scholarly journals Comparative inhibition analysis of wild type and G671S catalytic site mutant of the SARS-CoV-2 RNA-dependent RNA polymerase

2021 ◽  
Author(s):  
Faisal Khan ◽  
Zarrin Basharat
Keyword(s):  
Protein Structure ◽  
Mortality Rate ◽  
Severe Acute Respiratory Syndrome ◽  
Respiratory Tract ◽  
Rna Polymerase ◽  
Wild Type ◽  
Rna Dependent Rna Polymerase ◽  
Medicinal Compound ◽  
Sowa Rigpa ◽  
Inhibition Analysis

Abstract Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) has wrecked a global havoc, after its emergence in Wuhan, Hubei province of China. It is known to have a distressing effect on the respiratory tract and has a high mortality rate. Till to date (25 Dec 2021), total number of cases (279,362,428) have been reported across the globe. Variable mutations have been reported on various geographical levels. In this study, we have analyzed G671S mutation of RdRp of the SARS-CoV-2, which has been reported in various strains globally, but importantly delta variant cases of Pakistan and has a serious impact on the protein structure. To study the conformational impact, we screened a Tibetan medicinal compound/Sowa Rigpa library against RdRp and compared the best docked compound (Kaempferol 3-O-gentiobioside) to the wild type and mutant RdRp against Remdesivir. A short simulation was used to validate the findings. Both remdesivir and our screened compound showed better inhibition for mutant, compared to the wild type RdRp.

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.

scholarly journals Thermolabile L-A virus-like particles from pet18 mutants of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (2) ◽  
pp. 404-410 ◽  
Author(s):  
T Fujimura ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Polymerase Activity ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Rna Dependent Rna Polymerase ◽  
Virus Like Particles ◽  
Rna Polymerase Activity ◽  
Lines Of Evidence

pet18 mutations in Saccharomyces cerevisiae confer on the cell the inability to maintain either L-A or M double-stranded RNAs (dsRNAs) at the nonpermissive temperature. In in vitro experiments, we examined the effects of pet18 mutations on the RNA-dependent RNA polymerase activity associated with virus-like particles (VLPs). pet18 mutations caused thermolabile RNA polymerase activity of L-A VLPs, and this thermolability was found to be due to the instability of the L-A VLP structure. The pet18 mutations did not affect RNA polymerase activity of M VLPs. Furthermore, the temperature sensitivity of wild-type L-A RNA polymerase differed substantially from that of M RNA polymerase. From these results, and from other genetic and biochemical lines of evidence which suggest that replication of M dsRNA requires the presence of L-A dsRNA, we propose that the primary effect of the pet18 mutation is on the L-A VLP structure and that the inability of pet18 mutants to maintain M dsRNA comes from the loss of L-A dsRNA.

scholarly journals Specific mutations in SARS-CoV2 RNA dependent RNA polymerase and helicase alter protein structure, dynamics and thus function: Effect on viral RNA replication

2020 ◽  
Author(s):  
Feroza Begum ◽  
Debica Mukherjee ◽  
Sandeepan Das ◽  
Dluya Thagriki ◽  
Prem Prakash Tripathi ◽  
...  
Keyword(s):  
Protein Structure ◽  
Rna Polymerase ◽  
Rna Replication ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Stem Loop ◽  
Reading Frame ◽  
Replication Complex ◽  
Structural Modifications ◽  
Mutation Analyses

1.AbstractThe open reading frame (ORF) 1ab of SARS-CoV2 encodes non-structural proteins involved in viral RNA functions like translation and replication including nsp1-4; 3C like proteinase; nsp6-10; RNA dependent RNA polymerase (RdRp); helicase and 3’-5’ exonuclease. Sequence analyses of ORF1ab unravelled emergence of mutations especially in the viral RdRp and helicase at specific positions, both of which are important in mediating viral RNA replication. Since proteins are dynamic in nature and their functions are governed by the molecular motions, we performed normal mode analyses of the SARS-CoV2 wild type and mutant RdRp and helicases to understand the effect of mutations on their structure, conformation, dynamics and thus function. Structural analyses revealed that mutation of RdRp (at position 4715 in the context of the polyprotein/ at position 323 of RdRp) leads to rigidification of structure and that mutation in the helicase (at position 5828 of polyprotein/ position 504) leads to destabilization increasing the flexibility of the protein structure. Such structural modifications and protein dynamics alterations might alter unwinding of complex RNA stem loop structures, the affinity/ avidity of polymerase RNA interactions and in turn the viral RNA replication. The mutation analyses of proteins of the SARS-CoV2 RNA replication complex would help targeting RdRp better for therapeutic intervention.

scholarly journals Prediction of Small Molecule Inhibitors Targeting the Severe Acute Respiratory Syndrome Coronavirus-2 RNA-dependent RNA Polymerase

ACS Omega ◽  
2020 ◽  
Vol 5 (29) ◽  
pp. 18356-18366 ◽  
Author(s):  
Mohammed Ahmad ◽  
Abhisek Dwivedy ◽  
Richard Mariadasse ◽  
Satish Tiwari ◽  
Deepsikha Kar ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Rna Polymerase ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Rna Dependent Rna Polymerase

scholarly journals Early combination treatment with existing HIV antivirals: an effective treatment for COVID-19? 

