scholarly journals Probing remdesivir nucleotide analogue insertion to SARS-CoV-2 RNA dependent RNA polymerase in viral replication

2021 ◽  
Author(s):  
Moises Ernesto Romero ◽  
Chunhong Long ◽  
Daniel La Rocco ◽  
Anusha Mysore Keerthi ◽  
Dajun Xu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Viral Replication ◽  
Active Site ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogue

Remdesivir (RDV) prodrug can be metabolized into a triphosphate form nucleotide analogue (RDV-TP) to bind and insert into the active site of viral RNA dependent RNA polymerase (RdRp) to further...

scholarly journals Probing remdesivir nucleotide analogue insertion to SARS-CoV-2 RNA dependent RNA polymerase in viral replication

2021 ◽  
Author(s):  
Moises Ernesto Romero ◽  
Chunhong Long ◽  
Daniel La Rocco ◽  
Anusha Mysore Keerthi ◽  
Dajun Xu ◽  
...  
Keyword(s):  
Free Energy ◽  
Rna Polymerase ◽  
Active Site ◽  
Energy Barrier ◽  
Umbrella Sampling ◽  
Dynamics Simulation ◽  
Free Energy Barrier ◽  
Base Stacking ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogue

Remdesivir (RDV) prodrug can be metabolized into a triphosphate form nucleotide analogue (RDV-TP) to bind and insert into the active site of viral RNA dependent RNA polymerase (RdRp) to further interfere with the viral genome replication. In this work, we computationally studied how RDV-TP binds and inserts to the SARS-CoV-2 RdRp active site, in comparison with natural nucleotide substrate adenosine triphosphate (ATP). To do that, we first constructed atomic structural models of an initial binding complex (active site open) and a substrate insertion complex (active site closed), based on high-resolution cryo-EM structures determined recently for SARS-CoV-2 RdRp or non-structural protein (nsp) 12, in complex with accessory protein factors nsp7 and nsp8. By conducting all-atom molecular dynamics simulation with umbrella sampling strategies on the nucleotide insertion between the open and closed state RdRp complexes, our studies show that RDV-TP can bind comparatively stabilized to the viral RdRp active site, as it primarily forms base stacking with the template Uracil nucleotide (at +1), which is under freely fluctuations and supports a low free energy barrier of the RDV-TP insertion (~ 1.5 kcal/mol). In comparison, the corresponding natural substrate ATP binds to the RdRp active site in Watson-Crick base pairing with the template nt, and inserts into the active site with a medium low free energy barrier (~ 2.6 kcal/mol), when the fluctuations of the template nt are well quenched. The simulations also show that the initial base stacking of RDV-TP with the template can be particularly stabilized by motif B-N691, S682, and motif F-K500 with the sugar, base, and the template backbone, respectively. Although the RDV-TP insertion can be hindered by motif-F R555/R553 interaction with the triphosphate, the ATP insertion seems to be facilitated by such interactions. The inserted RDV-TP and ATP can be further distinguished by specific sugar interaction with motif B-T687 and motif-A D623, respectively.

scholarly journals 2′-C-methylated nucleotides terminate virus RNA synthesis by preventing active site closure of the viral RNA-dependent RNA polymerase

2019 ◽  
Vol 294 (45) ◽  
pp. 16897-16907 ◽  
Author(s):  
Alyson K. Boehr ◽  
Jamie J. Arnold ◽  
Hyung S. Oh ◽  
Craig E. Cameron ◽  
David D. Boehr
Keyword(s):  
Rna Polymerase ◽  
Active Site ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Virus Rna

scholarly journals The Plant Noncanonical Antiviral Resistance Protein JAX1 Inhibits Potexviral Replication by Targeting the Viral RNA-Dependent RNA Polymerase

2018 ◽  
Vol 93 (3) ◽  
Author(s):  
Tetsuya Yoshida ◽  
Takuya Shiraishi ◽  
Yuka Hagiwara-Komoda ◽  
Ken Komatsu ◽  
Kensaku Maejima ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Viral Replication ◽  
Resistance Genes ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Antiviral Resistance ◽  
Wild Type ◽  
Rna Dependent Rna Polymerase ◽  
Resistance Breaking ◽  
Lectin Protein

