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Abstract: The RNA-dependent RNA polymerase (RdRp) of SARS coronavirus (SARS-CoV) is essential for viral replication and a potential target for anti-SARS drugs. We report here the cloning, expression, and purification of the N-terminal GST-fused SARS-CoV RdRp and its polymerase catalytic domain in Escherichia coli. During purification, the full-length GST-RdRp was found to cleave into three main fragments: an N-terminal p12 fragment, a middle p30 fragment, and a C-terminal p64 fragment comprising the catalytic domain, presumably due to bacterial proteases. Biochemical assays show that the full-length GST-RdRp has RdRp activity and the p64 and p12 fragments form a complex that exhibits comparable RdRp activity, whereas the GST-p64 protein has no activity, suggesting that the p12 domain is required for polymerase activity possibly via involvement in template-primer binding. Nonnucleoside HIV-1 RT inhibitors are shown to have no evident inhibitory effect on SARS-CoV RdRp activity. This work provides a basis for biochemical and structural studies of SARS-CoV RdRp and for development of anti-SARS drugs. [Copyright &y& Elsevier]

Severe acute respiratory syndrome (SARS) is a highly contagious and sometimes a lethal disease, which spread over five continents in 2002–2003. Laboratory analysis showed that the etiologic agent for SARS is a new type of coronavirus. Currently, there is no specific treatment for this disease. RNA interference (RNAi) is a recently discovered antiviral mechanism in plant and animal cells that induces a specific degradation of double-stranded RNA. Here, we provide evidences that RNAi targeting at coronavirus RNA-dependent RNA polymerase (RDRP) using short hairpin RNA (shRNA) expression plasmids can specifically inhibit expression of extraneous coronavirus RDRP in 293 and HeLa cells. Moreover, this construct significantly reduced the plaque formation of SARS coronaviruses in Vero-E6 cells. The data may suggest a new approach for treatment of SARS patients. [Copyright &y& Elsevier]

Heterochromatin mediates various nuclear processes including centromere function, gene silencing and nuclear organization. Although it was discovered nearly 75 years ago, the pathways involved in heterochromatin establishment, assembly and epigenetic maintenance have been elusive. Recent reports have demonstrated that distinct and novel chromatin-associated factors, including DNA, RNA and histone modifications, are involved in each of these events. These new findings define a novel conserved mechanism of heterochromatin formation that is likely to have an impact on all eukaryotic silencing pathways. [Copyright &y& Elsevier]

RNA interference (RNAi) is a post-transcriptional gene-silencing phenomenon induced by double-stranded RNA. It has been widely used as a knockdown technology to analyze gene function in various organisms. Although RNAi was first discovered in worms, related phenomena such as post-transcriptional gene silencing and coat protein mediated protection from viral infection had been observed in plants prior to this. In plants, RNAi is often achieved through transgenes that produce hairpin RNA. For genetic improvement of crop plants, RNAi has advantages over antisense-mediated gene silencing and co-suppression, in terms of its efficiency and stability. It also offers advantages over mutation-based reverse genetics in its ability to suppress transgene expression in multigene families in a regulated manner. [Copyright &y& Elsevier]

microRNAs (miRNAs) are a newly identified and surprisingly large class of endogenous tiny regulatory RNAs. They exhibit various expressional patterns and are highly conserved across species. Recently, several regulatory targets of miRNAs have been predicted. Functional analysis of the potential targets indicated that miRNAs may be involved in a wide range of pivotally biological events. The nature of miRNAs and their intersection with small interfering RNAs endow them with many regulatory advantages over proteins and make them a potent and novel means to regulate gene expression at almost all levels. Here we argue that miRNAs are key participants in gene regulatory network. [Copyright &y& Elsevier]

The discovery of hundreds of plant micro RNAs (miRNAs) has triggered much speculation about their potential roles in plant development. The search for plant genes involved in miRNA processing has revealed common factors such as DICER, and new molecules, including HEN1. Progress is also being made toward identifying miRNA target genes and understanding the mechanisms of miRNA-mediated gene regulation in plants. This work has lead to a reexamination of many previously characterized mutations that are now known to affect components or targets of miRNA-mediated pathways. [Copyright &y& Elsevier]