2020 ◽  
Author(s):  
Roberto Nico Dallocchio ◽  
Alessandro Dessì ◽  
Andrea De Vito ◽  
Giovanna Delogu ◽  
Pier Andrea ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Molecular Docking ◽  
Severe Acute Respiratory Syndrome ◽  
Rna Polymerase ◽  
Protease Inhibitor ◽  
Combination Treatment ◽  
Binding Sites ◽  
Nucleoside Analogues ◽  
Rna Dependent Rna Polymerase ◽  
Protein Pocket

Abstract Since no effective therapy exists, we aimed to test existing HIV antivirals for combination treatment of Coronavirus disease 19 (COVID-19). Our molecular docking findings suggest that lopinavir, ritonavir, darunavir, and atazanavir activated interactions with the key binding sites of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) protease with a better Ki for lopinavir, ritonavir, and darunavir. Furthermore, we evidenced the ability of remdesivir, tenofovir, emtricitabine, and lamivudine to be incorporated in SARS-CoV-2 RNA-dependent RNA polymerase in the same protein pocket where poses the corresponding natural nucleoside substrates with comparable Ki and activating similar interactions. In principle, the four antiviral nucleotides might be used effectively against SARS-CoV-2. The combination of a protease inhibitor and two nucleoside analogues should be evaluated in clinical trials for the treatment of COVID-19.

scholarly journals TMPRSS2 and RNA-Dependent RNA Polymerase Are Effective Targets of Therapeutic Intervention for Treatment of COVID-19 Caused by SARS-CoV-2 Variants (B.1.1.7 and B.1.351)

2021 ◽  
Author(s):  
Jihye Lee ◽  
JinAh Lee ◽  
Hyeon Ju Kim ◽  
Meehyun Ko ◽  
Youngmee Jee ◽  
...  
Keyword(s):  
Public Health ◽  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Rna Polymerase ◽  
Causative Agent ◽  
Therapeutic Intervention ◽  
Human Society ◽  
Rna Dependent Rna Polymerase ◽  
Global Problems

The coronavirus disease 2019 (COVID-19) pandemic is causing unprecedented global problems in both public health and human society. While some vaccines and monoclonal antibodies were successfully developed very quickly and are currently being used, numerous variants of the causative agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are emerging and threatening the efficacy of vaccines and monoclonal antibodies.

scholarly journals Development of a Challenge-Protective Vaccine Concept by Modification of the Viral RNA-Dependent RNA Polymerase of Canine Distemper Virus

2007 ◽  
Vol 81 (24) ◽  
pp. 13649-13658 ◽  
Author(s):  
D. Silin ◽  
O. Lyubomska ◽  
M. Ludlow ◽  
W. P. Duprex ◽  
B. K. Rima
Keyword(s):  
Rna Polymerase ◽  
Neutralizing Antibodies ◽  
Canine Distemper Virus ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Hinge Region ◽  
Canine Distemper ◽  
Wild Type ◽  
Rna Dependent Rna Polymerase ◽  
L Protein

ABSTRACT We demonstrate that insertion of the open reading frame of enhanced green fluorescent protein (EGFP) into the coding sequence for the second hinge region of the viral L (large) protein (RNA-dependent RNA polymerase) attenuates a wild-type canine distemper virus. Moreover, we show that single intranasal immunization with this recombinant virus provides significant protection against challenge with the virulent parental virus. Protection against wild-type challenge was gained either after recovery of cellular immunity postimmunization or after development of neutralizing antibodies. Insertion of EGFP seems to result in overattenuation of the virus, while our previous experiments demonstrated that the insertion of an epitope tag into a similar position did not affect L protein function. Thus, a desirable level of attenuation could be reached by manipulating the length of the insert (in the second hinge region of the L protein), providing additional tools for optimization of controlled attenuation. This strategy for controlled attenuation may be useful for a “quick response” in vaccine development against well-known and “new” viral infections and could be combined efficiently with other strategies of vaccine development and delivery systems.

scholarly journals RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 968 ◽  
Author(s):  
Hyejeong Kim ◽  
Victor D. Ellis ◽  
Andrew Woodman ◽  
Yan Zhao ◽  
Jamie J. Arnold ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Double Mutant ◽  
Biochemical Properties ◽  
Model System ◽  
Viral Population ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Rna Dependent Rna Polymerase ◽  
Or Efficiency ◽  
Coding Sequence

Using the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV) as our model system, we have shown that Lys-359 in motif-D functions as a general acid in the mechanism of nucleotidyl transfer. A K359H (KH) RdRp derivative is slow and faithful relative to wild-type enzyme. In the context of the KH virus, RdRp-coding sequence evolves, selecting for the following substitutions: I331F (IF, motif-C) and P356S (PS, motif-D). We have evaluated IF-KH, PS-KH, and IF-PS-KH viruses and enzymes. The speed and fidelity of each double mutant are equivalent. Each exhibits a unique recombination phenotype, with IF-KH being competent for copy-choice recombination and PS-KH being competent for forced-copy-choice recombination. Although the IF-PS-KH RdRp exhibits biochemical properties within twofold of wild type, the virus is impaired substantially for recombination in cells. We conclude that there are biochemical properties of the RdRp in addition to speed and fidelity that determine the mechanism and efficiency of recombination. The interwoven nature of speed, fidelity, the undefined property suggested here, and recombination makes it impossible to attribute a single property of the RdRp to fitness. However, the derivatives described here may permit elucidation of the importance of recombination on the fitness of the viral population in a background of constant polymerase speed and fidelity.

Sign in / Sign up

Export Citation Format

Share Document