Template-primer RNA Research Articles

RNA-DEPENDENT RNA POLYMERASE (RDRP) INHIBITOR DRUGS AGAINST SARS-COV-2: A MOLECULAR DOCKING STUDY

Objective: SARS-CoV-2 associated viral pandemic was first reported in Wuhan, China, in December 2019. Due to the rapid increase in its pathogenicity, SARS-CoV-2 was declared a global pandemic by WHO on March 11, 2020. For that reason, determining the most attractive viral protein targets became a must. One of the most important target proteins is SARS-COV-2 RNA-dependent RNA polymerase (RdRp) on which COVID-19 depends in its replication process. This study aimed to examine the possible interactions between RdRp and the most promising RdRp nucleoside inhibitors especially Purine nucleoside analogs, to detect the most important residues that commonly interact with RdRp's inhibitors and to investigate whether if there any mutations have been observed so far in these residues or not. Material and Method: Molecular docking studies were carried out using AutoDock Vina between SARS-CoV-2 RdRp and drugs approved against different viral RdRps (Galidesivir, Remdesivir, Ribavirin, Sofosbuvir, and Favipiravir) as well as physiological nucleotides (ATP and GTP). Based on the obtained results, a detailed surface-interaction analysis was also performed using Pymol and Discovery Studio Visualizer software for the models that exhibited the most suitable location and configuration in space. Result and Discussion: All the tested molecules were able to bind to SARS-CoV-2 RdRp successfully. Also, they all commonly interact with 9 different amino acids (Arg553, Arg555, Asp618, Asp623, Ser682, Asn691, Ser759, Asp760, and Asp761), and 3 different Template-primer RNA nucleotides (U10, A11, and U20) causing inhibition of viral RdRp via non obligate RNA chain termination.

Exploring the Mechanisms of Antiviral Therapeutics: A 3D Model of SARS‐CoV‐2 RdRp in Complex with Remdesivir

The recently discovered severe acute respiratory syndrome coronavirus 2 (SARS‐Cov‐2) causes coronavirus disease 2019 (COVID‐19), a respiratory disease affecting the human population worldwide. SARS‐CoV‐2 replication requires the RNA dependent RNA polymerase (RdRp) complex, composed of non‐structural proteins (nsp) 7, 8, and 12. Nsp 7 and 8 function as a primase, whereas nsp12 functions as RdRp for replication and transcription. This polymerase is the target of the antiviral drug remdesivir, an adenosine monophosphate analog. During RNA replication catalyzed by RdRp, remdesivir is covalently attached to the growing RNA strand, resulting in chain termination. Here, we designed a 3‐dimensional (3D) model of the SARS‐COV‐2 nsp12‐nsp7‐nsp8 RdRp complex bound to template‐primer double‐stranded (ds)RNA and remdesivir using Jmol and PDB 7BV2 cryo‐EM structure (Yin et al., 2020). The 3D model shows the nsp12 subunit bound to dsRNA template and growing RNA strand that forms a corkscrew‐like structure within the center channel. The model highlights specific residues aspartic acid 760, valine557, and serine861 within the active site and the interactions of the template‐primer RNA strands with remdesivir. An additional 3D model illustrates the structural similarity of remdesivir to adenosine monophosphate. These 3D models enable students to visualize complex biomolecules and understand mechanisms of therapeutics.

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

Since no effective therapy exists, we aimed to test existing HIV antivirals for combination treatment of Coronavirus disease 19 (COVID-19). The crystal structures of SARS-CoV-2 main protein (Mpro) (PDB ID: 6Y2F) and SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) (PDB ID: 7BV2) both available from Protein Data Bank were used in the study. Automated Docking by using blind and standard method both on Mpro and RdRp bound to the modified template-primer RNA was performed with AutoDock 4.2.6 program suite. Lamarckian genetic algorithm (LGA) was used for structures docking. All inhibitors were docked with all bonds completely free to rotate. Our molecular docking findings suggest that lopinavir, ritonavir, darunavir, and atazanavir activated interactions with the key binding sites of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) protease with a better inhibition constant (Ki) for lopinavir, ritonavir, and darunavir. Furthermore, we evidenced the ability of remdesivir, tenofovir, emtricitabine, and lamivudine to be incorporated in SARS-CoV-2 RdRp in the same protein pocket where poses the corresponding natural nucleoside substrates with comparable Ki and activating similar interactions. In principle, the four antiviral nucleotides might be used effectively against SARS-CoV-2. The combination of a protease inhibitor and two nucleoside analogues, drugs widely used to treat HIV infection, could be evaluated in clinical trials for the treatment of COVID-19.

