Showing papers on "RNA-dependent RNA polymerase published in 2023"
TL;DR: In this article , the authors extended these previous findings to prove the universal activities of Ensitrelvir against any coronavirus, irrespective of its type, through synchronously acting on most of its main unchanged replication enzymes/proteins, including (in addition to the Mpro) the highly conserved RNA-dependent RNA polymerase (RdRp) and 3′-to-5′ exoribonuclease (ExoN).
Abstract: Lately, nitrogenous heterocyclic antivirals, such as nucleoside-like compounds, oxadiazoles, thiadiazoles, triazoles, quinolines, and isoquinolines, topped the therapeutic scene as promising agents of choice for the treatment of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and their accompanying ailment, the coronavirus disease 2019 (COVID-19). At the same time, the continuous emergence of new strains of SARS-CoV-2, like the Omicron variant and its multiple sublineages, resulted in a new defiance in the enduring COVID-19 battle. Ensitrelvir (S-217622) is a newly discovered orally active noncovalent nonpeptidic agent with potential strong broad-spectrum anticoronaviral activities, exhibiting promising nanomolar potencies against the different SARS-CoV-2 variants. S-217622 effectively and nonspecifically hits the main protease (Mpro) enzyme of a broad scope of coronaviruses. Herein, in the present computational/biological study, we tried to extend these previous findings to prove the universal activities of this investigational agent against any coronavirus, irrespective of its type, through synchronously acting on most of its main unchanged replication enzymes/proteins, including (in addition to the Mpro), e.g., the highly conserved RNA-dependent RNA polymerase (RdRp) and 3′-to-5′ exoribonuclease (ExoN). Biochemical evaluation proved, using the in vitro anti-RdRp/ExoN bioassay, that S-217622 can potently inhibit the replication of coronaviruses, including the new virulent strains of SARS-CoV-2, with extremely minute in vitro anti-RdRp and anti-RdRp/ExoN half-maximal effective concentration (EC50) values of 0.17 and 0.27 μM, respectively, transcending the anti-COVID-19 drug molnupiravir. The preliminary in silico results greatly supported these biochemical results, proposing that the S-217622 molecule strongly and stabilizingly strikes the key catalytic pockets of the SARS-CoV-2 RdRp’s and ExoN’s principal active sites predictably via the nucleoside analogism mode of anti-RNA action (since the S-217622 molecule can be considered as a uridine analog). Moreover, the idealistic druglikeness and pharmacokinetic characteristics of S-217622 make it ready for pharmaceutical formulation with the expected very good clinical behavior as a drug for the infections caused by coronaviruses, e.g., COVID-19. To cut it short, the current critical findings of this extension work significantly potentiate and extend the S-217622’s previous in vitro/in vivo (preclinical) results since they showed that the striking inhibitory activities of this novel anti-SARS-CoV-2 agent on the Mpro could be extended to other replication enzymes like RdRp and ExoN, unveiling the possible universal use of the compound against the next versions of the virus (i.e., disclosing the nonspecific anticoronaviral properties of this compound against almost any coronavirus strain), e.g., SARS-CoV-3, and encouraging us to rapidly start the compound’s vast clinical anti-COVID-19 evaluations.
10 citations
TL;DR: In this article , the authors proposed a dual pathway to hinder SARS-CoV-2 reproduction and stop COVID-19 progression by hitting the two principal coronaviral-2 multiplication enzymes RNA-dependent RNA polymerase (RdRp) and 3-to-5' exoribonuclease (ExoN) synchronously using the same ligand.
Abstract: Recently, natural and synthetic nitrogenous heterocyclic antivirals topped the scene as first choices for the treatment of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and their accompanying disease, the coronavirus disease 2019 (COVID-19). Meanwhile, the mysterious evolution of a new strain of SARS-CoV-2, the Omicron variant and its sublineages, caused a new defiance in the continual COVID-19 battle. Hitting the two principal coronaviral-2 multiplication enzymes RNA-dependent RNA polymerase (RdRp) and 3'-to-5' exoribonuclease (ExoN) synchronously using the same ligand is a highly effective novel dual pathway to hinder SARS-CoV-2 reproduction and stop COVID-19 progression irrespective of the SARS-CoV-2 variant type since RdRps and ExoNs are widely conserved among all SARS-CoV-2 strains. Herein, the present computational/biological study screened our previous small libraries of nitrogenous heterocyclic compounds, searching for the most ideal drug candidates predictably able to efficiently act through this double approach. Theoretical filtration gave rise to three promising antioxidant nitrogenous heterocyclic compounds of the 1,3,4-thiadiazole type, which are CoViTris2022, Taroxaz-26, and ChloViD2022. Further experimental evaluation proved for the first time, utilizing the in vitro anti-RdRp/ExoN and anti-SARS-CoV-2 bioassays, that ChloViD2022, CoViTris2022, and Taroxaz-26 could effectively inhibit the replication of the new virulent strains of SARS-CoV-2 with extremely minute in vitro