RESUMEN
Human respiratory syncytial virus (HRSV) envelope glycoproteins traffic to assembly sites through the secretory pathway, while nonglycosylated proteins M and N are present in HRSV inclusion bodies but must reach the plasma membrane, where HRSV assembly happens. Little is known about how nonglycosylated HRSV proteins reach assembly sites. Here, we show that HRSV M and N proteins partially colocalize with the Golgi marker giantin, and the glycosylated F and nonglycosylated N proteins are closely located in the trans-Golgi, suggesting their interaction in that compartment. Brefeldin A compromised the trafficking of HRSV F and N proteins and inclusion body sizes, indicating that the Golgi is important for both glycosylated and nonglycosylated HRSV protein traffic. HRSV N and M proteins colocalized and interacted with sorting nexin 2 (SNX2), a retromer component that shapes endosomes in tubular structures. Glycosylated F and nonglycosylated N HRSV proteins are detected in SNX2-laden aggregates with intracellular filaments projecting from their outer surfaces, and VPS26, another retromer component, was also found in inclusion bodies and filament-shaped structures. Similar to SNX2, TGN46 also colocalized with HRSV M and N proteins in filamentous structures at the plasma membrane. Cell fractionation showed enrichment of SNX2 in fractions containing HRSV M and N proteins. Silencing of SNX1 and 2 was associated with reduction in viral proteins, HRSV inclusion body size, syncytium formation, and progeny production. The results indicate that HRSV structural proteins M and N are in the secretory pathway, and SNX2 plays an important role in the traffic of HRSV structural proteins toward assembly sites.IMPORTANCE The present study contributes new knowledge to understand HRSV assembly by providing evidence that nonglycosylated structural proteins M and N interact with elements of the secretory pathway, shedding light on their intracellular traffic. To the best of our knowledge, the present contribution is important given the scarcity of studies about the traffic of HRSV nonglycosylated proteins, especially by pointing to the involvement of SNX2, a retromer component, in the HRSV assembly process.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Interacciones Microbiota-Huesped , Proteínas de la Nucleocápside/metabolismo , Virus Sincitial Respiratorio Humano/fisiología , Proteínas Virales/metabolismo , Ensamble de Virus , Precursor de Proteína beta-Amiloide/genética , Proteínas Portadoras , Aparato de Golgi/metabolismo , Proteínas de la Matriz de Golgi/metabolismo , Células HeLa , Humanos , Transporte de ProteínasRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Leishmania RNA virus (LRV) is an important virulence factor associated with the development of mucocutaneous Leishmaniasis, a severe form of the disease. LRV-mediated disease exacerbation relies on TLR3 activation, but downstream mechanisms remain largely unexplored. Here, we combine human and mouse data to demonstrate that LRV triggers TLR3 and TRIF to induce type I IFN production, which induces autophagy. This process results in ATG5-mediated degradation of NLRP3 and ASC, thereby limiting NLRP3 inflammasome activation in macrophages. Consistent with the known restricting role of NLRP3 for Leishmania replication, the signaling pathway triggered by LRV results in increased parasite survival and disease progression. In support of this data, we find that lesions in patients infected with LRV+ Leishmania are associated with reduced inflammasome activation and the development of mucocutaneous disease. Our findings reveal the mechanisms triggered by LRV that contribute to the development of the debilitating mucocutaneous form of Leishmaniasis.
Asunto(s)
Inmunidad Innata/inmunología , Inflamasomas/inmunología , Leishmania/inmunología , Leishmaniasis Mucocutánea/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Virus ARN/inmunología , Receptor Toll-Like 3/inmunología , Animales , Autofagia/inmunología , Humanos , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Leishmania/fisiología , Leishmania/virología , Leishmaniasis Mucocutánea/parasitología , Leishmaniasis Mucocutánea/virología , Macrófagos/inmunología , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Virus ARN/fisiología , Transducción de Señal/inmunología , Receptor Toll-Like 3/metabolismoRESUMEN
Zika virus (ZIKV) has been associated with serious health conditions, and an intense search to discover different ways to prevent and treat ZIKV infection is underway. Berberine and emodin possess several pharmacological properties and have been shown to be particularly effective against the entry and replication of several viruses. We show that emodin and berberine trigger a virucidal effect on ZIKV. When the virus was exposed to 160 µM of berberine, a reduction of 77.6% in the infectivity was observed; when emodin was used (40 µM), this reduction was approximately 83.3%. Dynamic light scattering data showed that both compounds significantly reduce the hydrodynamic radius of virus particle in solution. We report here that berberine and emodin, two natural compounds, have strong virucidal effect in Zika virus.
Asunto(s)
Antivirales/farmacología , Productos Biológicos/farmacología , Plantas Medicinales/química , Virus Zika/efectos de los fármacos , Animales , Antivirales/aislamiento & purificación , Berberina/farmacología , Productos Biológicos/aislamiento & purificación , Chlorocebus aethiops , Emodina/farmacología , Medicina Tradicional de Asia Oriental , Células Vero , Virión/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Leishmania RNA Virus (LRV, Totiviridae) infect Leishmania cells and subvert mice immune response, probably promoting parasite persistence, suggesting significant roles for LRV in host-parasite interaction. Here we describe a new LRV1-4 purification protocol, enabling capsid visualization by negatively stained electron microscopy representing a significant contribution to future LRV investigations.
Asunto(s)
Leishmaniavirus/aislamiento & purificación , Virión/aislamiento & purificación , Virología/métodos , Leishmaniavirus/ultraestructura , Microscopía Electrónica de Transmisión , Coloración y Etiquetado/métodos , Virión/ultraestructuraRESUMEN
In bacteria selenocysteyl-tRNA(sec) (SelC) is synthesized by selenocysteine synthase (SelA). Here we show by fluorescence anisotropy binding assays and electron microscopical symmetry analysis that the SelA-tRNA(sec) binding stoichiometry is of one tRNA(sec) molecule per SelA monomer (1:1) rather than the 1:2 value proposed previously. Negative stain transmission electron microscopy revealed a D5 pointgroup symmetry for the SelA-tRNA(sec) assembly both with and without tRNA(sec) bound. Furthermore, SelA can associate forming a supramolecular complex of stacked decamer rings, which does not occur in the presence of tRNA(sec). We discuss the structure-function relationships of these assemblies and their regulatory role in bacterial selenocysteyl-tRNA(sec) synthesis.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Aminoacil-ARN de Transferencia/genética , Transferasas/genética , Secuencia de Bases , Unión Competitiva , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Polarización de Fluorescencia , Cinética , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Sustancias Macromoleculares/ultraestructura , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Protozoario/química , ARN Protozoario/genética , ARN Protozoario/metabolismo , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismo , Selenocisteína/genética , Selenocisteína/metabolismo , Transcripción Genética , Transferasas/química , Transferasas/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
Leishmania RNA Virus (LRV, Totiviridae) infect Leishmania cells and subvert mice immune response, probably promoting parasite persistence, suggesting significant roles for LRV in host-parasite interaction. Here we describe a new LRV1-4 purification protocol, enabling capsid visualization by negatively stained electron microscopy representing a significant contribution to future LRV investigations.