We have previously determined the crystal structure of a non-structural 5B (NS5B) protein, an RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV). NS5B protein with the hydrophobic C-terminal 21 amino acid residues truncated, designated NS5B570, shows a typical nucleotide polymerase structure resembling a right-hand shape. In the crystal structure, a C-terminal region between Leu545 and His562 occupies a putative RNA-binding cleft of this polymerase and seems to inhibit the polymerase activity. Varieties of recombinant NS5B proteins (NS5B552, NS5B544, NS5B536 or NS5B531, with C-terminal 39, 47, 55 or 60 amino acid residues truncated, respectively) were systematically constructed to elucidate effects of the region on the polymerase activity. NS5B544, NS5B536 and NS5B531 showed markedly higher RdRp activities compared to the activities of NS5B570 or NS5B552. Furthermore, when the hydrophobic amino acid residues Leu547, Trp550 and Phe551 (LWF) in NS5B570 and NS5B552 were changed to alanine, their activities were higher than that of the original NS5B570. The crystal structures of the various recombinant NS5B proteins were also determined. Structural comparison of the NS5B proteins indicates that the activation was caused by elimination of a unique hydrophobic interaction between the three C-terminal residues and a shallowly concave pocket consisting of thumb and palm domains. [Copyright &y& Elsevier]

For a long time, RNA has been merely regarded as a molecule that can either function as a messenger (mRNA) or as part of the translational machinery (tRNA, rRNA). Meanwhile, it became clear that RNAs are versatile molecules that do not only play key roles in many important biological processes like splicing, editing, protein export and others, but can also—like enzymes—act catalytically. Two important aspects of RNA function—antisense-RNA control and RNA interference (RNAi)—are emphasized in this review. Antisense-RNA control functions in all three kingdoms of life—although the majority of examples are known from bacteria. In contrast, RNAi, gene silencing triggered by double-stranded RNA, the oldest and most ubiquitous antiviral system, is exclusively found in eukaryotes. Our current knowledge about occurrence, biological roles and mechanisms of action of antisense RNAs as well as the recent findings about involved genes/enzymes and the putative mechanism of RNAi are summarized. An interesting intersection between both regulatory mechanisms is briefly discussed. [Copyright &y& Elsevier]

Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family and cannot be propagated in vitro, which has impeded the progress of investigating its replication mechanism. Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells. Here, aided by this replicon system and using two-step affinity purification, we purified the RHDV replicase and identified its associated host factors. We identified rabbit nucleolin (NCL) as a physical link, which mediating the interaction between other RNA-dependent RNA polymerase (RdRp)-related host proteins and the viral replicase RdRp. We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 ​cells, respectively. NCL was identified to directly interact with RHDV RdRp, p16, and p23. Furthermore, NCL knockdown severely impaired the binding of RdRp to RdRp-related host factors. Collectively, these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2022 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

For coronaviruses, RNA-dependent RNA polymerase (RdRp) is an essential enzyme that catalyses the replication from RNA template and therefore remains an attractive therapeutic target for anti-COVID drug discovery. In the present study, we performed a comprehensive in silico screening for 16,776 potential molecules from recently established drug libraries based on two important pharmacophores (3-amino-4-phenylbutan-2-ol and piperazine). Based on initial assessment, 4042 molecules were obtained suitable as drug candidates, which were following Lipinski's rule. Molecular docking implemented for the analysis of molecular interactions narrowed this number of compounds down to 19. Subsequent to screening filtering criteria and considering the critical parameters viz. docking score and MM-GBSA binding free energy, 1-(4-((2 S ,3 S )-3-amino-2-hydroxy-4-phenylbutyl)piperazin-1-yl)-3-phenylurea (compound 1 ) was accomplished to score highest in comparison to the remaining 18 shortlisted drug candidates. Notably, compound 1 displayed higher docking score (-8.069 kcal/mol) and MM-GBSA binding free energy (-49.56 kcal/mol) than the control drug, remdesivir triphosphate, the active form of remdesivir as well as adenosine triphosphate. Furthermore, a molecular dynamics simulation was carried out (100 ns), which substantiated the candidacy of compound 1 as better inhibitor. Overall, our systematic in silico study predicts the potential of compound 1 to exhibit a more favourable specific activity than remdesivir triphosphate. Hence, we suggest compound 1 as a novel potential drug candidate, which should be considered for further exploration and validation of its potential against SARS-CoV-2 in wet lab experimental studies.Communicated by Ramasawamy H. Sarma.

