Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase.

Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here, we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas there are highly conserved residues in nsp12, positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanism of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription and replication machinery.

Point mutations at specific sites of the nsp12-nsp8 interface dramatically affect the RNA polymerization activity of SARS-CoV-2.

In a recent characterization of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variability present in 30 diagnostic samples from patients of the first COVID-19 pandemic wave, 41 amino acid substitutions were documented in the RNA-dependent RNA polymerase (RdRp) nsp12. Eight substitutions were selected in this work to determine whether they had an impact on the RdRp activity of the SARS-CoV-2 nsp12-nsp8-nsp7 replication complex. Three of these substitutions were found around the polymerase central cavity, in the template entry channel (D499G and M668V), and within the motif B (V560A), and they showed polymerization rates similar to the wild type RdRp. The remaining five mutations (P323L, L372F, L372P, V373A, and L527H) were placed near the nsp12-nsp8F contact surface; residues L372, V373, and L527 participated in a large hydrophobic cluster involving contacts between two helices in the nsp12 fingers and the long α-helix of nsp8F. The presence of any of these five amino acid substitutions resulted in important alterations in the RNA polymerization activity. Comparative primer elongation assays showed different behavior depending on the hydrophobicity of their side chains. The substitution of L by the bulkier F side chain at position 372 slightly promoted RdRp activity. However, this activity was dramatically reduced with the L372P, and L527H mutations, and to a lesser extent with V373A, all of which weaken the hydrophobic interactions within the cluster. Additional mutations, specifically designed to disrupt the nsp12-nsp8F interactions (nsp12-V330S, nsp12-V341S, and nsp8-R111A/D112A), also resulted in an impaired RdRp activity, further illustrating the importance of this contact interface in the regulation of RNA synthesis.

A Biochemical and Biophysical Analysis of the Interaction of nsp9 with nsp12 from SARS-CoV-2-Implications for Future Drug Discovery Efforts.

The ongoing global pandemic of the coronavirus 2019 (COVID-19) disease is caused by the virus SARS-CoV-2, with very few highly effective antiviral treatments currently available. The machinery responsible for the replication and transcription of viral RNA during infection is made up of several important proteins. Two of these are nsp12, the catalytic subunit of the viral polymerase, and nsp9, a cofactor of nsp12 involved in the capping and priming of viral RNA. While several recent studies have determined the structural details of the interaction of nsp9 with nsp12 in the context of RNA capping, very few biochemical or biophysical details are currently available. In this study, we have used a combination of surface plasmon resonance (SPR) experiments, size exclusion chromatography (SEC) experiments, and biochemical assays to identify specific nsp9 residues that are critical for nsp12 binding as well as RNAylation, both of which are essential for the RNA capping process. Our data indicate that nsp9 dimerization is unlikely to play a significant functional role in the virus. We confirm that a set of recently discovered antiviral peptides inhibit nsp9-nsp12 interaction by specifically binding to nsp9; however, we find that these peptides do not impact RNAylation. In summary, our results have important implications for future drug discovery efforts to combat SARS-CoV-2 and any newly emerging coronaviruses.

PSMB1 Inhibits the Replication of Porcine Reproductive and Respiratory Syndrome Virus by Recruiting NBR1 To Degrade Nonstructural Protein 12 by Autophagy.