anti-RdRp and anti-SARS-CoV-2 EC50 values of 0.17 and 0.41 μM for ChloViD2022, 0.21 and 0.69 μM for CoViTris2022, and 0.23 and 0.73 μM for Taroxaz-26, respectively, transcending the anti-COVID-19 drug molnupiravir. The preliminary in silico outcomes greatly supported these biochemical results, proposing that the three molecules potently strike the key catalytic pockets of the SARS-CoV-2 (Omicron variant) RdRp's and ExoN's vital active sites. Moreover, the idealistic pharmacophoric hallmarks of CoViTris2022, Taroxaz-26, and ChloViD2022 molecules relatively make them typical dual-action inhibitors of SARS-CoV-2 replication and proofreading, with their highly flexible structures open for various kinds of chemical derivatization. To cut it short, the present pivotal findings of this comprehensive work disclosed the promising repositioning potentials of the three 2-aminothiadiazoles, CoViTris2022, Taroxaz-26, and ChloViD2022, to successfully interfere with the crucial biological interactions of the coronaviral-2 polymerase/exoribonuclease with the four principal RNA nucleotides, and, as a result, cure COVID-19 infection, encouraging us to rapidly start the three drugs' broad preclinical/clinical anti-COVID-19 evaluations. Dual SARS-CoV-2 polymerase (RdRp) and exoribonuclease (ExoN) inhibition via nucleoside mimicry is a very effective novel approach for COVID-19 infection therapy. Hydroxylated nitrogenous heterocyclic compounds are currently considered first choices in COVID-19 therapy. Extensive computational investigations disclosed three synthetic 5-substituted-2-amino-1,3,4-thiadiazoles, CoViTris2022, Taroxaz-26, and ChloViD2022, with ideal anti-RdRp/ExoN features. ChloViD2022 was ranked the top among the three NAs, with biochemical anti-RdRp EC50 value of 0.17 μM. ChloViD2022 accordingly displayed excellent anti-SARS-CoV-2 EC50 value of 0.41 μM against the Omicron variant.
5 citations
TL;DR: Tsukamoto et al. as discussed by the authors identified a derivative of a natural product from Streptomyces, called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S-adenosyl-lmethionine binding pocket to restrict influenza virus replication.
Abstract: Orthomyxo- and bunyaviruses steal the 5′ cap portion of host RNAs to prime their own transcription in a process called “cap snatching.” We report that RNA modification of the cap portion by host 2′-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces, called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S-adenosyl-l-methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs. Description Catching out cap snatching Some virus families hijack part of their hosts’ RNA to enable their own replication in a process called cap snatching. Before they can enact a snatch, influenza viruses specifically require host cap maturation by a host methyltransferase called MTr1. Tsukamoto et al. screened a compound library and found that trifluoromethyl-tubercidin (TFMT) inhibits host Mtr1 and suppresses virus replication. TFMT inhibits host cap RNA maturation and impedes binding of host cap RNAs with the viral polymerase, thus disabling viral replication. TFMT was not only effective in inhibiting viral replication in human lung cells, but was also effective in mice, displayed little toxicity, and acted in synergy with approved anti-influenza drugs. —CA Inhibition of a host methyltransferase interferes with binding of host cap RNAs to viral polymerase and inhibits influenza virus replication.
4 citations
TL;DR: In this paper , the authors summarized the knowledge about the inhibition of SARS-CoV-2 and influenza A virus propagation by targeting their RNA secondary structure, which is a natural and promising target for the development of inhibitors.
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic, whereas the influenza A virus (IAV) causes seasonal epidemics and occasional pandemics. Both viruses lead to widespread infection and death. SARS-CoV-2 and the influenza virus are RNA viruses. The SARS-CoV-2 genome is an approximately 30 kb, positive sense, 5′ capped single-stranded RNA molecule. The influenza A virus genome possesses eight single-stranded negative-sense segments. The RNA secondary structure in the untranslated and coding regions is crucial in the viral replication cycle. The secondary structure within the RNA of SARS-CoV-2 and the influenza virus has been intensively studied. Because the whole of the SARS-CoV-2 and influenza virus replication cycles are dependent on RNA with no DNA intermediate, the RNA is a natural and promising target for the development of inhibitors. There are a lot of RNA-targeting strategies for regulating pathogenic RNA, such as small interfering RNA for RNA interference, antisense oligonucleotides, catalytic nucleic acids, and small molecules. In this review, we summarized the knowledge about the inhibition of SARS-CoV-2 and influenza A virus propagation by targeting their RNA secondary structure.
3 citations
TL;DR: ChloViD2022, Taroxaz-26, and CoViTris2022 molecules were presented in-silico/in-vitro research winnowed our own small libraries of antioxidant nitrogenous heterocyclic compounds, inspecting for the utmost convenient drug candidates expectedly capable of effectively working through this dual tactic as mentioned in this paper .
