RESUMEN

The coronavirus disease 2019 (COVID-19), the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents the most disastrous infectious disease pandemic of the past century. As a member of the Betacoronavirus genus, the SARS-CoV-2 genome encodes a total of 29 proteins. The spike protein, RNA-dependent RNA polymerase, and proteases play crucial roles in the virus replication process and are promising targets for drug development. In recent years, structural studies of these viral proteins and of their complexes with antibodies and inhibitors have provided valuable insights into their functions and laid a solid foundation for drug development. In this review, we summarize the structural features of these proteins and discuss recent progress in research regarding therapeutic development, highlighting mechanistically representative molecules and those that have already been approved or are under clinical investigation.

Asunto(s)

Antivirales , SARS-CoV-2 , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/metabolismo , Antivirales/farmacología , Antivirales/química , Humanos , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Tratamiento Farmacológico de COVID-19 , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , ARN Polimerasa Dependiente de ARN de Coronavirus/química , ARN Polimerasa Dependiente de ARN de Coronavirus/antagonistas & inhibidores , Modelos Moleculares , COVID-19/virología , COVID-19/metabolismo , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo

RESUMEN

Rotavirus, a dsRNA virus in the Reoviridae family, shows a segmented genome. The VP1 gene encodes the RNA-dependent RNA polymerase (RdRp). This study aims to develop a multiepitope-based vaccine targeting RdRp using immunoinformatic approaches. In this study, 100 available nucleotide sequences of VP1-Rotavirus belonging to different strains across the world were retrieved from NCBI database. The selected sequences were aligned, and a global consensus sequence was developed by using CLC work bench. The study involved immunoinformatic approaches and molecular docking studies to reveal the promiscuous epitopes that can be eventually used as active vaccine candidates for Rotavirus. In total, 27 highly immunogenic, antigenic, and non-allergenic T-cell and B-cell epitopes were predicted for the Multiepitope vaccine (MEV) against rotavirus. It was also observed that MEV can prove to be effective worldwide due to its high population coverage, demonstrating the consistency of this vaccine. Moreover, there is a high docking interaction and immunological response with a binding score of -50.2 kcal/mol, suggesting the vaccine's efficacy. Toll-like receptors (TLRs) also suggest that the vaccine is physiologically and immunologically effective. Collectively, our data point to an effective MEV against rotavirus that can effectively reduce viral infections and improve the health status worldwide.

Asunto(s)

Simulación del Acoplamiento Molecular , Vacunas contra Rotavirus , Rotavirus , Vacunas de Subunidad , Rotavirus/inmunología , Rotavirus/genética , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/genética , Vacunas contra Rotavirus/inmunología , ARN Polimerasa Dependiente del ARN/inmunología , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/química , Biología Computacional , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/genética , Humanos , Epítopos/inmunología , Epítopos/genética , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/genética , Infecciones por Rotavirus/prevención & control , Infecciones por Rotavirus/inmunología , Inmunoinformática , Vacunas de Subunidades Proteicas

RESUMEN

Replication of influenza viral RNA depends on at least two viral polymerases, a parental replicase and an encapsidase, and cellular factor ANP32. ANP32 comprises an LRR domain and a long C-terminal low complexity acidic region (LCAR). Here we present evidence suggesting that ANP32 is recruited to the replication complex as an electrostatic chaperone that stabilises the encapsidase moiety within apo-polymerase symmetric dimers that are distinct for influenza A and B polymerases. The ANP32 bound encapsidase, then forms the asymmetric replication complex with the replicase, which is embedded in a parental ribonucleoprotein particle (RNP). Cryo-EM structures reveal the architecture of the influenza A and B replication complexes and the likely trajectory of the nascent RNA product into the encapsidase. The cryo-EM map of the FluB replication complex shows extra density attributable to the ANP32 LCAR wrapping around and stabilising the apo-encapsidase conformation. These structures give new insight into the various mutations that adapt avian strain polymerases to use the distinct ANP32 in mammalian cells.