ABSTRACTUnderstanding the innate immune mechanisms of plants is necessary for the breeding of disease-resistant lines. Previously, we identified the antiviral resistance geneJAX1fromArabidopsis thaliana, which inhibits infection by potexviruses.JAX1encodes a unique jacalin-type lectin protein. In this study, we analyzed the molecular mechanisms of JAX1-mediated resistance. JAX1 restricted the multiplication of a potexviral replicon lacking movement-associated proteins, suggesting inhibition of viral replication. Therefore, we developed anin vitropotato virus X (PVX) translation/replication system using vacuole- and nucleus-free lysates from tobacco protoplasts, and we revealed that JAX1 inhibits viral RNA synthesis but not the translation of the viral RNA-dependent RNA polymerase (RdRp). JAX1 did not affect the replication of a resistance-breaking mutant of PVX. Blue native polyacrylamide gel electrophoresis of fractions separated by sucrose gradient sedimentation showed that PVX RdRp constituted the high-molecular-weight complex that seems to be crucial for viral replication. JAX1 was detected in this complex of the wild-type PVX replicon but not in that of the resistance-breaking mutant. In addition, JAX1 interacted with the RdRp of the wild-type virus but not with that of a virus with a point mutation at the resistance-breaking residue. These results suggest that JAX1 targets RdRp to inhibit potexviral replication.IMPORTANCEResistance genes play a crucial role in plant antiviral innate immunity. The roles of conventional nucleotide-binding leucine-rich repeat (NLR) proteins and the associated defense pathways have long been studied. In contrast, recently discovered resistance genes that do not encode NLR proteins (non-NLR resistance genes) have not been investigated extensively. Here we report that the non-NLR resistance factor JAX1, a unique jacalin-type lectin protein, inhibitsde novopotexviral RNA synthesis by targeting the huge complex of viral replicase. This is unlike other known antiviral resistance mechanisms. Molecular elucidation of the target in lectin-type protein-mediated antiviral immunity will enhance our understanding of the non-NLR-mediated plant resistance system.

scholarly journals Zinc Salts Block Hepatitis E Virus Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Nidhi Kaushik ◽  
Chandru Subramani ◽  
Saumya Anang ◽  
Rajagopalan Muthumohan ◽  
Shalimar ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Hepatitis E Virus ◽  
Zinc Supplementation ◽  
Hepatitis E ◽  
Viral Rna ◽  
Health Concern ◽  
Healthy Individuals ◽  
Genomic Rna ◽  
Rna Dependent Rna Polymerase ◽  
Zinc Salts

ABSTRACT Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going up to 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource-starved countries. No specific antiviral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In a human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-Huh7 cell line-based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveal that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection. IMPORTANCE Hepatitis E virus (HEV) is a public health concern in resource-starved countries due to frequent outbreaks. It is also emerging as a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as ribavirin have been used to treat HEV cases, there are known side effects and limitations of such therapy. Our discovery of the ability of zinc salts to block HEV replication by virtue of their ability to inhibit the activity of viral RdRp is important because these findings pave the way to test the efficacy of zinc supplementation therapy in HEV-infected patients. Since zinc supplementation therapy is known to be safe in healthy individuals and since high-dose zinc is used in the treatment of Wilson's disease, it may be possible to control HEV-associated health problems following a similar treatment regimen.

scholarly journals Crystal structures of murine norovirus-1 RNA-dependent RNA polymerase

2011 ◽  
Vol 92 (7) ◽  
pp. 1607-1616 ◽  
Author(s):  
Ji-Hye Lee ◽  
Intekhab Alam ◽  
Kang Rok Han ◽  
Sunyoung Cho ◽  
Sungho Shin ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Crystal Structures ◽  
Active Site ◽  
Metal Ion ◽  
Health Concern ◽  
Murine Norovirus ◽  
Active Site Residues ◽  
Rna Dependent Rna Polymerase ◽  
Close Proximity ◽  
Functional Features

Norovirus is one of the leading agents of gastroenteritis and is a major public health concern. In this study, the crystal structures of recombinant RNA-dependent RNA polymerase (RdRp) from murine norovirus-1 (MNV-1) and its complex with 5-fluorouracil (5FU) were determined at 2.5 Å resolution. Crystals with C2 symmetry revealed a dimer with half a dimer in the asymmetrical unit, and the protein exists predominantly as a monomer in solution, in equilibrium with a smaller population of dimers, trimers and hexamers. MNV-1 RdRp exhibited polymerization activity with a right-hand fold typical of polynucleotide polymerases. The metal ion modelled in close proximity to the active site was found to be coordinated tetrahedrally to the carboxyl groups of aspartate clusters. The orientation of 5FU observed in three molecules in the asymmetrical unit was found to be slightly different, but it was stabilized by a network of favourable interactions with the conserved active-site residues Arg185, Asp245, Asp346, Asp347 and Arg395. The information gained on the structural and functional features of MNV-1 RdRp will be helpful in understanding replication of norovirus and in designing novel therapeutic agents against this important pathogen.