The nonstructural proteins (Nsps) of porcine reproductive and respiratory syndrome virus (PRRSV) play essential roles in virus replication-a multistep process that requires the participation of host factors. It is of great significance for the development of antiviral drugs to characterize the host proteins that interact with PRRSV Nsps and their functions in PRRSV replication. Here, we determined that proteasome subunit ß type 1 (PSMB1) interacted with viral Nsp12 to inhibit PRRSV replication in target and permissive cells. PSMB1 could be downregulated by PRRSV infection through interaction with the transcription factor EBF1. Proteasome and autophagy inhibitor assays showed that PSMB1 was regulated by the autophagic pathway to degrade Nsp12. Cotransfection of PSMB1 and Nsp12 increased the level of intracellular autophagy; both molecules were colocated in lysosomes. We also found that the selective autophagy cargo receptor protein NBR1 and E3 ubiquitin ligase STUB1 interacted with PSMB1 and Nsp12, respectively, in the autophagic degradation of Nsp12. Furthermore, the degradation of Nsp12 by PSMB1 was mainly dependent on the ubiquitination of Nsp12 at lysine site 130. Our results indicate for the first time that PSMB1 is an anti-PRRSV host protein that inhibits the replication of PRRSV by degradation of Nsp12 through the selective autophagy pathway. IMPORTANCE PRRS is a major threat to the global pig industry and urgently requires an effective and sustainable control strategy. PRRSV Nsps have important roles in viral RNA synthesis, proteinase activity, induction of replication-associated membrane rearrangements, replicative endoribonuclease activity, determination of virulence, and regulation of host immune response. Research associated with PRRSV Nsps can provide vital guidance to modify the PRRSV genome through reverse genetics in the development of vaccines and diagnostics. The function of Nsp12, which generally plays essential roles in virus replication, remains unclear. We demonstrated that PSMB1 interacted with and degraded Nsp12 through an autophagic pathway to inhibit PRRSV replication. Our data confirmed a novel antiviral function of PSMB1 and allowed us to elaborate on the roles of Nsp12 in PRRSV pathogenesis. These findings suggest a valid and highly conserved candidate target for the development of novel therapies and more effective vaccines and demonstrate the complex cross talk between selective autophagy and PRRSV infection.

SARS-CoV-2 NSP12 associates with TRiC and the P323L substitution acts as a host adaption.

IMPORTANCE: SARS-CoV-2 has caused a worldwide health and economic crisis. During the course of the pandemic, genetic changes occurred in the virus, which have resulted in new properties of the virus-particularly around gains in transmission and the ability to partially evade either natural or vaccine-acquired immunity. Some of these viruses have been labeled Variants of Concern (VoCs). At the root of all VoCs are two mutations, one in the viral spike protein that has been very well characterized and the other in the virus polymerase (NSP12). This is the viral protein responsible for replicating the genome. We show that NSP12 associates with host cell proteins that act as a scaffold to facilitate the function of this protein. Furthermore, we found that different variants of NSP12 interact with host cell proteins in subtle and different ways, which affect function.

Sequence analysis of the Spike, RNA-dependent RNA polymerase, and protease genes reveals a distinct evolutionary pattern of SARS-CoV-2 variants circulating in Yogyakarta and Central Java provinces, Indonesia.

During the Covid-19 pandemic, the resurgence of SARS-CoV-2 was due to the development of novel variants of concern (VOC). Thus, genomic surveillance is essential to monitor continuing evolution of SARS-CoV-2 and to track the emergence of novel variants. In this study, we performed phylogenetic, mutation, and selection pressure analyses of the Spike, nsp12, nsp3, and nsp5 genes of SARS-CoV-2 isolates circulating in Yogyakarta and Central Java provinces, Indonesia from May 2021 to February 2022. Various bioinformatics tools were employed to investigate the evolutionary dynamics of distinct SARS-CoV-2 isolates. During the study period, 213 and 139 isolates of Omicron and Delta variants were identified, respectively. Particularly in the Spike gene, mutations were significantly more abundant in Omicron than in Delta variants. Consistently, in all of four genes studied, the substitution rates of Omicron were higher than that of Delta variants, especially in the Spike and nsp12 genes. In addition, selective pressure analysis revealed several sites that were positively selected in particular genes, implying that these sites were functionally essential for virus evolution. In conclusion, our study demonstrated a distinct evolutionary pattern of SARS-CoV-2 variants circulating in Yogyakarta and Central Java provinces, Indonesia.

Redefining pseudokinases: A look at the untapped enzymatic potential of pseudokinases.