Abstract: Currently, nitrogen-containing heterocyclic virucides take the lead as top options for treating the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and their escorting disease, the coronavirus disease 2019 (COVID-19). But unfortunately, the sudden emergence of a new strain of SARS-CoV-2, the Omicron variant and its lineages, complicated matters in the incessant COVID-19 battle. Goaling the two paramount coronaviral-2 multiplication enzymes RNA-dependent RNA polymerase (RdRp) and 3′-to-5′ exoribonuclease (ExoN) at synchronous times using single ligand is a quite effective new binary avenue to restrain SARS-CoV-2 reproduction and cease COVID-19 progression irrespective of the SARS-CoV-2 strain type, as RdRps and ExoNs are vastly conserved in all SARS-CoV-2 strains. The presented in-silico/in-vitro research winnowed our own small libraries of antioxidant nitrogenous heterocyclic compounds, inspecting for the utmost convenient drug candidates expectedly capable of effectively working through this dual tactic. Computational screening afforded three promising compounds of the antioxidant 1,3,4-thiadiazole class, which were named ChloViD2022, Taroxaz-26, and CoViTris2022. Subsequent biological examination, employing the in-vitro anti-RdRp/anti-ExoN and anti-SARS-CoV-2 assays, exclusively demonstrated that ChloViD2022, CoViTris2022, and Taroxaz-26 could efficiently block the replication of the new lineages of SARS-CoV-2 with considerably minute anti-RdRp and anti-SARS-CoV-2 EC50 values of about 0.18 and 0.44 μM for ChloViD2022, 0.22 and 0.72 μM for CoViTris2022, and 0.25 and 0.78 μM for Taroxaz-26, in the order, overtaking the standard anti-SARS-CoV-2 drug molnupiravir. These biochemical findings were optimally presupported by the results of the prior in-silico screening, suggesting that the three compounds might potently hit the catalytic active sites of the virus's RdRp and ExoN enzymes. Furthermore, the perfect pharmacophoric features of ChloViD2022, Taroxaz-26, and CoViTris2022 molecules make them typical dual inhibitors of SARS-CoV-2 replication and proofreading, with their relatively flexible structures eligible for diverse forms of chemical modification. In sum, the current important results of this thorough research work exposed the interesting repurposing potential of the three 2-amino-1,3,4-thiadiazole ligands, ChloViD2022, Taroxaz-26, and CoViTris2022, to effectively conflict with the vital biointeractions between the coronavirus's polymerase/exoribonuclease and the four essential RNA nucleotides, and, accordingly, arrest COVID-19 disease, persuading the relevant investigators to quickly begin the three agents' comprehensive preclinical and clinical anti-COVID-19 assessments.
2 citations
TL;DR: In this paper , a comprehensive evolutionary analysis of the norovirus GI RdRp gene was presented, and it was shown that the most recent common ancestor was 1484 and that the overall evolutionary rate of GI RDRp is 1.821 × 10−3 substitutions/site/year.
Abstract: Norovirus is the leading viral agent of gastroenteritis in humans. RNA-dependent RNA polymerase (RdRp) is essential in the replication of norovirus RNA. Here, we present a comprehensive evolutionary analysis of the norovirus GI RdRp gene. Our results show that the norovirus GI RdRp gene can be divided into three groups, and that the most recent common ancestor was 1484. The overall evolutionary rate of GI RdRp is 1.821 × 10−3 substitutions/site/year. Most of the amino acids of the GI RdRp gene were under negative selection, and only a few positively selected sites were recognized. Amino acid substitutions in the GI RdRp gene accumulated slowly over time. GI.P1, GI.P3 and GI.P6 owned the higher evolutionary rates. GI.P11 and GI.P13 had the faster accumulation rate of amino acid substitutions. GI.P2, GI.P3, GI.P4, GI.P6 and GI.P13 presented a strong linear evolution. These results reveal that the norovirus GI RdRp gene evolves conservatively, and that the molecular evolutionary characteristics of each P-genotype are diverse. Sequencing in RdRp and VP1 of norovirus should be advocated in the surveillance system to explore the effect of RdRp on norovirus activity.
2 citations
TL;DR: Trans-amplifying RNA (TaRNA) as mentioned in this paper is a split-vector derivative of self-ambulating RNA (SARNA), which is a promising vaccine platform for immunization.
2 citations
TL;DR: In this article , the structure of the Hantaan virus polymerase core and conditions for in vitro replication activity were established for future development of antivirals against this group of emerging pathogens.
Abstract: Abstract Hantaviruses are causing life-threatening zoonotic infections in humans. Their tripartite negative-stranded RNA genome is replicated by the multi-functional viral RNA-dependent RNA-polymerase. Here we describe the structure of the Hantaan virus polymerase core and establish conditions for in vitro replication activity. The apo structure adopts an inactive conformation that involves substantial folding rearrangement of polymerase motifs. Binding of the 5′ viral RNA promoter triggers Hantaan virus polymerase reorganization and activation. It induces the recruitment of the 3′ viral RNA towards the polymerase active site for prime-and-realign initiation. The elongation structure reveals the formation of a template/product duplex in the active site cavity concomitant with polymerase core widening and the opening of a 3′ viral RNA secondary binding site. Altogether, these elements reveal the molecular specificities of Hantaviridae polymerase structure and uncover the mechanisms underlying replication. They provide a solid framework for future development of antivirals against this group of emerging pathogens.
2 citations
TL;DR: In this paper , the RNA-dependent RNA polymerase (RdRp) of the severe fever with thrombocytopenia syndrome virus (SFTSV) was studied.