The bovine ephemeral fever virus (BEFV) is an enzootic agent that affects millions of bovines and causes major economic losses. Though the virus is seasonally reported with a very high morbidity rate (80-100%) from African, Australian, and Asiatic continents, it remains a neglected pathogen in many of its endemic areas, with no proper therapeutic drugs or vaccines presently available for treatment. The RNA-dependent RNA polymerase (RdRp) catalyzes the viral RNA synthesis and is an appropriate candidate for antiviral drug developments. We utilized integrated computational tools to build the 3D model of BEFV-RdRp and then predicted its probable active binding sites. The virtual screening and optimization against these active sites, using several small-molecule inhibitors from a different category of Life Chemical database and FDA-approved drugs from the ZINC database, was performed. We found nine molecules that have docking scores varying between -6.84 to -10.43 kcal/mol. Furthermore, these complexes were analyzed for their conformational dynamics and thermodynamic stability using molecular dynamics simulations in conjunction with the molecular mechanics generalized Born surface area (MM-GBSA) scheme. The binding free energy calculations depict that the electrostatic interactions play a dominant role in the RdRp-inhibitor binding. The hot spot residues, such as Arg565, Asp631, Glu633, Asp740, and Glu707, were found to control the RdRp-inhibitor interaction. The ADMET analysis strongly suggests favorable pharmacokinetics of these compounds that may prove useful for treating the BEFV ailment. Overall, we anticipate that these findings would help explore and develop a wide range of anti-BEFV therapy.Communicated by Ramaswamy H. Sarma.

[Display omitted] • Coronaviral RdRp inhibition is the most effective strategy for COVID-19 treatment. • Cyanorona-20 is the first favipiravir derivative with anti-SARS-CoV-2 RdRp activity. • Cyanorona-20 is the first extremely potent specific anti-COVID-19 compound. • Cyanorona-20 selectively inhibits SARS-CoV-2 replication with EC 50 = 0.45 μM. • Cyanorona-20 is a coronaviral-2 infection antidote and it has very high CC 50 & SI. Explicit hindrance and blockade of the viral RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is considered one of the most promising and efficient approaches for developing highly potent remedies for COVID-19. However, almost all of the reported viral RdRp inhibitors (either repurposed or new antiviral drugs) lack specific selectivity against the novel coronaviral RdRp and still at a beginning phase of advancement. Herein, I discovered and introduce a new pyrazine derivative, (E)- N -(4-cyanobenzylidene)-6-fluoro-3-hydroxypyrazine-2-carboxamide (cyanorona-20), as the first potent SARS-CoV-2 RdRp inhibitor with very high selectivity (209- and 45-fold more potent than favipiravir and remdesivir, respectively). This promising selective specific anti-COVID-19 compound is also deemed to be the first distinctive derivative of favipiravir. Cyanorona-20, the unprecedented nucleoside/nucleotide analog, was designed, synthesized, characterized, computationally studied, and biologically evaluated for its anti-COVID-19 actions (through a precise in vitro anti-COVID-19 assay). The results of the biological assay displayed that cyanorona-20 surprisingly exhibited very high and largely significant anti-COVID-19 activities (anti-SARS-CoV-2 EC 50 = 0.45 μM), and, in addition, it could be also a very promising guide and lead compound for the design and synthesis of new anti-SARS-CoV-2 and anti-COVID-19 agents through structural modifications and further computational studies. Further appraisal for the improvement of cyanorona-20 medication is a prerequisite requirement in the coming days. In a word, the ascent of the second member (cyanorona-20 "Corona Antidote") of the novel and promising class of anti-COVID-19 pyrazine derivatives would drastically make a medical uprising in the pharmacotherapeutic treatment regimens and protocols of the recently-emerged SARS-CoV-2 infection and its accompanying COVID-19. [ABSTRACT FROM AUTHOR]