Asunto(s)

Microscopía por Crioelectrón , Virus de la Influenza A , Chaperonas Moleculares , Proteínas de Unión al ARN , Electricidad Estática , Replicación Viral , Humanos , Animales , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Virus de la Influenza A/metabolismo , Virus de la Influenza A/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/química , ARN Viral/metabolismo , ARN Viral/química , ARN Viral/genética , Aves/virología , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Gripe Aviar/virología , Gripe Aviar/metabolismo , Modelos Moleculares , Gripe Humana/virología

RESUMEN

RNA polymerase IV (Pol IV) forms a complex with RNA-directed RNA polymerase 2 (RDR2) to produce double-stranded RNA (dsRNA) precursors essential for plant gene silencing. In the "backtracking-triggered RNA channeling" model, Pol IV backtracks and delivers its transcript's 3' terminus to RDR2, which synthesizes dsRNA. However, the mechanisms underlying Pol IV backtracking and RNA protection from cleavage are unclear. Here, we determined cryo-electron microscopy structures of Pol IV elongation complexes at four states of its nucleotide addition cycle (NAC): posttranslocation, guanosine triphosphate-bound, pretranslocation, and backtracked states. The structures reveal that Pol IV maintains an open DNA cleft and kinked bridge helix in all NAC states, loosely interacts with the nucleoside triphosphate substrate, and barely contacts proximal backtracked nucleotides. Biochemical data indicate that Pol IV is inefficient in forward translocation and RNA cleavage. These findings suggest that Pol IV transcription elongation is prone to backtracking and incapable of RNA hydrolysis, ensuring efficient dsRNA production by Pol IV-RDR2.

Asunto(s)

Microscopía por Crioelectrón , ARN Polimerasas Dirigidas por ADN , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/química , Modelos Moleculares , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/química , Elongación de la Transcripción Genética , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/química , ARN Bicatenario/metabolismo , Unión Proteica , Transcripción Genética

RESUMEN

Viral diseases pose a serious global health threat due to their rapid transmission and widespread impact. The RNA-dependent RNA polymerase (RdRp) participates in the synthesis, transcription, and replication of viral RNA in host. The current study investigates the antiviral potential of secondary metabolites particularly those derived from bacteria, fungi, and plants to develop novel medicines. Using a virtual screening approach that combines molecular docking and molecular dynamics (MD) simulations, we aimed to discover compounds with strong interactions with RdRp of five different retroviruses. The top five compounds were selected for each viral RdRp based on their docking scores, binding patterns, molecular interactions, and drug-likeness properties. The molecular docking study uncovered several metabolites with antiviral activity against RdRp. For instance, cytochalasin Z8 had the lowest docking score of -8.9 (kcal/mol) against RdRp of SARS-CoV-2, aspulvinone D (-9.2 kcal/mol) against HIV-1, talaromyolide D (-9.9 kcal/mol) for hepatitis C, aspulvinone D (-9.9 kcal/mol) against Ebola and talaromyolide D also maintained the lowest docking score of -9.2 kcal/mol against RdRp enzyme of dengue virus. These compounds showed remarkable antiviral potential comparable to standard drug (remdesivir -7.4 kcal/mol) approved to target RdRp and possess no significant toxicity. The molecular dynamics simulation confirmed that the best selected ligands were firmly bound to their respective target proteins for a simulation time of 200 ns. The identified lead compounds possess distinctive pharmacological characteristics, making them potential candidates for repurposing as antiviral drugs against SARS-CoV-2. Further experimental evaluation and investigation are recommended to ascertain their efficacy and potential.

Asunto(s)

Antivirales , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , ARN Polimerasa Dependiente del ARN , Antivirales/farmacología , Antivirales/química , Antivirales/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Humanos , VIH-1/efectos de los fármacos , VIH-1/enzimología , Metabolismo Secundario , Productos Biológicos/química , Productos Biológicos/farmacología , Productos Biológicos/metabolismo

RESUMEN

Since the emergence of SARS-CoV-2, mutations in all subunits of the RNA-dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano-differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13°C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS-CoV-2 subvariants.