Abstract: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a phenuivirus that has rapidly become endemic in several East Asian countries. The large (L) protein of SFTSV, which includes the RNA-dependent RNA polymerase (RdRp), is responsible for catalysing viral genome replication and transcription. Here, we present 5 cryo-electron microscopy (cryo-EM) structures of the L protein in several states of the genome replication process, from pre-initiation to late-stage elongation, at a resolution of up to 2.6 Å. We identify how the L protein binds the 5' viral RNA in a hook-like conformation and show how the distal 5' and 3' RNA ends form a duplex positioning the 3' RNA terminus in the RdRp active site ready for initiation. We also observe the L protein stalled in the early and late stages of elongation with the RdRp core accommodating a 10-bp product-template duplex. This duplex ultimately splits with the template binding to a designated 3' secondary binding site. The structural data and observations are complemented by in vitro biochemical and cell-based mini-replicon assays. Altogether, our data provide novel key insights into the mechanism of viral genome replication by the SFTSV L protein and will aid drug development against segmented negative-strand RNA viruses.
2 citations
TL;DR: Zhang et al. as discussed by the authors used virtual screening to identify potential anti-ZIKV agents targeting RNA-dependent RNA polymerase (RdRp) and found that posaconazole (POS) could effectively inhibit both RdRp activity with an IC50 of 4.29 μM and the ZIKV replication with an EC50 of 0.59 μM.
Abstract: The Zika virus (ZIKV) epidemic poses a significant threat to human health globally. Thus, there is an urgent need for developing effective anti-ZIKV agents. ZIKV non-structural protein 5 RNA-dependent RNA polymerase (RdRp), a viral enzyme for viral replication, has been considered an attractive drug target. In this work, we screened an anti-infection compound library and a natural product library by virtual screening to identify potential candidates targeting RdRp. Then, five selected candidates were further applied for RdRp enzymatic analysis, cytotoxicity, and binding examination by SPR. Finally, posaconazole (POS) was confirmed to effectively inhibit both RdRp activity with an IC50 of 4.29 μM and the ZIKV replication with an EC50 of 0.59 μM. Moreover, POS was shown to reduce RdRp activity by binding with the key amino acid D666 through molecular docking and site-directed mutation analysis. For the first time, our work found that POS could inhibit ZIKV replication with a stronger inhibitory activity than chloroquine. This work also demonstrated fast anti-ZIKV screening for inhibitors of RdRp and provided POS as a potential anti-ZIKV agent.
1 citations
TL;DR: Perkovic et al. as mentioned in this paper improved trans-amplifying RNA (taRNA), a bipartite vector system aiming to accelerate RNA vaccine adaptation by using an mRNA-encoded alphaviral replicase that amplifies minimal amounts of antigen coding transreplicon RNA.
TL;DR: Wang et al. as discussed by the authors determined the biological significance of nsp2 in viral pathogenesis by constructing interlineage chimeric mutants between the Chinese highly pathogenic PRRSV (HP-PRRSV) strain JXwn06 (lineage 8) and the low-virulent NADC30-like strain CHsx1401-SPJX.
Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) has been a major threat to the world swine industry. In the field, rapid genetic variations (e.g., deletion, mutation, recombination, etc.) within the nsp2 region present an intriguing conundrum to PRRSV biology and pathogenesis. ABSTRACT Fast evolution in the field of the replicase nsp2 represents a most prominent feature of porcine reproductive and respiratory syndrome virus (PRRSV). Here, we determined its biological significance in viral pathogenesis by constructing interlineage chimeric mutants between the Chinese highly pathogenic PRRSV (HP-PRRSV) strain JXwn06 (lineage 8) and the low-virulent NADC30-like strain CHsx1401 (lineage 1). Replacement with nsp2 from JXwn06 was surprisingly lethal to the backbone virus CHsx1401, but combined substitution with the structural protein-coding region (SP) gave rise to viable virus CHsx1401-SPnsp2JX. Meanwhile, a derivative carrying only the SP region (CHsx1401-SPJX) served as a control. Subsequent animal experiments revealed that acquisition of SP alone (CHsx1401-SPJX) did not allow CHsx1401 to gain much virulence, but additional swapping of HP-PRRSV nsp2 (CHsx1401-SPnsp2JX) enabled CHsx1401 to acquire some properties of HP-PRRSV, exemplified by prolonged high fever, microscopic lung hemorrhage, and a significant increase in proinflammatory cytokines in the acute stage. Consistent with this was the transcriptomic analysis of persistently infected secondary lymphoid tissues that revealed a much stronger induction of host cellular immune responses in this group and identified several core immune genes (e.g., TLR4, IL-1β, MPO, etc.) regulated by HP-PRRSV nsp2. Interestingly, immune activation status in the individual groups correlated well with the rate of viremia clearance and viral tissue load reduction. Overall, the above results suggest that the Chinese HP-PRRSV nsp2 is a critical virulence regulator and highlight the importance of nsp2 genetic variation in modulating PRRSV virulence and persistence via immune modulation. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) has been a major threat to the world swine industry. In the field, rapid genetic variations (e.g., deletion, mutation, recombination, etc.) within the nsp2 region present an intriguing conundrum to PRRSV biology and pathogenesis. By making chimeric mutants, here, we show that the Chinese highly pathogenic PRRSV (HP-PRRSV) nsp2 is a virulence factor and a much stronger inducer of host immune responses (e.g., inflammation) than its counterpart, currently epidemic, NADC30-like strains. Differences in the ability to modulate host immunity provide insight into the mechanisms of why NADC30-like strains and their derivatives are rising to be the dominant viruses, whereas the Chinese HP-PRRSV strains gradually give away center stage in the field. Our results have important implications in understanding PRRSV evolution, interlineage recombination, and persistence.
