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1.
Nature ; 577(7789): 275-279, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31698413

RESUMEN

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) cause severe respiratory diseases in infants and elderly adults1. No vaccine or effective antiviral therapy currently exists to control RSV or HMPV infections. During viral genome replication and transcription, the tetrameric phosphoprotein P serves as a crucial adaptor between the ribonucleoprotein template and the L protein, which has RNA-dependent RNA polymerase (RdRp), GDP polyribonucleotidyltransferase and cap-specific methyltransferase activities2,3. How P interacts with L and mediates the association with the free form of N and with the ribonucleoprotein is not clear for HMPV or other major human pathogens, including the viruses that cause measles, Ebola and rabies. Here we report a cryo-electron microscopy reconstruction that shows the ring-shaped structure of the polymerase and capping domains of HMPV-L bound to a tetramer of P. The connector and methyltransferase domains of L are mobile with respect to the core. The putative priming loop that is important for the initiation of RNA synthesis is fully retracted, which leaves space in the active-site cavity for RNA elongation. P interacts extensively with the N-terminal region of L, burying more than 4,016 Å2 of the molecular surface area in the interface. Two of the four helices that form the coiled-coil tetramerization domain of P, and long C-terminal extensions projecting from these two helices, wrap around the L protein in a manner similar to tentacles. The structural versatility of the four P protomers-which are largely disordered in their free state-demonstrates an example of a 'folding-upon-partner-binding' mechanism for carrying out P adaptor functions. The structure shows that P has the potential to modulate multiple functions of L and these results should accelerate the design of specific antiviral drugs.


Asunto(s)
Metapneumovirus/enzimología , Fosfoproteínas/química , ARN Polimerasa Dependiente del ARN/química , Secuencia de Aminoácidos , Animales , Línea Celular , Microscopía por Crioelectrón , Metapneumovirus/genética , Modelos Moleculares , Fosfoproteínas/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo
2.
PLoS Pathog ; 17(12): e1010151, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34914795

RESUMEN

It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3´ terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties of polymerases of respiratory syncytial virus (RSV), a pneumovirus, human parainfluenza virus type 3 (PIV-3), a paramyxovirus, and Marburg virus (MARV), a filovirus, both on their cognate promoters and on promoters of other viruses. We found that in contrast to the RSV polymerase, which initiated at positions 1 and 3 of its promoter, the PIV-3 and MARV polymerases initiated exclusively at position 1 on their cognate promoters. However, all three polymerases could recognize and initiate from heterologous promoters, with the promoter sequence playing a key role in determining initiation site selection. In addition to examining de novo initiation, we also compared the ability of the RSV and PIV-3 polymerases to engage in back-priming, an activity in which the promoter template is folded into a secondary structure and nucleotides are added to the template 3´ end. This analysis showed that whereas the RSV polymerase was promiscuous in back-priming activity, the PIV-3 polymerase generated barely detectable levels of back-primed product, irrespective of promoter template sequence. Overall, this study shows that the polymerases from these three nsNSV families are fundamentally similar in their initiation properties, but have differences in their abilities to engage in back-priming.


Asunto(s)
Marburgvirus/enzimología , Virus de la Parainfluenza 3 Humana/enzimología , ARN Polimerasa Dependiente del ARN/metabolismo , Virus Sincitiales Respiratorios/enzimología , Proteinas del Complejo de Replicasa Viral/metabolismo , Animales , Células Cultivadas
3.
PLoS Pathog ; 16(10): e1008987, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-33031461

RESUMEN

The ribonucleocapsid complex of respiratory syncytial virus (RSV) is responsible for both viral mRNA transcription and viral replication during infection, though little is known about how this dual function is achieved. Here, we report the use of a recombinant RSV virus with a FLAG-tagged large polymerase protein, L, to characterize and localize RSV ribonucleocapsid structures during the early and late stages of viral infection. Through proximity ligation assays and super-resolution microscopy, viral RNA and proteins in the ribonucleocapsid complex were revealed to dynamically rearrange over time, particularly between 6 and 8 hours post infection, suggesting a connection between the ribonucleocapsid structure and its function. The timing of ribonucleocapsid rearrangement corresponded with an increase in RSV genome RNA accumulation, indicating that this rearrangement is likely involved with the onset of RNA replication and secondary transcription. Additionally, early overexpression of RSV M2-2 from in vitro transcribed mRNA was shown to inhibit virus infection by rearranging the ribonucleocapsid complex. Collectively, these results detail a critical understanding into the localization and activity of RSV L and the ribonucleocapsid complex during RSV infection.