Asunto(s)

ARN Polimerasa Dependiente de ARN de Coronavirus , Mutación , SARS-CoV-2 , Proteínas no Estructurales Virales , SARS-CoV-2/genética , SARS-CoV-2/enzimología , ARN Polimerasa Dependiente de ARN de Coronavirus/genética , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , ARN Polimerasa Dependiente de ARN de Coronavirus/química , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Humanos , COVID-19/virología , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Estabilidad Proteica , Unión Proteica

RESUMEN

The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness programs. Here, we studied the mechanism of action of a newly discovered nucleotide analog against diverse RNA-dependent RNA polymerases (RdRps) of prototypic respiratory viruses. GS-646939 is the active 5'-triphosphate metabolite of a 4'-cyano modified C-adenosine analog phosphoramidate prodrug GS-7682. Enzyme kinetics show that the RdRps of human rhinovirus type 16 (HRV-16) and enterovirus 71 incorporate GS-646939 with unprecedented selectivity; GS-646939 is incorporated 20-50-fold more efficiently than its natural ATP counterpart. The RdRp complex of respiratory syncytial virus and human metapneumovirus incorporate GS-646939 and ATP with similar efficiency. In contrast, influenza B RdRp shows a clear preference for ATP and human mitochondrial RNA polymerase does not show significant incorporation of GS-646939. Once incorporated into the nascent RNA strand, GS-646939 acts as a chain terminator although higher NTP concentrations can partially overcome inhibition for some polymerases. Modeling and biochemical data suggest that the 4'-modification inhibits RdRp translocation. Comparative studies with GS-443902, the active triphosphate form of the 1'-cyano modified prodrugs remdesivir and obeldesivir, reveal not only different mechanisms of inhibition, but also differences in the spectrum of inhibition of viral polymerases. In conclusion, 1'-cyano and 4'-cyano modifications of nucleotide analogs provide complementary strategies to target the polymerase of several families of respiratory RNA viruses.

Asunto(s)

Antivirales , ARN Polimerasa Dependiente del ARN , Humanos , Antivirales/farmacología , Antivirales/química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/química , Virus ARN/efectos de los fármacos , Virus ARN/enzimología , Metapneumovirus/efectos de los fármacos , Nucleótidos/química , Nucleótidos/farmacología , Nucleótidos/metabolismo

RESUMEN

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.

Asunto(s)

Metiltransferasas , Modelos Moleculares , ARN Polimerasa Dependiente del ARN , Proteínas no Estructurales Virales , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/química , Metiltransferasas/metabolismo , Metiltransferasas/química , Cristalografía por Rayos X , Flavivirus/enzimología , Flavivirus/metabolismo , Unión Proteica , Secuencia de Aminoácidos , Dominios Proteicos , Virus de la Encefalitis Transmitidos por Garrapatas/metabolismo

RESUMEN

The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein's host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In Arabidopsis thaliana, if 2b-induced inhibition of AGO1 is too efficient, it induces reinforcement of antiviral silencing by AGO2 and triggers increased resistance against aphids, CMV's insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein, which sequesters sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated, and red and green fluorescent protein fusions were used to investigate subcellular colocalization with AGO1 and the 1a protein. The effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b protein's interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b protein's ability to interact with AGO1, the 1a protein, and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.

Asunto(s)

Proteínas de Arabidopsis , Proteínas Argonautas , Interacciones Huésped-Patógeno , Proteínas Virales , Arabidopsis/metabolismo , Arabidopsis/virología , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Cucumovirus/metabolismo , Cucumovirus/genética , Cucumovirus/fisiología , Metiltransferasas , Enfermedades de las Plantas/virología , Unión Proteica , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/química , Proteínas Virales/metabolismo , Proteínas Virales/genética , Proteinas del Complejo de Replicasa Viral/metabolismo , Proteinas del Complejo de Replicasa Viral/genética