TL;DR: In this paper , the structure and function of the HEV ORF1 polyprotein were investigated and it was shown that it operates as a multifunctional protein, which is not subject to proteolytic processing.
Abstract: Hepatitis E virus (HEV) is an RNA virus responsible for over 20 million infections annually. HEV’s open reading frame (ORF)1 polyprotein is essential for genome replication, though it is unknown how the different subdomains function within a structural context. Our data show that ORF1 operates as a multifunctional protein, which is not subject to proteolytic processing. Supporting this model, scanning mutagenesis performed on the putative papain-like cysteine protease (pPCP) domain revealed six cysteines essential for viral replication. Our data are consistent with their role in divalent metal ion coordination, which governs local and interdomain interactions that are critical for the overall structure of ORF1; furthermore, the ‘pPCP’ domain can only rescue viral genome replication in trans when expressed in the context of the full-length ORF1 protein but not as an individual subdomain. Taken together, our work provides a comprehensive model of the structure and function of HEV ORF1.
TL;DR: Sarma et al. as mentioned in this paper used computer-based drug discovery to identify four potential compounds (Ushinsunine, Cassameridine, (+)-Epiexcelsin, (−)-Phanostenine) with good binding scores and allosteric interactions with the target protein.
Abstract: RNA-dependent RNA polymerase (RdRp) is considered a potential drug target for dengue virus (DENV) inhibition and has attracted attention in antiviral drug discovery. Here, we screened 121 natural compounds from Litsea cubeba against DENV RdRp using various approaches of computer-based drug discovery. Notably, we identified four potential compounds (Ushinsunine, Cassameridine, (+)-Epiexcelsin, (−)-Phanostenine) with good binding scores and allosteric interactions with the target protein. Moreover, molecular dynamics simulation studies were done to check the conformational stability of the complexes under given conditions. Additionally, we performed post-simulation analysis to find the stability of potential drugs in the target protein. The findings suggest Litsea cubeba-derived phytomolecules as a therapeutic solution to control DENV infection.Communicated by Ramaswamy H. Sarma
TL;DR: In this article , a selection strategy was used to identify endogenous RNAs from a transcriptome library derived from lung cells that interact with the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2.
TL;DR: In this article , the RNA polymerase from human influenza viruses is shown to possess the polymerization activity, whereas the PA subunits are found to be involved in the cap-snatching and proofreading (PR) activities, respectively.
Abstract: RNA polymerase from human influenza viruses is a heterotrimeric enzyme and performs the crucial functions of both transcription and replication for multiplication of the viruses in human cells. The heterotrimeric enzyme is made up of two basic protein subunits (PB1 and PB2) and an acidic protein subunit (PA). All the three subunits perform well-defined function(s) in the transcription and replication processes in the human cells. The basic protein subunit PB1 is shown to possess the polymerization activity, whereas the PB2 and the PA subunits are found to be involved in the cap-snatching and proofreading (PR) activities, respectively. The polymerase activity in the catalytic subunit, PB1, is found to be an RNA-dependent RNA polymerase (RdRp). Multiple sequence alignment (MSA) analysis of the PB1 subunits from all the three human influenza viruses, A, B and C shows large number of highly conserved peptides, amino acid motifs and invariant amino acids. Site-directed mutagenesis (SDM) analysis and X-ray crystallographic data have shown that two completely conserved motifs, viz. –GDN- and –SDD-, are involved in binding to the catalytic Mg2+ ion. These data are in close agreement with the MSA analysis data of the polymerases from all the three human influenza viruses. Furthermore, two highly conserved polymerase catalytic regions are identified in the PB1 subunits by sequence similarity to other DNA/RNA polymerases and hence, are proposed to function in the nucleotidyl transfer activities. Presence of the two catalytic regions suggest that the polymerase may function in a dual mode, i.e., in phase I, in association with the cap-snatching subunit PB2, it could be involved in the synthesis of mRNAs (transcription mode) and once enough proteins are made from the mRNAs, in the second phase, in association with PR exonuclease subunit PA, it could switch to the replication mode to synthesize error-free, exact copies of the viral genome. For both the activities, it could use the same invariant catalytic Mg2+-binding –GDN- and –SDD- motifs.
TL;DR: In this article , the authors developed 77 family-level Hidden Markov Model profiles for the viral RNA-dependent RNA polymerase (RdRp)-the only universal "hallmark" gene of RNA viruses.