Hand, Foot and Mouth Disease (HFMD) is usually a self-limiting, mild childhood disease that is caused mainly by Coxsackie virus A16 (CVA16) and Enterovirus A71 (EV-A71), both members of the Picornaviridae family. However, recurring HFMD outbreaks and epidemics due to EV-A71 infection in the Western Pacific region, and the propensity of EV-A71 strains to cause severe neurological complications have made this neurotropic virus a serious public health concern in afflicted countries. High mutation rate leading to viral quasispecies combined with frequent intra- and inter-typic recombination events amongst co-circulating EV-A71 strains have contributed to the great diversity and fast evolution of EV-A71 genomes, making impossible any accurate prediction of the next epidemic strain. Comparative genome sequence analyses and mutagenesis approaches have led to the identification of a number of viral determinants involved in EV-A71 fitness and virulence. These viral determinants include amino acid residues located in the structural proteins of the virus, affecting attachment to the host cell surface, receptor binding, and uncoating events. Critical residues in non-structural proteins have also been identified, including 2C, 3A, 3C proteases and the RNA-dependent RNA polymerase. Finally, mutations altering key secondary structures in the 5' untranslated region were also found to influence EV-A71 fitness and virulence. While our current understanding of EV-A71 pathogenesis remains fragmented, these studies may help in the rational design of effective treatments and broadly protective vaccine candidates.

[Display omitted] • The present review showcases the major pharmaceutical drug targets that can be used to fight against the SARS-CoV-2 pandemic. • Nonstructural protein inhibitors are essential in preventing host cell entry of coronaviruses and are thoroughly discussed. • Potential SARS-CoV-2 drug targets, including their structures, effects and potential impacts, are reviewed. • Small-molecule therapeutics and their modes of disease suppression are explained in detail. The SARS-CoV-2 outbreak and pandemic that began near the end of 2019 has posed a challenge to global health. At present, many candidate small-molecule therapeutics have been developed that can inhibit both the infection and replication of SARS-CoV-2 and even potentially relieve cytokine storms and other related complications. Meanwhile, host-targeted drugs that inhibit cellular transmembrane serine protease (TMPRSS2) can prevent SARS-CoV-2 from entering cells, and its combination with chloroquine and dihydroorotate dehydrogenase (DHODH) inhibitors can limit the spread of SARS-CoV-2 and reduce the morbidity and mortality of patients with COVID-19. The present article provides an overview of these small-molecule therapeutics based on insights from medicinal chemistry research and focuses on RNA-dependent RNA polymerase (RdRp) inhibitors, such as the nucleoside analogues remdesivir, favipiravir and ribavirin. This review also covers inhibitors of 3C-like protease (3CLpro), papain-like protease (PLpro) and other potentially innovative active ingredient molecules, describing their potential targets, activities, clinical status and side effects. [ABSTRACT FROM AUTHOR]