scholarly journals Spatial Perturbations within an RNA Promoter Specifically Recognized by a Viral RNA-Dependent RNA Polymerase (RdRp) Reveal That RdRp Can Adjust Its Promoter Binding Sites

1999 ◽  
Vol 73 (1) ◽  
pp. 198-204 ◽  
Author(s):  
Scott Stevenson Stawicki ◽  
C. Cheng Kao
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Dna And Rna ◽  
In The Wild ◽  
Nucleotide Insertions

ABSTRACT RNA synthesis during viral replication requires specific recognition of RNA promoters by the viral RNA-dependent RNA polymerase (RdRp). Four nucleotides (−17, −14, −13, and −11) within the brome mosaic virus (BMV) subgenomic core promoter are required for RNA synthesis by the BMV RdRp (R. W. Siegel et al., Proc. Natl. Acad. Sci. USA 94:11238–11243, 1997). The spatial requirements for these four nucleotides and the initiation (+1) cytidylate were examined in RNAs containing nucleotide insertions and deletions within the BMV subgenomic core promoter. Spatial perturbations between nucleotides −17 and −11 resulted in decreased RNA synthesis in vitro. However, synthesis was still dependent on the key nucleotides identified in the wild-type core promoter and the initiation cytidylate. In contrast, changes between nucleotides −11 and +1 had a less severe effect on RNA synthesis but resulted in RNA products initiated at alternative locations in addition to the +1 cytidylate. The results suggest a degree of flexibility in the recognition of the subgenomic promoter by the BMV RdRp and are compared with functional regions in other DNA and RNA promoters.

scholarly journals (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 ◽  
2019 ◽  
Vol 24 (13) ◽  
pp. 2360 ◽  
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.

scholarly journals Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency

2020 ◽  
Vol 295 (20) ◽  
pp. 6785-6797 ◽  
Author(s):  
Calvin J. Gordon ◽  
Egor P. Tchesnokov ◽  
Emma Woolner ◽  
Jason K. Perry ◽  
Joy Y. Feng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Lassa Virus ◽  
Nonstructural Proteins ◽  
Rna Dependent Rna Polymerase ◽  
Direct Acting Antiviral ◽  
Nucleotide Analogue ◽  
Direct Acting

Effective treatments for coronavirus disease 2019 (COVID-19) are urgently needed to control this current pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Replication of SARS-CoV-2 depends on the viral RNA-dependent RNA polymerase (RdRp), which is the likely target of the investigational nucleotide analogue remdesivir (RDV). RDV shows broad-spectrum antiviral activity against RNA viruses, and previous studies with RdRps from Ebola virus and Middle East respiratory syndrome coronavirus (MERS-CoV) have revealed that delayed chain termination is RDV's plausible mechanism of action. Here, we expressed and purified active SARS-CoV-2 RdRp composed of the nonstructural proteins nsp8 and nsp12. Enzyme kinetics indicated that this RdRp efficiently incorporates the active triphosphate form of RDV (RDV-TP) into RNA. Incorporation of RDV-TP at position i caused termination of RNA synthesis at position i+3. We obtained almost identical results with SARS-CoV, MERS-CoV, and SARS-CoV-2 RdRps. A unique property of RDV-TP is its high selectivity over incorporation of its natural nucleotide counterpart ATP. In this regard, the triphosphate forms of 2′-C-methylated compounds, including sofosbuvir, approved for the management of hepatitis C virus infection, and the broad-acting antivirals favipiravir and ribavirin, exhibited significant deficits. Furthermore, we provide evidence for the target specificity of RDV, as RDV-TP was less efficiently incorporated by the distantly related Lassa virus RdRp, and termination of RNA synthesis was not observed. These results collectively provide a unifying, refined mechanism of RDV-mediated RNA synthesis inhibition in coronaviruses and define this nucleotide analogue as a direct-acting antiviral.

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