Asunto(s)
Proteínas de la Nucleocápside/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/fisiología , Ribonucleoproteínas/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Células A549 , Animales , Chlorocebus aethiops , Humanos , Proteínas de la Nucleocápside/genética , ARN Viral/genética , ARN Viral/metabolismo , Infecciones por Virus Sincitial Respiratorio/genética , Infecciones por Virus Sincitial Respiratorio/metabolismo , Ribonucleoproteínas/genética , Transcripción Genética , Células Vero , Proteínas Virales/genética
4.
J Biol Chem ; 293(43): 16761-16777, 2018 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-30206124

RESUMEN

Respiratory syncytial virus (RSV) represents a significant health threat to infants and to elderly or immunocompromised individuals. There are currently no vaccines available to prevent RSV infections, and disease management is largely limited to supportive care, making the identification and development of effective antiviral therapeutics against RSV a priority. To identify effective chemical scaffolds for managing RSV disease, we conducted a high-throughput anti-RSV screen of a 57,000-compound library. We identified a hit compound that specifically blocked activity of the RSV RNA-dependent RNA polymerase (RdRp) complex, initially with moderate low-micromolar potency. Mechanistic characterization in an in vitro RSV RdRp assay indicated that representatives of this compound class block elongation of RSV RNA products after initial extension by up to three nucleotides. Synthetic hit-to-lead exploration yielded an informative 3D quantitative structure-activity relationship (3D-QSAR) model and resulted in analogs with more than 20-fold improved potency and selectivity indices (SIs) of >1,000. However, first-generation leads exhibited limited water solubility and poor metabolic stability. A second optimization strategy informed by the 3D-QSAR model combined with in silico pharmacokinetics (PK) predictions yielded an advanced lead, AVG-233, that demonstrated nanomolar activity against both laboratory-adapted RSV strains and clinical RSV isolates. This anti-RSV activity extended to infection of established cell lines and primary human airway cells. PK profiling in mice revealed 34% oral bioavailability of AVG-233 and sustained high drug levels in the circulation after a single oral dose of 20 mg/kg. This promising first-in-class lead warrants further development as an anti-RSV drug.


Asunto(s)
Antivirales/farmacología , ARN Polimerasa Dependiente del ARN/metabolismo , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Regulación Alostérica , Animales , Células Cultivadas , Humanos , Masculino , Ratones , ARN Polimerasa Dependiente del ARN/genética , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Proteínas Virales/metabolismo
5.
Artículo en Inglés | MEDLINE | ID: mdl-29891600

RESUMEN

Morbidity and mortality resulting from influenza-like disease are a threat, especially for older adults. To improve case management, next-generation broad-spectrum antiviral therapeutics that are efficacious against major drivers of influenza-like disease, including influenza viruses and respiratory syncytial virus (RSV), are urgently needed. Using a dual-pathogen high-throughput screening protocol for influenza A virus (IAV) and RSV inhibitors, we have identified N4-hydroxycytidine (NHC) as a potent inhibitor of RSV, influenza B viruses, and IAVs of human, avian, and swine origins. Biochemical in vitro polymerase assays and viral RNA sequencing revealed that the ribonucleotide analog is incorporated into nascent viral RNAs in place of cytidine, increasing the frequency of viral mutagenesis. Viral passaging in cell culture in the presence of an inhibitor did not induce robust resistance. Pharmacokinetic profiling demonstrated dose-dependent oral bioavailability of 36 to 56%, sustained levels of the active 5'-triphosphate anabolite in primary human airway cells and mouse lung tissue, and good tolerability after extended dosing at 800 mg/kg of body weight/day. The compound was orally efficacious against RSV and both seasonal and highly pathogenic avian IAVs in mouse models, reducing lung virus loads and alleviating disease biomarkers. Oral dosing reduced IAV burdens in a guinea pig transmission model and suppressed virus spread to uninfected contact animals through direct transmission. Based on its broad-spectrum efficacy and pharmacokinetic properties, NHC is a promising candidate for future clinical development as a treatment option for influenza-like diseases.