The virus responsible for the current COVID-19 pandemic is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): a new virus with high infectivity and moderate mortality. The major clinical manifestation of COVID-19 is interstitial pneumonia, which may progress to acute respiratory distress syndrome (ARDS). However, the disease causes a potent systemic hyperin-flammatory response, i.e., a cytokine storm or macrophage activation syndrome (MAS), which is associated with thrombotic complications. The complexity of the disease requires appropriate intensive treatment. One of promising treatment is statin administration, these being 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors that exert pleiotropic anti-inflammatory effects. Recent studies indicate that statin therapy is associated with decreased mortality in COVID-19, which may be caused by direct and indirect mechanisms. According to literature data, statins can limit SARS-CoV-2 cell entry and replication by inhibiting the main protease (Mpro) and RNA-dependent RNA polymerase (RdRp). The cytokine storm can be ameliorated by lowering serum IL-6 levels; this can be achieved by inhibiting Toll-like receptor 4 (TLR4) and modulating macrophage activity. Statins can also reduce the complications of COVID-19, such as thrombosis and pulmonary fibrosis, by reducing serum PAI-1 levels, attenuating TGF-β and VEGF in lung tissue, and improving endothelial function. Despite these benefits, statin therapy may have side effects that should be considered, such as elevated creatinine kinase (CK), liver enzyme and serum glucose levels, which are already elevated in severe COVID-19 infection. The present study analyzes the latest findings regarding the benefits and limitations of statin therapy in patients with COVID-19. [ABSTRACT FROM AUTHOR]

The widespread nature of several viruses is greatly credited to their rapidly altering RNA genomes that enable the infection to persist despite challenges presented by host cells. Within the RNA genome of infections is RNA-dependent RNA polymerase (RdRp), which is an essential enzyme that helps in RNA synthesis by catalysing the RNA template-dependent development of phosphodiester bonds. Therefore, RdRp is an important therapeutic target in RNA virus-caused diseases, including SARS-CoV-2. In this review, we describe the promising RdRp inhibitors that have been launched or are currently in clinical studies for the treatment of RNA virus infections. Structurally, nucleoside inhibitors (NIs) bind to the RdRp protein at the enzyme active site, and nonnucleoside inhibitors (NNIs) bind to the RdRp protein at allosteric sites. By reviewing these inhibitors, more precise guidelines for the development of more promising anti-RNA virus drugs should be set, and due to the current health emergency, they will eventually be used for COVID-19 treatment. Image 1 • This article provides an overview of RdRp inhibitors that have been launched or are in clinical studies for the treatment of RNA virus infections, including COVID-19. • Nucleoside inhibitors (NIs) exhibit broad-spectrum antiviral activity but pose a more serious toxicity risk than non-nucleoside inhibitors (NNIs) because high dosages of NIs result in accumulation in plasma. • Structurally diverse NNIs are endowed with high potential activities but tend to induce drug resistance due to the RdRp allosteric site. [ABSTRACT FROM AUTHOR]

• Lurasidone and Lurasidone exo inhibit SARS-CoV-2 via ' one drug multiple targets ' strategy. • MD simulation revealed their strong binding affinity against multiple targets of SARS-CoV-2. • Both the drugs exhibit favourable pharmacokinetic properties as predicted by ADMET parameters. • Their unique multitargeting feature warrants further in-vitro and in-vivo experiments. The emerging paradigm shift from 'one molecule, one target, for one disease' towards 'multi-targeted small molecules' has paved an ingenious pathway in drug discovery in recent years. We extracted this idea for the investigation of drugs for COVID-19. Perceiving the importance of organosulfur compounds, seventy-six known organosulfur compounds were screened and studied for the interaction with multiple SARS-CoV-2 target proteins by molecular dynamics simulation. Lurasidone and its derivatives displayed substantial binding affinity against five proteins (Mpro, PLpro, Spro, helicase and RdRp). The pharmacokinetics, ADMET properties and target prediction studies performed in this work further potentiates the effectiveness against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