RESUMEN

Dengue virus (DENV) nonstructural protein 5 (NS5), consisting of methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, is critical for viral RNA synthesis within endoplasmic reticulum-derived replication complexes in the cytoplasm. However, a significant proportion of NS5 is localized to the nucleus of infected cells for DENV2, 3, and 4, whereas DENV1 NS5 is localized diffusely in the cytoplasm. We still have an incomplete understanding of how the DENV NS5 subcellular localization is regulated. Within NS5, two putative nuclear localization signal (NLS) sequences have been identified: NLSCentral residing in the palm of the RdRp domain as well as the recently discovered NLSC-term residing in the flexible region at the C-terminal of the RdRp domain. We have previously shown that DENV2 NS5 nuclear localization can be significantly reduced by single-point mutations to the NLSC-term. Here, we present biochemical, virological, and structural data demonstrating that the relative importance of either NLS in NS5 nuclear localization is unique to each of the four DENV serotypes. DENV1 NS5's cytoplasmic localization appears to be due to a functionally weak interaction between its NLSCentral and importin-α (IMPα), while DENV2 NS5 is almost exclusively nuclear through its NLSC-term's strong interaction with IMPα. Both NLSs of DENV3 NS5 appear to contribute to directing its nuclear localization. Lastly, in the case of DENV4, the regulation of its NS5 nuclear localization remains an enigma but appears to be associated with its NLSC-term.

Asunto(s)

Núcleo Celular , Virus del Dengue , Señales de Localización Nuclear , Serogrupo , Proteínas no Estructurales Virales , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/química , Virus del Dengue/genética , Virus del Dengue/fisiología , Núcleo Celular/metabolismo , Humanos , Citoplasma/metabolismo , Replicación Viral , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/química , Animales , Dengue/virología , Transporte de Proteínas

RESUMEN

Avian influenza A viruses (IAVs) pose a public health threat, as they are capable of triggering pandemics by crossing species barriers. Replication of avian IAVs in mammalian cells is hindered by species-specific variation in acidic nuclear phosphoprotein 32 (ANP32) proteins, which are essential for viral RNA genome replication. Adaptive mutations enable the IAV RNA polymerase (FluPolA) to surmount this barrier. Here, we present cryo-electron microscopy structures of monomeric and dimeric avian H5N1 FluPolA with human ANP32B. ANP32B interacts with the PA subunit of FluPolA in the monomeric form, at the site used for its docking onto the C-terminal domain of host RNA polymerase II during viral transcription. ANP32B acts as a chaperone, guiding FluPolA towards a ribonucleoprotein-associated FluPolA to form an asymmetric dimer-the replication platform for the viral genome. These findings offer insights into the molecular mechanisms governing IAV genome replication, while enhancing our understanding of the molecular processes underpinning mammalian adaptations in avian-origin FluPolA.

Asunto(s)

Microscopía por Crioelectrón , Genoma Viral , Subtipo H5N1 del Virus de la Influenza A , Proteínas Nucleares , ARN Polimerasa Dependiente del ARN , Replicación Viral , Humanos , Adaptación Fisiológica/genética , Células HEK293 , Subtipo H5N1 del Virus de la Influenza A/genética , Gripe Humana/virología , Modelos Moleculares , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/química , Multimerización de Proteína , ARN Viral/metabolismo , ARN Viral/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/química , Proteínas Virales/metabolismo , Proteínas Virales/genética , Proteínas Virales/química , Replicación Viral/genética

RESUMEN

The viral polymerase complex, comprising the large protein (L) and phosphoprotein (P), is crucial for both genome replication and transcription in non-segmented negative-strand RNA viruses (nsNSVs), while structures corresponding to these activities remain obscure. Here, we resolved two L-P complex conformations from the mumps virus (MuV), a typical member of nsNSVs, via cryogenic-electron microscopy. One conformation presents all five domains of L forming a continuous RNA tunnel to the methyltransferase domain (MTase), preferably as a transcription state. The other conformation has the appendage averaged out, which is inaccessible to MTase. In both conformations, parallel P tetramers are revealed around MuV L, which, together with structures of other nsNSVs, demonstrates the diverse origins of the L-binding X domain of P. Our study links varying structures of nsNSV polymerase complexes with genome replication and transcription and points to a sliding model for polymerase complexes to advance along the RNA templates.