Mechanistic insight on the remdesivir binding to RNA-Dependent RNA polymerase (RdRp) of SARS-cov-2

The RNA-dependent RNA polymerase (RdRp) is a key enzyme which regulates the viral replication of SARS-CoV-2. Remdesivir (RDV) is clinically used drug which targets RdRp, however its mechanism of action remains elusive. This study aims to find out the binding dynamics of active Remdesivir-triphosphate (RDV-TP) to RdRp by means of molecular dynamics (MD) simulation. We built a homology model of RdRp along with RNA and manganese ion using RdRp hepatitis C virus and recent SARS-CoV-2 structures. We determined that the model was stable during the 500 ns MD simulations. We then employed the model to study the binding of RDV-TP to RdRp during three independent 500 ns MD simulations. It was revealed that the interactions of protein and template-primer RNA were dominated by salt bridge interactions with phosphate groups of RNA, while interactions with base pairs of template-primer RNA were minimal. The binding of RDV-TP showed that the position of phosphate groups was at the entry of the NTP channel and it was stabilized by the interactions with K551, R553, and K621, while the adenosine group on RDV-TP was pairing with U2 of the template strand. The manganese ion was located close to D618, D760, and D761, and helps in stabilization of the phosphate groups of RDV-TP. Further we identified three hits from the natural product database that pose similar to RDV-TP while having lower binding energies than that of RDV-TP, and that SN00359915 had binding free energy about three times lower than that of RDV-TP.

Architecture of a SARS-CoV-2 mini replication and transcription complex

Non-structural proteins (nsp) constitute the SARS-CoV-2 replication and transcription complex (RTC) to play a pivotal role in the virus life cycle. Here we determine the atomic structure of a SARS-CoV-2 mini RTC, assembled by viral RNA-dependent RNA polymerase (RdRp, nsp12) with a template-primer RNA, nsp7 and nsp8, and two helicase molecules (nsp13-1 and nsp13-2), by cryo-electron microscopy. Two groups of mini RTCs with different conformations of nsp13-1 are identified. In both of them, nsp13-1 stabilizes overall architecture of the mini RTC by contacting with nsp13-2, which anchors the 5′-extension of RNA template, as well as interacting with nsp7-nsp8-nsp12-RNA. Orientation shifts of nsp13-1 results in its variable interactions with other components in two forms of mini RTC. The mutations on nsp13-1:nsp12 and nsp13-1:nsp13-2 interfaces prohibit the enhancement of helicase activity achieved by mini RTCs. These results provide an insight into how helicase couples with polymerase to facilitate its function in virus replication and transcription.

Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5′ extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.

Mutant Viral Polymerase in the Transition of Virus to Error Catastrophe Identifies a Critical Site for RNA Binding