The four circulating serotypes of dengue virus (DENV) occasionally cause potentially fetal symptoms of severe dengue, which there is currently no specific treatment available. Extensive efforts have been made to inhibit viral replication processes by impeding the activity of an exclusive RNA-dependent RNA polymerase (RdRp) in the viral non-structural protein 5 (NS5). In our earlier work, we identified the characteristic, specific interaction between the C-terminal thumb subdomain of RdRp and an apical loop in the 3′ stem-loop (SL) element in the DENV RNA genome, which is fundamental for viral replication. Here, we demonstrated a new approach for interfering viral replication via blocking of 3′ SL RNA binding to RdRp by the single-chain variable fragments (scFvs). We isolated and cloned 3 different human scFvs that bound to RdRp from DENV serotype 2 and interfered with 3′ SL-binding, utilizing a combination of phage-display panning and Alpha methods. When tagged with a cell penetrating peptide, a selected scFv clone, 2E3, entered cells and partially colocalized with NS5 in the cytoplasm of infected HuH-7 cells. 2E3 significantly inhibited DENV RNA replication with sub-nanomolar EC50 values and significantly reduced the production of infectious particles. The molecular docking models suggested that 2E3 recognized both palm and thumb subdomains of RdRp, and interacted with Lys841, a key residue involved in RNA binding. Our results provide a new potential therapeutic molecule specific for flaviviral infection. • Human scFvs specific to DENV RdRp were identified via phage display and Alpha. • The 2E3 scFv blocked the binding of the 3′ SL RNA to the RdRp thumb subdomain. • Cell-penetrable 2E3 inhibited DENV-2 RNA synthesis with sub-nanomolar EC50 values. • Our work highlights scFvs as a new potential therapeutic mean for virus infection. [ABSTRACT FROM AUTHOR]

A coronavirus disease (COVID-19) outbreak associated with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading widely through person-to-person transmission. Various detection approaches have been developed involving quantitative polymerase chain reaction (qPCR) methods, CRISPR-based systems, and direct targeting of specific coronavirus proteins. However, there have only been a few reports on the detection of RNA-dependent RNA polymerase (RdRP), the primer-independent RNA-replicable protein produced by the RNA genes of coronavirus. Here, we introduce a novel diagnostic methodology for COVID-19 using the RNA-directed and de novo RNA replicable function of RdRP. We devised an RNA platform for RdRP-induced transcription (RPRIT) that includes an RNA template that can be directly transcribed by RdRP. By utilizing RPRIT, the presence of RdRP can be readily confirmed within 30 min using isothermal incubation without PCR. This RdRP detection method can provide a new route for rapid diagnosis of RNA virus-infected patients.

The coronavirus disease of 2019 (COVID-19) pandemic speaks to the need for drugs that not only are effective but also remain effective given the mutation rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To this end, we describe structural binding-site insights for facilitating COVID-19 drug design when targeting RNA-dependent RNA polymerase (RDRP), a common conserved component of RNA viruses. We combined an RDRP structure data set, including 384 RDRP PDB structures and all corresponding RDRP-ligand interaction fingerprints, thereby revealing the structural characteristics of the active sites for application to RDRP-targeted drug discovery. Specifically, we revealed the intrinsic ligand-binding modes and associated RDRP structural characteristics. Four types of binding modes with corresponding binding pockets were determined, suggesting two major subpockets available for drug discovery. We screened a drug data set of 7894 compounds against these binding pockets and presented the top-10 small molecules as a starting point in further exploring the prevention of virus replication. In summary, the binding characteristics determined here help rationalize RDRP-targeted drug discovery and provide insights into the specific binding mechanisms important for containing the SARS-CoV-2 virus.

Viperin plays an important and multifaceted role in the innate immune response to viral infection. Viperin is also notable as one of very few radical SAM-dependent enzymes present in higher animals; however, the enzyme appears broadly conserved across all kingdoms of life, which suggests that it represents an ancient defense mechanism against viral infections. Although viperin was discovered some 20 years ago, only recently was the enzyme's structure determined and its catalytic activity elucidated. The enzyme converts CTP to 3'-deoxy-3',4'-didehydro-CTP, which functions as novel chain-terminating antiviral nucleotide when misincorporated by viral RNA-dependent RNA polymerases. Moreover, in higher animals, viperin interacts with numerous other host and viral proteins, and it is apparent that this complex network of interactions constitutes another important aspect of the protein's antiviral activity. An emerging theme is that viperin appears to facilitate ubiquitin-dependent proteasomal degradation of some of the proteins it interacts with. Viperin-targeted protein degradation contributes to the antiviral response either by down-regulating various metabolic pathways important for viral replication or by directly targeting viral proteins for degradation. Here, we review recent advances in our understanding of the structure and catalytic activity of viperin, together with studies investigating the interactions between viperin and its target proteins. These studies have provided detailed insights into the biochemical processes underpinning this unusual enzyme's wide-ranging antiviral activity. We also highlight recent intriguing reports that implicate a broader role for viperin in regulating nonpathological cellular processes, including thermogenesis and protein secretion., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Ghosh and Marsh.)