Catalytically inactive kinases, known as pseudokinases, are conserved in all three domains of life. Due to the lack of catalytic residues, pseudokinases are considered to act as allosteric regulators and scaffolding proteins with no enzymatic function. However, since these "dead" kinases are conserved along with their active counterparts, a role for pseudokinases may have been overlooked. In this review, we will discuss the recently characterized pseudokinases Selenoprotein O, Legionella effector SidJ, and the SARS-CoV2 protein nsp12 which catalyze AMPylation, glutamylation, and RNAylation, respectively. These studies provide structural and mechanistic insight into the versatility and diversity of the kinase fold.

Development and characterization of a new monoclonal antibody against SARS-CoV-2 NSP12 (RdRp).

SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication.

In Vitro Selection of Remdesivir-Resistant SARS-CoV-2 Demonstrates High Barrier to Resistance.

In vitro selection of remdesivir-resistant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed the emergence of a V166L substitution, located outside of the polymerase active site of the Nsp12 protein, after 9 passages of a single lineage. V166L remained the only Nsp12 substitution after 17 passages (10 µM remdesivir), conferring a 2.3-fold increase in 50% effective concentration (EC50). When V166L was introduced into a recombinant SARS-CoV-2 virus, a 1.5-fold increase in EC50 was observed, indicating a high in vitro barrier to remdesivir resistance.

Remdesivir and GS-441524 Retain Antiviral Activity against Delta, Omicron, and Other Emergent SARS-CoV-2 Variants.

Genetic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence and rapid spread of multiple variants throughout the pandemic, of which Omicron is currently the predominant variant circulating worldwide. SARS-CoV-2 variants of concern/variants of interest (VOC/VOI) have evidence of increased viral transmission, disease severity, or decreased effectiveness of vaccines and neutralizing antibodies. Remdesivir (RDV [VEKLURY]) is a nucleoside analog prodrug and the first FDA-approved antiviral treatment of COVID-19. Here, we present a comprehensive antiviral activity assessment of RDV and its parent nucleoside, GS-441524, against 10 current and former SARS-CoV-2 VOC/VOI clinical isolates by nucleoprotein enzyme-linked immunosorbent assay (ELISA) and plaque reduction assay. Delta and Omicron variants remained susceptible to RDV and GS-441524, with 50% effective concentration (EC50) values 0.30- to 0.62-fold of those observed against the ancestral WA1 isolate. All other tested variants exhibited EC50 values ranging from 0.13- to 2.3-fold of the observed EC50 values against WA1. Analysis of nearly 6 million publicly available variant isolate sequences confirmed that Nsp12, the RNA-dependent RNA polymerase (RdRp) target of RDV and GS-441524, is highly conserved across variants, with only 2 prevalent changes (P323L and G671S). Using recombinant viruses, both RDV and GS-441524 retained potency against all viruses containing frequent variant substitutions or their combination. Taken together, these results highlight the conserved nature of SARS-CoV-2 Nsp12 and provide evidence of sustained SARS-CoV-2 antiviral activity of RDV and GS-441524 across the tested variants. The observed pan-variant activity of RDV supports its continued use for the treatment of COVID-19 regardless of the SARS-CoV-2 variant.

SARS-CoV-2 NSP12 Protein Is Not an Interferon-ß Antagonist.