Abstract: RNA viruses are abundant and highly diverse and infect all or most eukaryotic organisms. However, only a tiny fraction of the number and diversity of RNA virus species have been catalogued. To cost-effectively expand the diversity of known RNA virus sequences, we mined publicly available transcriptomic data sets. We developed 77 family-level Hidden Markov Model profiles for the viral RNA-dependent RNA polymerase (RdRp)-the only universal "hallmark" gene of RNA viruses. By using these to search the National Center for Biotechnology Information Transcriptome Shotgun Assembly database, we identified 5,867 contigs encoding RNA virus RdRps or fragments thereof and analyzed their diversity, taxonomic classification, phylogeny, and host associations. Our study expands the known diversity of RNA viruses, and the 77 curated RdRp Profile Hidden Markov Models provide a useful resource for the virus discovery community.
TL;DR: In this paper , the authors designed potent derivatives of remdesivir through functional group modification of the parent drug targeting RNA-dependent RNA polymerase (RdRp) and main protease (MPro) of SARS-CoV-2.
Abstract: The coronavirus disease of 2019 (COVID-19) is a highly contagious respiratory illness that has become a global health crisis with new variants, an unprecedented number of infections, and deaths and demands urgent manufacturing of potent therapeutics. Despite the success of vaccination campaigns around the globe, there is no particular therapeutics approved to date for efficiently treating infected individuals. Repositioning or repurposing previously effective antivirals against RNA viruses to treat COVID-19 patients is a feasible option. Remdesivir is a broad-spectrum antiviral drug that the Food and Drug Administration (FDA) licenses for treating COVID-19 patients who are critically ill patients. Remdesivir’s low efficacy, which has been shown in some clinical trials, possible adverse effects, and dose-related toxicities are issues with its use in clinical use. Our study aimed to design potent derivatives of remdesivir through the functional group modification of the parent drug targeting RNA-dependent RNA polymerase (RdRp) and main protease (MPro) of SARS-CoV-2. The efficacy and stability of the proposed derivatives were assessed by molecular docking and extended molecular dynamics simulation analyses. Furthermore, the pharmacokinetic activity was measured to ensure the safety and drug potential of the designed derivatives. The derivatives were non-carcinogenic, chemically reactive, highly interactive, and stable with the target proteins. D-CF3 is one of the designed derivatives that finally showed stronger interaction than the parent drug, according to the docking and dynamics simulation analyses, with both target proteins. However, in vitro and in vivo investigations are guaranteed to validate the findings in the future.
TL;DR: In this article , the Ntaya virus MTase and RdRp domains were analyzed and compared to other flaviviral NS5s, and the enzymatic centers were well conserved across Flaviviridae, suggesting that the development of drugs targeting all flaviviruses is feasible.
Abstract: Flaviviruses are single-stranded positive-sense RNA (+RNA) viruses that are responsible for several (re)emerging diseases such as Yellow, Dengue or West Nile fevers. The Zika epidemic highlighted their dangerousness when a relatively benign virus known since the 1950s turned into a deadly pathogen. The central protein for their replication is NS5 (non-structural protein 5), which is composed of the N-terminal methyltransferase (MTase) domain and the C-terminal RNA-dependent RNA-polymerase (RdRp) domain. It is responsible for both, RNA replication and installation of the 5’ RNA cap. We structurally and biochemically analyzed the Ntaya virus MTase and RdRp domains and we compared their properties to other flaviviral NS5s. The enzymatic centers are well conserved across Flaviviridae, suggesting that the development of drugs targeting all flaviviruses is feasible. However, the enzymatic activities of the isolated proteins were significantly different for the MTase domains.
TL;DR: In this paper , a mini-RNA replicon encoded the β subunit, one of the subunits of the Qβ replicase derived from the positive-sense single-stranded Qβ RNA phage and is replicated by the replicases in E. coli.
Abstract: Abstract How the ribonucleic acid (RNA) world transited to the deoxyribonucleic acid (DNA) world has remained controversial in evolutionary biology. At a certain time point in the transition from the RNA world to the DNA world, ‘RNA replicons’, in which RNAs produce proteins to replicate their coding RNA, and ‘DNA replicons’, in which DNAs produce RNA to synthesize proteins that replicate their coding DNA, can be assumed to coexist. The coexistent state of RNA replicons and DNA replicons is desired for experimental approaches to determine how the DNA world overtook the RNA world. We constructed a mini-RNA replicon in Escherichia coli. This mini-RNA replicon encoded the β subunit, one of the subunits of the Qβ replicase derived from the positive-sense single-stranded Qβ RNA phage and is replicated by the replicase in E. coli. To maintain the mini-RNA replicon persistently in E. coli cells, we employed a system of α complementation of LacZ that was dependent on the Qβ replicase, allowing the cells carrying the RNA replicon to grow in the lactose minimal medium selectively. The coexistent state of the mini-RNA replicon and DNA replicon (E. coli genome) was successively synthesized. The coexistent state can be used as a starting system to experimentally demonstrate the transition from the RNA–protein world to the DNA world, which will contribute to progress in the research field of the origin of life.
TL;DR: In this article , the authors performed deep mutational scanning (DMS) on the NS5 of dengue virus serotype 2 in mammalian cells, and the comprehensive single amino acid mutational data corroborated key residues and interactions involved in enzymatic functions of the replicase and suggested potential plasticity in NS5 guanylyl transferase.