Picornaviral RNA-dependent RNA polymerases (RdRPs) have low replication fidelity that is essential for viral fitness and evolution. Their global fold consists of the classical "cupped right hand" structure with palm, fingers, and thumb domains, and these RdRPs also possess a unique contact between the fingers and thumb domains. This interaction restricts movements of the fingers, and RdRPs use a subtle conformational change within the palm domain to close their active sites for catalysis. We have previously shown that this core RdRP structure and mechanism provide a platform for polymerases to fine-tune replication rates and fidelity to optimize virus fitness. Here, we further elucidated the structural basis for differences in replication rates and fidelity among different viruses by generating chimeric RdRPs from poliovirus and coxsackievirus B3. We designed these chimeric polymerases by exchanging the fingers, pinky finger, or thumb domains. The results of biochemical, rapid-quench, and stopped-flow assays revealed that differences in biochemical activity map to individual modular domains of this polymerase. We found that the pinky finger subdomain is a major regulator of initiation and that the palm domain is the major determinant of catalytic rate and nucleotide discrimination. We further noted that thumb domain interactions with product RNA regulate translocation and that the palm and thumb domains coordinately control elongation complex stability. Several RdRP chimeras supported the growth of infectious poliovirus, providing insights into enterovirus species-specific protein-protein interactions required for virus replication., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Watkins et al.)

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., (© 2020 Gordon et al.)

Antiviral drugs for managing infections with human coronaviruses are not yet approved, posing a serious challenge to current global efforts aimed at containing the outbreak of severe acute respiratory syndrome-coronavirus 2 (CoV-2). Remdesivir (RDV) is an investigational compound with a broad spectrum of antiviral activities against RNA viruses, including severe acute respiratory syndrome-CoV and Middle East respiratory syndrome (MERS-CoV). RDV is a nucleotide analog inhibitor of RNA-dependent RNA polymerases (RdRps). Here, we co-expressed the MERS-CoV nonstructural proteins nsp5, nsp7, nsp8, and nsp12 (RdRp) in insect cells as a part a polyprotein to study the mechanism of inhibition of MERS-CoV RdRp by RDV. We initially demonstrated that nsp8 and nsp12 form an active complex. The triphosphate form of the inhibitor (RDV-TP) competes with its natural counterpart ATP. Of note, the selectivity value for RDV-TP obtained here with a steady-state approach suggests that it is more efficiently incorporated than ATP and two other nucleotide analogs. Once incorporated at position i , the inhibitor caused RNA synthesis arrest at position i + 3. Hence, the likely mechanism of action is delayed RNA chain termination. The additional three nucleotides may protect the inhibitor from excision by the viral 3'-5' exonuclease activity. Together, these results help to explain the high potency of RDV against RNA viruses in cell-based assays., (© 2020 Gordon et al.)