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is bringing an unprecedented health crisis to the world. To date, our understanding of the interaction between SARS-CoV-2 and host innate immunity is still limited. Previous studies reported that SARS-CoV-2 nonstructural protein 12 (NSP12) was able to suppress interferon-ß (IFN-ß) activation in IFN-ß promoter luciferase reporter assays, which provided insights into the pathogenesis of COVID-19. In this study, we demonstrated that IFN-ß promoter-mediated luciferase activity was reduced during coexpression of NSP12. However, we could show NSP12 did not affect IRF3 or NF-κB activation. Moreover, IFN-ß production induced by Sendai virus (SeV) infection or other stimulus was not affected by NSP12 at mRNA or protein level. Additionally, the type I IFN signaling pathway was not affected by NSP12, as demonstrated by the expression of interferon-stimulated genes (ISGs). Further experiments revealed that different experiment systems, including protein tags and plasmid backbones, could affect the readouts of IFN-ß promoter luciferase assays. In conclusion, unlike as previously reported, our study showed SARS-CoV-2 NSP12 protein is not an IFN-ß antagonist. It also rings the alarm on the general usage of luciferase reporter assays in studying SARS-CoV-2. IMPORTANCE Previous studies investigated the interaction between SARS-CoV-2 viral proteins and interferon signaling and proposed that several SARS-CoV-2 viral proteins, including NSP12, could suppress IFN-ß activation. However, most of these results were generated from IFN-ß promoter luciferase reporter assay and have not been validated functionally. In our study, we found that, although NSP12 could suppress IFN-ß promoter luciferase activity, it showed no inhibitory effect on IFN-ß production or its downstream signaling. Further study revealed that contradictory results could be generated from different experiment systems. On one hand, we demonstrated that SARS-CoV-2 NSP12 could not suppress IFN-ß signaling. On the other hand, our study suggests that caution needs to be taken with the interpretation of SARS-CoV-2-related luciferase assays.

Development of novel monoclonal antibodies against nsp12 of SARS-CoV-2.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a global pandemic of coronavirus disease 19. Coronaviruses, including SARS-CoV-2, use RNA-dependent RNA polymerase (RdRP) for viral replication and transcription. Since RdRP is a promising therapeutic target for infection of SARS-CoV-2, it would be beneficial to develop new experimental tools for analysis of the RdRP reaction of SARS-CoV-2. Here, we succeeded to develop novel mouse monoclonal antibodies (mAbs) that recognize SARS-CoV-2 nsp12, catalytic subunit of the RdRP. These anti-nsp12 mAbs, RdMab-2, -13, and -20, specifically recognize SARS-CoV-2 nsp12 by western blotting analysis, while they exhibit less or no cross-reactivity to SARS-CoV nsp12. In addition, SARS-CoV-2 nsp12 was successfully immunoprecipitated using RdMab-2 from lysates of cells overexpressing SARS-CoV-2 nsp12. RdMab-2 was able to detect SARS-CoV-2 nsp12 transiently expressed in established culture cells such as HEK293T cells by indirect immunofluorescence technique. These novel mAbs against SARS-CoV-2 nsp12 are useful to elucidate the RdRP reaction of SARS-CoV-2 and biological cell response against it.

Plant-derived exosomal microRNAs inhibit lung inflammation induced by exosomes SARS-CoV-2 Nsp12.

Lung inflammation is a hallmark of coronavirus disease 2019 (COVID-19). In this study, we show that mice develop inflamed lung tissue after being administered exosomes released from the lung epithelial cells exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Nsp12 and Nsp13 (exosomesNsp12Nsp13). Mechanistically, we show that exosomesNsp12Nsp13 are taken up by lung macrophages, leading to activation of nuclear factor κB (NF-κB) and the subsequent induction of an array of inflammatory cytokines. Induction of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß from exosomesNsp12Nsp13-activated lung macrophages contributes to inducing apoptosis in lung epithelial cells. Induction of exosomesNsp12Nsp13-mediated lung inflammation was abolished with ginger exosome-like nanoparticle (GELN) microRNA (miRNA aly-miR396a-5p. The role of GELNs in inhibition of the SARS-CoV-2-induced cytopathic effect (CPE) was further demonstrated via GELN aly-miR396a-5p- and rlcv-miR-rL1-28-3p-mediated inhibition of expression of Nsp12 and spike genes, respectively. Taken together, our results reveal exosomesNsp12Nsp13 as potentially important contributors to the development of lung inflammation, and GELNs are a potential therapeutic agent to treat COVID-19.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase.

The coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologues in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified three novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.

Inspection on the Mechanism of SARS-CoV-2 Inhibition by Penciclovir: A Molecular Dynamic Study.