Asunto(s)

Microscopía por Crioelectrón , Virus de la Parotiditis , Proteínas Virales , Virus de la Parotiditis/genética , Virus de la Parotiditis/ultraestructura , Virus de la Parotiditis/metabolismo , Proteínas Virales/metabolismo , Proteínas Virales/ultraestructura , Proteínas Virales/química , Proteínas Virales/genética , Modelos Moleculares , ARN Viral/metabolismo , ARN Viral/ultraestructura , ARN Viral/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/ultraestructura , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Dominios Proteicos , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/ultraestructura , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/ultraestructura , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Replicación Viral , Transcripción Genética , Conformación Proteica

RESUMEN

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.

Asunto(s)

Antivirales , Virus del Dengue , Litsea , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Fitoquímicos , ARN Polimerasa Dependiente del ARN , Virus del Dengue/efectos de los fármacos , Virus del Dengue/enzimología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Antivirales/farmacología , Antivirales/química , Fitoquímicos/farmacología , Fitoquímicos/química , Regulación Alostérica/efectos de los fármacos , Litsea/química , Unión Proteica

RESUMEN

Human norovirus (HuNoV), a primary cause of non-bacterial gastroenteritis, currently lacks approved treatment. RdRp is vital for virus replication, making it an attractive target for therapeutic intervention. By application of structure-based virtual screening procedure, we present CX-6258 hydrochloride hydrate as a potent RdRp non-nucleoside inhibitor, effectively inhibiting HuNoV RdRp activity with an IC50 of 3.61 µM. Importantly, this compound inhibits viral replication in cell culture, with an EC50 of 0.88 µM. In vitro binding assay validate that CX-6258 hydrochloride hydrate binds to RdRp through interaction with the "B-site" binding pocket. Interestingly, CX-6258-contacting residues such as R392, Q439, and Q414 are highly conserved among major norovirus GI and GII variants, suggesting that it may be a general inhibitor of norovirus RdRp. Given that CX-6258 hydrochloride hydrate is already utilized as an orally efficacious pan-Pim kinase inhibitor, it may serve as a potential lead compound in the effort to control HuNoV infections.

Asunto(s)

Antivirales , Norovirus , ARN Polimerasa Dependiente del ARN , Replicación Viral , Norovirus/efectos de los fármacos , Norovirus/enzimología , Norovirus/genética , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/química , Antivirales/farmacología , Antivirales/química , Humanos , Replicación Viral/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Simulación del Acoplamiento Molecular , Sitios de Unión

RESUMEN

Influenza A viruses, causing seasonal epidemics and occasional pandemics, rely on interactions with host proteins for their RNA genome transcription and replication. The viral RNA polymerase utilizes host RNA polymerase II (Pol II) and interacts with the serine 5 phosphorylated (pS5) C-terminal domain (CTD) of Pol II to initiate transcription. Our study, using single-particle electron cryomicroscopy (cryo-EM), reveals the structure of the 1918 pandemic influenza A virus polymerase bound to a synthetic pS5 CTD peptide composed of four heptad repeats mimicking the 52 heptad repeat mammalian Pol II CTD. The structure shows that the CTD peptide binds at the C-terminal domain of the PA viral polymerase subunit (PA-C) and reveals a previously unobserved position of the 627 domain of the PB2 subunit near the CTD. We identify crucial residues of the CTD peptide that mediate interactions with positively charged cavities on PA-C, explaining the preference of the viral polymerase for pS5 CTD. Functional analysis of mutants targeting the CTD-binding site within PA-C reveals reduced transcriptional function or defects in replication, highlighting the multifunctional role of PA-C in viral RNA synthesis. Our study provides insights into the structural and functional aspects of the influenza virus polymerase-host Pol II interaction and identifies a target for antiviral development.IMPORTANCEUnderstanding the intricate interactions between influenza A viruses and host proteins is crucial for developing targeted antiviral strategies. This study employs advanced imaging techniques to uncover the structural nuances of the 1918 pandemic influenza A virus polymerase bound to a specific host protein, shedding light on the vital process of viral RNA synthesis. The study identifies key amino acid residues in the influenza polymerase involved in binding host polymerase II (Pol II) and highlights their role in both viral transcription and genome replication. These findings not only deepen our understanding of the influenza virus life cycle but also pinpoint a potential target for antiviral development. By elucidating the structural and functional aspects of the influenza virus polymerase-host Pol II interaction, this research provides a foundation for designing interventions to disrupt viral replication and transcription, offering promising avenues for future antiviral therapies.