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. [ABSTRACT FROM AUTHOR]

For many viruses, RNA is the holder of genetic information and serves as the template for both replication and translation. While host and viral proteins play important roles in viral decision‐making, the extent to which viral RNA (vRNA) actively participates in translation and replication might be surprising. Here, the focus is on flaviviruses, which include common human scourges such as dengue, West Nile, and Zika viruses, from an RNA‐centric viewpoint. In reviewing more recent findings, an attempt is made to fill knowledge gaps and revisit some canonical views of vRNA structures involved in replication. In particular, alternative views are offered on the nature of the flaviviral promoter and genome cyclization, and the feasibility of refining in vitro‐derived models with modern RNA probing and sequencing methods is pointed out. By tracing vRNA structures from translation through encapsidation, a dynamic molecule closely involved in the self‐regulation of viral replication is revealed. [ABSTRACT FROM AUTHOR]

RNA viruses synthesize new genomes in the infected host thanks to dedicated, virally-encoded RNA-dependent RNA polymerases (RdRps). As such, these enzymes are prime targets for antiviral therapy, as has recently been demonstrated for hepatitis C virus (HCV). However, peculiarities in the architecture and dynamics of RdRps raise fundamental questions about access to their active site during RNA polymerization. Here, we used molecular modeling and molecular dynamics simulations, starting from the available crystal structures of HCV NS5B in ternary complex with template-primer duplexes and nucleotides, to address the question of ribonucleotide entry into the active site of viral RdRp. Tracing the possible passage of incoming UTP or GTP through the RdRp-specific entry tunnel, we found two successive checkpoints that regulate nucleotide traffic to the active site. We observed that a magnesium-bound nucleotide first binds next to the tunnel entry, and interactions with the triphosphate moiety orient it such that its base moiety enters first. Dynamics of RdRp motifs F1 + F3 then allow the nucleotide to interrogate the RNA template base prior to nucleotide insertion into the active site. These dynamics are finely regulated by a second magnesium dication, thus coordinating the entry of a magnesium-bound nucleotide with shuttling of the second magnesium necessary for the two-metal ion catalysis. The findings of our work suggest that at least some of these features are general to viral RdRps and provide further details on the original nucleotide selection mechanism operating in RdRps of RNA viruses., (© 2019 Ben Ouirane et al.)

Dengue virus, an ∼10.7-kb positive-sense RNA virus, is the most common arthropod-communicated pathogen in the world. Despite dengue's clear epidemiological importance, mechanisms for its replication remain elusive. Here, we probed the entire dengue genome for interactions with viral RNA-dependent RNA polymerase (RdRp), and we identified the dominant interaction as a loop-forming ACAG motif in the 3' positive-stranded terminus, complicating the prevailing model of replication. A subset of interactions coincides with known flaviviral recombination sites inside the viral protein-coding region. Specific recognition of the RNA element occurs via an arginine patch in the C-terminal thumb domain of RdRp. We also show that the highly conserved nature of the consensus RNA motif may relate to its tolerance to various mutations in the interacting region of RdRp. Disruption of the interaction resulted in loss of viral replication ability in cells. This unique RdRp-RNA interface is found throughout flaviviruses, implying possibilities for broad disease interventions., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Nodaviruses are a family of positive-stranded RNA viruses with a bipartite genome of RNAs. In nodaviruses, genomic RNA1 encodes protein A, which is recognized as an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for its RNA replication. Although nodaviral RNA replication has been studied in considerable detail, and nodaviruses are well recognized models for investigating viral RNA replication, the mechanism(s) governing the initiation of nodaviral RNA synthesis have not been determined. In this study, we characterized the RdRP activity of Wuhan nodavirus (WhNV) protein A in detail and determined that this nodaviral protein A initiates RNA synthesis via a de novo mechanism, and this RNA synthesis initiation could be independent of other viral or cellular factors. Moreover, we uncovered that WhNV protein A contains a terminal nucleotidyltransferase (TNTase) activity, which is the first time such an activity has been identified in nodaviruses. We subsequently found that the TNTase activity could function in vitro to repair the 3' initiation site, which may be digested by cellular exonucleases, to ensure the efficiency and accuracy of viral RNA synthesis initiation. Furthermore, we determined the cis-acting elements for RdRP or TNTase activity at the 3'-end of positive or negative strand RNA1. Taken together, our data establish the de novo synthesis initiation mechanism and the TNTase activity of WhNV protein A, and this work represents an important advance toward understanding the mechanism(s) of nodaviral RNA replication.