Asunto(s)
Antivirales/farmacología , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Animales , Células Cultivadas , Cobayas , Humanos , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/genética , Virus de la Influenza B/efectos de los fármacos , Virus de la Influenza B/genética , Ratones , ARN Viral/genética , Virus Sincitial Respiratorio Humano/genética , Virus Sincitiales Respiratorios/efectos de los fármacos , Virus Sincitiales Respiratorios/genética
6.
Nat Commun ; 15(1): 3163, 2024 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-38605025

RESUMEN

The transcription and replication processes of non-segmented, negative-strand RNA viruses (nsNSVs) are catalyzed by a multi-functional polymerase complex composed of the large protein (L) and a cofactor protein, such as phosphoprotein (P). Previous studies have shown that the nsNSV polymerase can adopt a dimeric form, however, the structure of the dimer and its function are poorly understood. Here we determine a 2.7 Å cryo-EM structure of human parainfluenza virus type 3 (hPIV3) L-P complex with the connector domain (CD') of a second L built, while reconstruction of the rest of the second L-P obtains a low-resolution map of the ring-like L core region. This study reveals detailed atomic features of nsNSV polymerase active site and distinct conformation of hPIV3 L with a unique ß-strand latch. Furthermore, we report the structural basis of L-L dimerization, with CD' located at the putative template entry of the adjoining L. Disruption of the L-L interface causes a defect in RNA replication that can be overcome by complementation, demonstrating that L dimerization is necessary for hPIV3 genome replication. These findings provide further insight into how nsNSV polymerases perform their functions, and suggest a new avenue for rational drug design.


Asunto(s)
Nucleotidiltransferasas , Virus ARN , Humanos , Dimerización , Dominio Catalítico , Replicación Viral
7.
Virology ; 540: 66-74, 2020 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-31739186

RESUMEN

Respiratory syncytial virus (RSV) is significant for public health, capable of causing respiratory tract disease in infants, the elderly and the immunocompromised. The RSV polymerase is an attractive target for antiviral drug development, but as yet, there is no high throughput assay for analyzing RSV polymerase activity, specifically. In this study, using a primer elongation assay as a basis, we analyzed the tolerance of the RSV polymerase for modifications at the 5' end of the primer, and nucleotide analogs. The RSV polymerase was found to accept primers containing 5' biotin or digoxygenin modifications, and nucleotide analogs that are reactive or fluorescent, including 5-ethynyl UTP, 8-azido ATP, 2-amino PTP, and thieno-GTP. These findings provide a menu of options for developing non-isotopic high throughput assays for RSV polymerase RNA synthesis activity, and yield insight regarding the molecular biology of the polymerase complex.


Asunto(s)
Nucleótidos/metabolismo , ARN/biosíntesis , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/fisiología , Cartilla de ADN , Humanos , Nucleótidos/química , ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Moldes Genéticos , Replicación Viral
8.
Antiviral Res ; 131: 19-25, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27059228

RESUMEN

Respiratory syncytial virus (RSV) infections affect millions of children and adults every year. Despite the significant disease burden, there are currently no safe and effective vaccines or therapeutics. We employed a replicon-based high throughput screen combined with live-virus triaging assays to identify three novel diversity-oriented synthesis-derived scaffolds with activity against RSV. One of these small molecules is shown to target the RSV polymerase (L protein) to inhibit viral replication and transcription; the mechanisms of action of the other small molecules are currently unknown. The compounds described herein may provide attractive inhibitors for lead optimization campaigns.


Asunto(s)
Antivirales/farmacología , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Pruebas de Sensibilidad Microbiana , Replicón/efectos de los fármacos , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Antivirales/química , Antivirales/aislamiento & purificación , Células Hep G2 , Humanos , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Infecciones por Virus Sincitial Respiratorio/terapia , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/enzimología , Virus Sincitial Respiratorio Humano/fisiología , Proteínas Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos
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