Asunto(s)

Microscopía por Crioelectrón , Virus de la Influenza A , ARN Polimerasa II , ARN Polimerasa Dependiente del ARN , Proteínas Virales , Humanos , Virus de la Influenza A/metabolismo , Virus de la Influenza A/genética , Virus de la Influenza A/enzimología , Gripe Humana/virología , Modelos Moleculares , Fosforilación , Unión Proteica , Dominios Proteicos , ARN Polimerasa II/metabolismo , ARN Polimerasa II/química , ARN Viral/metabolismo , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/química , Transcripción Genética , Proteínas Virales/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Replicación Viral

RESUMEN

An optimized protocol has been developed to express and purify the core RNA-dependent RNA polymerase (RdRP) complex from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The expression and purification of active core SARS-CoV-2 RdRp complex is challenging due to the complex multidomain fold of nsp12, and the assembly of the multimeric complex involving nsp7, nsp8, and nsp12. Our approach adapts a previously published method to express the core SARS-CoV-2 RdRP complex in Escherichia coli and combines it with a procedure to express the nsp12 fusion with maltose-binding protein in insect cells to promote the efficient assembly and purification of an enzymatically active core polymerase complex. The resulting method provides a reliable platform to produce milligram amounts of the polymerase complex with the expected 1:2:1 stoichiometry for nsp7, nsp8, and nsp12, respectively, following the removal of all affinity tags. This approach addresses some of the limitations of previously reported methods to provide a reliable source of the active polymerase complex for structure, function, and inhibition studies of the SARS-CoV-2 RdRP complex using recombinant plasmid constructs that have been deposited in the widely accessible Addgene repository. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and production of SARS-CoV-2 nsp7, nsp8, and nsp12 in E. coli cells Support Protocol: Establishment and maintenance of insect cell cultures Basic Protocol 2: Generation of recombinant baculovirus in Sf9 cells and production of nsp12 fusion protein in T. ni cells Basic Protocol 3: Purification of the SARS-CoV-2 core polymerase complex.

Asunto(s)

COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Escherichia coli/genética , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo

RESUMEN

Aim: Mucormycosis has been associated with SARS-CoV-2 infections during the last year. The aim of this study was to triple-hit viral and fungal RNA-dependent RNA polymerases (RdRps) and human inosine monophosphate dehydrogenase (IMPDH). Materials & methods: Molecular docking and molecular dynamics simulation were used to test nucleotide inhibitors (NIs) against the RdRps of SARS-CoV-2 and Rhizopus oryzae RdRp. These same inhibitors targeted IMPDH. Results: Four NIs revealed a comparable binding affinity to the two drugs, remdesivir and sofosbuvir. Binding energies were calculated using the most abundant conformations of the RdRps after 100-ns molecular dynamics simulation. Conclusion: We suggest the triple-inhibition potential of four NIs against pathogenic RdRps and IMPDH, which is worth experimental validation.

Asunto(s)

COVID-19 , SARS-CoV-2 , Humanos , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/química , Antivirales/uso terapéutico , Rhizopus oryzae , Simulación del Acoplamiento Molecular , Nucleótidos , ARN Viral