Abstract: Flavivirus NS5 is multi-functional viral protein that play critical roles in virus replication, evolution, and immune antagonism against the hosts. Its error-prone replicase activity copies viral RNA for progeny virus particles and shapes virus evolution. Its methyltransferase activity and STAT2-targeting activity compromise type-I interferon signalling, dampening protective immune response during infection. It interacts with several host factors to shape the host-cell environment for virus replication. Thus, NS5 represents a critical target for both vaccine and antiviral drug development. Here, we performed deep mutational scanning (DMS) on the NS5 of dengue virus serotype 2 in mammalian cells. In combination with available structural and biochemical data, the comprehensive single amino-acid mutational data corroborated key residues and interactions involved in enzymatic functions of the replicase and suggested potential plasticity in NS5 guanylyl transferase. Strikingly, we identified that a set of strictly conserved residues in the motifs lining the replicase active site could tolerate mutations, suggesting additional roles of the priming loop in viral RNA synthesis and possible strategies to modulate the error rate of viral replicase activity through active-site engineering. Our DMS dataset and NS5 libraries could provide a framework and a resource to investigate molecular, evolutionary, and immunological aspects of NS5 functions, with relevance to vaccine and antiviral drug development.
TL;DR: In this article , RNA-dependent RNA polymerase (RdRp) residues were shown to be phosphorylated by host kinases in several human, animal or plant viruses including flavivirus, picornaviruses, coronavirus, influenza viruses and tymoviruses.
Abstract: RNA viruses encode an RNA-dependent RNA polymerase (RdRp), which is essential for transcription and replication of their genome since host cells lack equivalent enzymes. RdRp residues were shown to be phosphorylated by host kinases in several human, animal or plant viruses including flaviviruses, picornaviruses, coronaviruses, influenza viruses and tymoviruses. RdRps can be phosphorylated on several residues by distinct host kinases. Phosphomimetic mutations of identified phosphorylated residues either positively or negatively regulate RNA synthesis or association of RdRps with RNA or other proteins. Interestingly, some RdRps evolved to recruit cellular kinases through direct protein-protein interaction, likely to promote or to tightly control their own phosphorylation. Given the essential nature of RdRps for RNA virus replication, a better knowledge of RdRps’ phosphorylation is expected to facilitate the design of future drugs that strongly affect polymerase activity.
TL;DR: In this paper , the double-stranded RNA of chikungunya virus has been analyzed in terms of the organization of the replication intermediate, and it is shown that the intermediate has a shorter apparent persistence length as compared to unconstrained double-standed RNA.
Abstract: Alphaviruses are mosquito-borne, positive-sense single-stranded RNA viruses. Amongst the alphaviruses, chikungunya virus is notable as a large source of human illness, especially in tropical and subtropical regions. When they invade a cell, alphaviruses generate dedicated organelles for viral genome replication, so-called spherules. Spherules form as outward-facing buds at the plasma membrane, and it has recently been shown that the thin membrane neck that connects this membrane bud with the cytoplasm is guarded by a two-megadalton protein complex that contains all the enzymatic functions necessary for RNA replication. The lumen of the spherules contains a single copy of the negative-strand template RNA, present in a duplex with newly synthesized positive-sense RNA. Less is known about the organization of this double-stranded RNA as compared to the protein components of the spherule. Here, we analyzed cryo-electron tomograms of chikungunya virus spherules in terms of the organization of the double-stranded RNA replication intermediate. We find that the double-stranded RNA has a shortened apparent persistence length as compared to unconstrained double-stranded RNA. Around half of the genome is present in either of five conformations identified by subtomogram classification, each representing a relatively straight segment of ~25-32 nm. Finally, the RNA occupies the spherule lumen at a homogeneous density, but has a preferred orientation to be perpendicular to a vector pointing from the membrane neck towards the spherule center. Taken together, this analysis lays another piece of the puzzle of the highly coordinated alphavirus genome replication.
TL;DR: ZINC66112069 and ZINC69481850 as mentioned in this paper showed good stability during the molecular dynamic simulation of 100 ns and could be proven as potential inhibitors of the HNoV RdRp in future antiviral medication development investigations.
Abstract: Norovirus (HNoV) is a leading cause of gastroenteritis globally, and there are currently no treatment options or vaccines available to combat it. RNA-dependent RNA polymerase (RdRp), one of the viral proteins that direct viral replication, is a feasible target for therapeutic development. Despite the discovery of a small number of HNoV RdRp inhibitors, the majority of them have been found to possess a little effect on viral replication, owing to low cell penetrability and drug-likeness. Therefore, antiviral agents that target RdRp are in high demand. For this purpose, we used in silico screening of a library of 473 natural compounds targeting the RdRp active site. The top two compounds, ZINC66112069 and ZINC69481850, were chosen based on their binding energy (BE), physicochemical and drug-likeness properties, and molecular interactions. ZINC66112069 and ZINC69481850 interacted with key residues of RdRp with BEs of −9.7, and −9.4 kcal/mol, respectively, while the positive control had a BE of −9.0 kcal/mol with RdRp. In addition, hits interacted with key residues of RdRp and shared several residues with the PPNDS, the positive control. Furthermore, the docked complexes showed good stability during the molecular dynamic simulation of 100 ns. ZINC66112069 and ZINC69481850 could be proven as potential inhibitors of the HNoV RdRp in future antiviral medication development investigations.