A foot-and-mouth disease virus (FMDV) polymerase (3D) with amino acid replacements G118D, V239M and G373D (triple DMD mutant) was obtained from a molecular clone derived from a virus population treated with ribavirin, in the transition to error catastrophe (virus extinction through lethal mutagenesis). DMD 3D was expressed in Escherichia coli, purified, and its activity compared with that of wild-type enzyme and mutant enzymes with either replacement G118D, G118A or D338A (the latter affecting the catalytic motif YGDD), generated by site-directed mutagenesis. No differences among the enzymes were noted in their interaction with monoclonal antibodies specific for the FMDV polymerase. Mutant enzymes with G118D or G118A showed a 100-fold decrease in polymerization activity relative to wild-type 3D, using poly(A)/oligo(dT)15 and poly(A)/VPg as template-primers, under several reaction conditions. As expected, the activity of 3D with D338A was undetectable (<0.01 times the value for wild-type 3D). DMD and the G118 mutants showed impaired binding to template-primer RNA whereas the D338A mutant showed a binding similar to wild-type 3D. Transfection of cells with FMDV RNA encoding DMD 3D resulted in selection of revertant viruses that maintained only substitutions V239M and G373D. Consistently, when infectious transcripts encoded 3D with either G118D, G118A or D338A, viruses with reversions to the wild-type sequence were isolated. The implication of G118 in template-primer binding is supported by the location of this residue in the template-binding groove of the FMDV polymerase. In addition to identifying an amino acid residue that is critical for the binding of polymerase to RNA, the results document the presence of defective genomes in the transition of virus to error catastrophe.

Structure of Foot-and-Mouth Disease Virus RNA-dependent RNA Polymerase and Its Complex with a Template-Primer RNA

Genome replication in picornaviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. The enzyme performs this operation, together with other viral and probably host proteins, in the cytoplasm of their host cells. The crystal structure of the 3D polymerase of foot-and-mouth disease virus, one of the most important animal pathogens, has been determined unliganded and bound to a template-primer RNA decanucleotide. The enzyme folds in the characteristic fingers, palm and thumb subdomains, with the presence of an NH2-terminal segment that encircles the active site. In the complex, several conserved amino acid side chains bind to the template-primer, likely mediating the initiation of RNA synthesis. The structure provides essential information for studies on RNA replication and the design of antiviral compounds.

Utilization of mammalian 70S RNA by a purified reverse transcriptase from human myelocytic leukemic cells

A DNA polymerase activity present in the 60,000 × g pellet of human acute myelocytic leukemic blood cells catalyzes an endogenous RNA-dependent (RNase sensitive) DNA synthesis. After purification, this enzyme transcribes heteropolymeric regions of 70S RNA from various RNA tumor viruses but prefers mammalian over avian viral RNA. DNA transcribed from the viral RNA is, like that from RNA tumor virus reverse transcriptase, hydrogen bonded and covalently attached to the RNA template-primer. The DNA transcript, after alkaline hydrolysis, hybridizes specifically to the template-primer RNA. This activity is not decreased in the presence of DNA polymerases from normal cells, suggesting that the inability of the latter polymerases to catalyze transcription of viral RNA is not due to the presence of inhibitor(s). This DNA polymerase is easily distinguished from a terminal addition deoxynucleotidyl transferase and has previously been shown to be immunologically closely related to primate type-C virus reverse transcriptase.

RNA-Directed DNA Synthesis and RNA Tumor Viruses

Publisher Summary The discovery of RNA-directed DNA synthesis in disrupted virions of RNA tumor viruses added strong support to the hypothesis that information transfer from RNA to DNA exists in biological system. The chapter discusses the properties of the endogenous reaction carried out by the virion DNA polymerase. To study the endogenous reaction disrupted, virions are incubated with substrates in the absence of any added template and synthesis of DNA is observed using the RNA present in the virions as template. The chapter also discusses the general implications of RNA-directed DNA synthesis in relation to tumor viruses, neoplastic cells, and normal cells. It is hypothesized that the leukovirus RNA-directed DNA polymerase activity is an integral part of the ribonucleoprotein core of the virions. The cores are synthesized in cells as precursor particles and then are incorporated into complete virions when the virions are assembled by budding at the cell surface. This core enzyme system contains not only the template-primer RNA, a DNA polymerase that can transfer information from RNA to double-stranded DNA, but ancillary enzymes, such as polynucleotide ligase and nucleases, which may aid in integrating the viral information with cellular DNA. This suggests that the virion enzyme activity is related to normal cellular DNA polymerases, and that there are homologies between the amino acid sequences of the viral enzyme and normal cellular enzymes. The relationship of RNA-directed DNA synthesis to neoplasia depends upon the relationship of RNA tumor viruses to neoplasia, which is supported by three general hypotheses: the provirus model, the oncogene model, and the protovirus model.