RESUMEN

BACKGROUND: Hepatitis C is an inflammatory condition of the liver caused by the hepatitis C virus, exhibiting acute and chronic manifestations with severity ranging from mild to severe and lifelong illnesses leading to liver cirrhosis and cancer. According to the World Health Organization's global estimates, a population of about 58 million have chronic hepatitis C virus infection, with around 1.5 million new infections occurring every year. OBJECTIVE: The present study aimed to identify novel molecules targeting the Hepatitis C viral RNA Dependent RNA polymerases, which play a crucial role in genome replication, mRNA synthesis, etc. Methods: Structure-based virtual screening of chemical libraries of small molecules was done using AutoDock/Vina. The top-ranking pose for every ligand was complexed with the protein and used for further protein-ligand interaction analysis using the Protein-ligand interaction Profiler. Molecules from virtual screening were further assessed using the pkCSM web server. The proteinligand interactions were further subjected to molecular dynamics simulation studies to establish dynamic stability. RESULTS: Molecular docking-based virtual screening of the database of small molecules, followed by screening based on pharmacokinetic and toxicity parameters, yielded eight probable RNA Dependent RNA polymerase inhibitors. The docking scores for the proposed candidates ranged from - 8.04 to -9.10 kcal/mol. The potential stability of the ligands bound to the target protein was demonstrated by molecular dynamics simulation studies. CONCLUSION: Data from exhaustive computational studies proposed eight molecules as potential anti-viral candidates, targeting Hepatitis C viral RNA Dependent RNA polymerases, which can be further evaluated for their biological potential.

Asunto(s)

Hepatitis C Crónica , Hepatitis C , Humanos , Hepacivirus , Simulación del Acoplamiento Molecular , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/uso terapéutico , Ligandos , Hepatitis C/tratamiento farmacológico , Simulación de Dinámica Molecular , ARN Viral , ARN Polimerasas Dirigidas por ADN/uso terapéutico , Antivirales/farmacología , Antivirales/química

RESUMEN

The respiratory syncytial virus (RSV) polymerase is a multifunctional RNA-dependent RNA polymerase composed of the large (L) protein and the phosphoprotein (P). It transcribes the RNA genome into ten viral mRNAs and replicates full-length viral genomic and antigenomic RNAs1. The RSV polymerase initiates RNA synthesis by binding to the conserved 3'-terminal RNA promoters of the genome or antigenome2. However, the lack of a structure of the RSV polymerase bound to the RNA promoter has impeded the mechanistic understanding of RSV RNA synthesis. Here we report cryogenic electron microscopy structures of the RSV polymerase bound to its genomic and antigenomic viral RNA promoters, representing two of the first structures of an RNA-dependent RNA polymerase in complex with its RNA promoters in non-segmented negative-sense RNA viruses. The overall structures of the promoter-bound RSV polymerases are similar to that of the unbound (apo) polymerase. Our structures illustrate the interactions between the RSV polymerase and the RNA promoters and provide the structural basis for the initiation of RNA synthesis at positions 1 and 3 of the RSV promoters. These structures offer a deeper understanding of the pre-initiation state of the RSV polymerase and could aid in antiviral research against RSV.

Asunto(s)

Regiones Promotoras Genéticas , ARN Polimerasa Dependiente del ARN , Virus Sincitial Respiratorio Humano , Regiones Promotoras Genéticas/genética , Virus Sincitial Respiratorio Humano/enzimología , Virus Sincitial Respiratorio Humano/genética , ARN Viral/biosíntesis , ARN Viral/genética , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/ultraestructura , Replicación Viral/genética , Microscopía por Crioelectrón , ARN Subgenómico/biosíntesis , ARN Subgenómico/genética , ARN Subgenómico/metabolismo

RESUMEN

IMPORTANCE: The influenza virus polymerase is important for adaptation to new hosts and, as a determinant of mutation rate, for the process of adaptation itself. We performed a deep mutational scan of the polymerase basic 1 (PB1) protein to gain insights into the structural and functional constraints on the influenza RNA-dependent RNA polymerase. We find that PB1 is highly constrained at specific sites that are only moderately predicted by the global structure or larger domain. We identified a number of beneficial mutations, many of which have been shown to be functionally important or observed in influenza virus' natural evolution. Overall, our atlas of PB1 mutations and their fitness impacts serves as an important resource for future studies of influenza replication and evolution.

Asunto(s)

Virus de la Influenza A , Mutación , ARN Polimerasa Dependiente del ARN , Proteínas Virales , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Mutación/genética , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/genética , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Evolución Molecular , Infecciones por Orthomyxoviridae/virología