TL;DR: In this article , a polyclonal antibody against RdRp was prepared by using a prokaryotic expression vector pET-28a-RdRp and provided a tool to investigate PEDV pathogenesis.
TL;DR: In this article , a substitution mutation in the nsp13-helicase of the betacoronavirus murine hepatitis virus (MHV) that was selected during passage with the RDV parent compound confers partial resistance independently and additively when expressed with co-selected RDV resistance mutations in nsp12-RdRp.
Abstract: ABSTRACT Coronaviruses (CoVs) encode nonstructural proteins 1–16 (nsps 1–16) which form replicase complexes that mediate viral RNA synthesis. Remdesivir (RDV) is an adenosine nucleoside analog antiviral that inhibits CoV RNA synthesis. RDV resistance mutations have been reported only in the nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp). We here show that a substitution mutation in the nsp13-helicase (nsp13-HEL A335V) of the betacoronavirus murine hepatitis virus (MHV) that was selected during passage with the RDV parent compound confers partial RDV resistance independently and additively when expressed with co-selected RDV resistance mutations in the nsp12-RdRp. The MHV A335V substitution did not enhance replication or competitive fitness compared to WT MHV and remained sensitive to the active form of the cytidine nucleoside analog antiviral molnupiravir (MOV). Biochemical analysis of the SARS-CoV-2 helicase encoding the homologous substitution (A336V) demonstrates that the mutant protein retained the ability to associate with the core replication proteins nsps 7, 8, and 12 but had impaired helicase unwinding and ATPase activity. Together, these data identify a novel determinant of nsp13-HEL enzymatic activity, define a new genetic pathway for RDV resistance, and demonstrate the importance of surveillance for and testing of helicase mutations that arise in SARS-CoV-2 genomes. IMPORTANCE Despite the development of effective vaccines against COVID-19, the continued circulation and emergence of new variants support the need for antivirals such as RDV. Understanding pathways of antiviral resistance is essential for surveillance of emerging variants, development of combination therapies, and for identifying potential new targets for viral inhibition. We here show a novel RDV resistance mutation in the CoV helicase also impairs helicase functions, supporting the importance of studying the individual and cooperative functions of the replicase nonstructural proteins 7–16 during CoV RNA synthesis. The homologous nsp13-HEL mutation (A336V) has been reported in the GISAID database of SARS-CoV-2 genomes, highlighting the importance of surveillance of and genetic testing for nucleoside analog resistance in the helicase. Despite the development of effective vaccines against COVID-19, the continued circulation and emergence of new variants support the need for antivirals such as RDV. Understanding pathways of antiviral resistance is essential for surveillance of emerging variants, development of combination therapies, and for identifying potential new targets for viral inhibition. We here show a novel RDV resistance mutation in the CoV helicase also impairs helicase functions, supporting the importance of studying the individual and cooperative functions of the replicase nonstructural proteins 7–16 during CoV RNA synthesis. The homologous nsp13-HEL mutation (A336V) has been reported in the GISAID database of SARS-CoV-2 genomes, highlighting the importance of surveillance of and genetic testing for nucleoside analog resistance in the helicase.
TL;DR: In this paper , the authors show that AT-9010 has several modes of action on DENV full-length NS5, including inhibition of RNA 2′-O-MTase and RNA dependent RNA polymerase (RdRp).
TL;DR: In this article , a review summarizes current perspectives on the therapeutic strategies adopted to target the DENV-NS5 (RdRp and MTase domains) at the host-pathogen interface and further discusses the directions to identify candidate drugs to combat DENV infection.
Abstract: Dengue virus (DENV) infection is one of the most emerging arboviral infections in humans. DENV is a positive-stranded RNA virus in the Flaviviridae family consisting of an 11 kb genome. DENV non-structural protein 5 (DENV-NS5) constitutes the largest among the non-structural proteins, which act as two domains, the RNA-dependent RNA polymerase (RdRp) and RNA methyltransferase enzyme (MTase). The DENV-NS5 RdRp domain contributes to the viral replication stages, whereas the MTase initiates viral RNA capping and facilitates polyprotein translation. Given the functions of both DENV-NS5 domains have made them an important druggable target. Possible therapeutic interventions and drug discoveries against DENV infection were thoroughly reviewed; however, a current update on the therapeutic strategies specific to DENV-NS5 or its active domains was not attempted. Since most potential compounds and drugs targeting the DENV-NS5 were evaluated in both in vitro cultures and animal models, a more detailed evaluation of molecules/drug candidates still requires investigation in randomized controlled clinical trials. This review summarizes current perspectives on the therapeutic strategies adopted to target the DENV-NS5 (RdRp and MTase domains) at the host-pathogen interface and further discusses the directions to identify candidate drugs to combat DENV infection.