza virus replication. IMPORTANCE Influenza viruses are responsible for up to 650,000 deaths per year through seasonal epidemics, and pandemics have caused tens of millions of deaths in the past. Most current therapeutics suffer from widespread resistance, creating a need for new drug targets against influenza virus. The virus encodes an RNA-dependent RNA polymerase, which replicates and transcribes the vRNA genome. The polymerase interacts with vRNA and the complementary replicative intermediate cRNA using several specific binding sites; however, the functions associated with these binding sites remain unknown. Here, we functionally characterize a binding site for the 3′ vRNA and cRNA promoters. Our data offer insight into the mechanism of viral genome transcription by the influenza virus polymerase and may be applicable to other related viruses.

scholarly journals Distinct Effects of T-705 (Favipiravir) and Ribavirin on Influenza Virus Replication and Viral RNA Synthesis

2016 ◽  
Vol 60 (11) ◽  
pp. 6679-6691 ◽  
Author(s):  
Evelien Vanderlinden ◽  
Bram Vrancken ◽  
Jeroen Van Houdt ◽  
Vivek K. Rajwanshi ◽  
Sarah Gillemot ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Viral Genome ◽  
Rna Synthesis ◽  
Antiviral Drug ◽  
Point Mutations ◽  
Viral Rna ◽  
Viral Polymerase ◽  
Cell Functions ◽  
Nucleotide Pools ◽  
Drug Concentrations

ABSTRACTT-705 (favipiravir) is a new antiviral agent in advanced clinical development for influenza therapy. It is supposed to act as an alternative substrate for the viral polymerase, causing inhibition of viral RNA synthesis or virus mutagenesis. These mechanisms were also proposed for ribavirin, an established and broad antiviral drug that shares structural similarity with T-705. We here performed a comparative analysis of the effects of T-705 and ribavirin on influenza virus and host cell functions. Influenza virus-infected cell cultures were exposed to T-705 or ribavirin during single or serial virus passaging. The effects on viral RNA synthesis and infectious virus yield were determined and mutations appearing in the viral genome were detected by whole-genome virus sequencing. In addition, the cellular nucleotide pools as well as direct inhibition of the viral polymerase enzyme were quantified. We demonstrate that the anti-influenza virus effect of ribavirin is based on IMP dehydrogenase inhibition, which results in fast and profound GTP depletion and an imbalance in the nucleotide pools. In contrast, T-705 acts as a potent and GTP-competitive inhibitor of the viral polymerase. In infected cells, viral RNA synthesis is completely inhibited by T-705 or ribavirin at ≥50 μM, whereas exposure to lower drug concentrations induces formation of noninfectious particles and accumulation of random point mutations in the viral genome. This mutagenic effect is 2-fold higher for T-705 than for ribavirin. Hence, T-705 and ribavirin both act as purine pseudobases but profoundly differ with regard to the mechanism behind their antiviral and mutagenic effects on influenza virus.

scholarly journals Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir

2020 ◽  
Author(s):  
Wanchao Yin ◽  
Chunyou Mao ◽  
Xiaodong Luan ◽  
Dan-Dan Shen ◽  
Qingya Shen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Virus Disease ◽  
Complex Structure ◽  
Antiviral Drug ◽  
Viral Rna ◽  
Chain Elongation ◽  
Structural Basis ◽  
Rna Dependent Rna Polymerase ◽  
Working Mechanism ◽  
Primer Rna

The pandemic of Corona Virus Disease 2019 (COVID-19) caused by SARS-CoV-2 has become a global crisis. The replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp), a direct target of the antiviral drug, Remdesivir. Here we report the structure of the SARS-CoV-2 RdRp either in the apo form or in complex with a 50-base template-primer RNA and Remdesivir at a resolution range of 2.5-2.8 Å. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp where Remdesivir is incorporated into the first replicated base pair and terminates the chain elongation. Our structures provide critical insights into the working mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.

scholarly journals Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir

Science ◽  
2020 ◽  
Vol 368 (6498) ◽  
pp. 1499-1504 ◽  
Author(s):  
Wanchao Yin ◽  
Chunyou Mao ◽  
Xiaodong Luan ◽  
Dan-Dan Shen ◽  
Qingya Shen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Complex Structure ◽  
Antiviral Drug ◽  
Viral Rna ◽  
Chain Elongation ◽  
Structural Basis ◽  
Rna Dependent Rna Polymerase ◽  
Double Stranded Rna ◽  
Cryo Electron Microscopy ◽  
Primer Rna

The pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global crisis. Replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp) enzyme, a target of the antiviral drug remdesivir. Here we report the cryo–electron microscopy structure of the SARS-CoV-2 RdRp, both in the apo form at 2.8-angstrom resolution and in complex with a 50-base template-primer RNA and remdesivir at 2.5-angstrom resolution. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp, where remdesivir is covalently incorporated into the primer strand at the first replicated base pair, and terminates chain elongation. Our structures provide insights into the mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.

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