In recent years, humanity has had to face a critical pandemic due to SARS-CoV-2. In the rapid search for effective drugs against this RNA-positive virus, the repurposing of already existing nucleotide/nucleoside analogs able to stop RNA replication by inhibiting the RNA-dependent RNA polymerase enzyme has been evaluated. In this process, a valid contribution has been the use of in silico experiments, which allow for a rapid evaluation of the possible effectiveness of the proposed drugs. Here we propose a molecular dynamic study to provide insight into the inhibition mechanism of Penciclovir, a nucleotide analog on the RNA-dependent RNA polymerase enzyme. Besides the presented results, in this article, for the first time, molecular dynamic simulations have been performed considering not only the RNA-dependent RNA polymerase protein, but also its cofactors (fundamental for RNA replication) and double-strand RNA.

Structural Homology-Based Drug Repurposing Approach for Targeting NSP12 SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2, also known as SARS-CoV-2, is the causative agent of the COVID-19 global pandemic. SARS-CoV-2 has a highly conserved non-structural protein 12 (NSP-12) involved in RNA-dependent RNA polymerase (RdRp) activity. For the identification of potential inhibitors for NSP-12, computational approaches such as the identification of homologous proteins that have been previously targeted by FDA-approved antivirals can be employed. Herein, homologous proteins of NSP-12 were retrieved from Protein DataBank (PDB) and the evolutionary conserved sequence and structure similarity of the active site of the RdRp domain of NSP-12 was characterized. The identified homologous structures of NSP-12 belonged to four viral families: Coronaviridae, Flaviviridae, Picornaviridae, and Caliciviridae, and shared evolutionary conserved relationships. The multiple sequences and structural alignment of homologous structures showed highly conserved amino acid residues that were located at the active site of the RdRp domain of NSP-12. The conserved active site of the RdRp domain of NSP-12 was evaluated for binding affinity with the FDA-approved antivirals, i.e., Sofosbuvir and Dasabuvir in a molecular docking study. The molecular docking of Sofosbuvir and Dasabuvir with the active site that contains conserved motifs (motif A-G) of the RdRp domain of NSP-12 revealed significant binding affinity. Furthermore, MD simulation also inferred the potency of Sofosbuvir and Dasabuvir. In conclusion, targeting the active site of the RdRp domain of NSP-12 with Dasabuvir and Sofosbuvir might reduce viral replication and pathogenicity and could be further studied for the treatment of SARS-CoV-2.

Repurposing nonnucleoside antivirals against SARS-CoV2 NSP12 (RNA dependent RNA polymerase): In silico-molecular insight.

The pandemic of SARS-CoV-2 has necessitated expedited research efforts towards finding potential antiviral targets and drug development measures. While new drug discovery is time consuming, drug repurposing has been a promising area for elaborate virtual screening and identification of existing FDA approved drugs that could possibly be used for targeting against functions of various proteins of SARS-CoV-2 virus. RNA dependent RNA polymerase (RdRp) is an important enzyme for the virus that mediates replication of the viral RNA. Inhibition of RdRp could inhibit viral RNA replication and thus new virus particle production. Here, we screened non-nucleoside antivirals and found three out of them to be strongest in binding to RdRp out of which two retained binding even using molecular dynamic simulations. We propose these two drugs as potential RdRp inhibitors which need further in-depth testing.

SARS-CoV-2 and SARS-CoV: Virtual screening of potential inhibitors targeting RNA-dependent RNA polymerase activity (NSP12).

Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the harm caused by coronaviruses to the world cannot be underestimated. Recently, a novel coronavirus (severe acute respiratory syndrome coronavirus-2 [SARS-CoV-2]) initially found to trigger human severe respiratory illness in Wuhan City of China in 2019, has infected more than six million people worldwide by 21 June 2020, and which has been recognized as a public health emergency of international concern as well. And the virus has spread to more than 200 countries around the world. However, the effective drug has not yet been officially licensed or approved to treat SARS-Cov-2 and SARS-Cov infection. NSP12-NSP7-NSP8 complex of SARS-CoV-2 or SARS-CoV, essential for viral replication and transcription, is generally regarded as a potential target to fight against the virus. According to the NSP12-NSP7-NSP8 complex (PDB ID: 7BW4) structure of SARS-CoV-2 and the NSP12-NSP7-NSP8 complex (PDB ID: 6NUR) structure of SARS-CoV, NSP12-NSP7 interface model, and NSP12-NSP8 interface model were established for virtual screening in the present study. Eight compounds (Nilotinib, Saquinavir, Tipranavir, Lonafarnib, Tegobuvir, Olysio, Filibuvir, and Cepharanthine) were selected for binding free energy calculations based on virtual screening and docking scores. All eight compounds can combine well with NSP12-NSP7-NSP8 in the crystal structure, providing drug candidates for the treatment and prevention of coronavirus disease 2019 and SARS.

Structures of SARS-CoV-2 RNA-Binding Proteins and Therapeutic Targets.

BACKGROUND: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) epidemic has resulted in thousands of infections and deaths worldwide. Several therapies are currently undergoing clinical trials for the treatment of SARS-CoV-2 infection. However, the development of new drugs and the repositioning of existing drugs can only be achieved after the identification of potential therapeutic targets within structures, as this strategy provides the most precise solution for developing treatments for sudden epidemic infectious diseases. SUMMARY: In the current investigation, crystal and cryo-electron microscopy structures encoded by the SARS-CoV-2 genome were systematically examined for the identification of potential drug targets. These structures include nonstructural proteins (Nsp-9; Nsp-12; and Nsp-15), nucleocapsid (N) proteins, and the main protease (Mpro). Key Message: The structural information reveals the presence of many potential alternative therapeutic targets, primarily involved in interaction between N protein and Nsp3, forming replication-transcription complexes (RTCs) which might be a potential drug target for effective control of current SARS-CoV-2 pandemic. RTCs consist of 16 nonstructural proteins (Nsp1-16) that play the most essential role in the synthesis of viral RNA. Targeting the physical linkage between the envelope and single-stranded positive RNA, a process facilitated by matrix proteins may provide a good alternative strategy. Our current study provides useful information for the development of new lead compounds against SARS-CoV-2 infections.

An in silico approach to target RNA-dependent RNA polymerase of COVID-19 with naturally occurring phytochemicals.

The novel coronavirus disease 2019 (COVID-19) was firstly reported from Wuhan city of China and found as a highly contagious, transmittable and pathogenic viral infection. The World Health Organization declared COVID-19 as a pandemic since its emergence from China. The RNA-dependent RNA polymerase (nsp-12) is a complex with nsp-7 and nsp-8 cofactors and is a major constituent of viral replication and RNA synthesis machinery. In the current study, the RdRp of the virus was selected as a receptor protein for computational drug discovery. Computational homology modelling was done in order to find the hidden secondary structures and structural assessment of the viral protein to target them via antiviral drugs. The study was based on molecular docking of different phytochemicals to check their potentials against viral replicative proteins. Out of 200 ligands used in this study from different plants, the best ten were selected based on drug discovery parameters such as S-score, ligand interactions, hydrophobic interactions and druglikeness. The ten best selected ligands were found to be verbenalin, epigallocatechin, swertisin, nobiletin, pinoresinol, caftaric acid, hesperetin, islandicin, neochlorogenic acid and sesamin that exploit the potency as antagonists of viral protein. Among binding interactions of all ligands, Arg339 centred as the main interacting residue among almost all the ligands. Till now, many antiviral agents have shown potency in only mild cases of SARS-CoV-2, but no effective drug has been found for critical pulmonary cases. In clinical trials, many broad-spectrum antiviral agents have been still in trial periods of testing against SARS-CoV-2. Till date, no effective drug or vaccine has been validated with significant efficacy and potency against the SARS-CoV-2; therefore, there is an urgent need to design effective vaccine against nCoV-19 infection.