RESUMEN

Birnaviruses are unconventional members of the group of double-stranded RNA (dsRNA) viruses that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the virally encoded RNA-dependent RNA polymerase (RdRp). This and other structural features suggest that birnaviruses may follow a completely different replication program from that followed by members of the Reoviridae family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses. Here we demonstrate that infectious bursal disease virus (IBDV), a prototypical member of the Birnaviridae family, hijacks endosomal membranes of infected cells through the interaction of a viral protein, VP3, with the phospholipids on the cytosolic leaflet of these compartments for replication. Employing a mutagenesis approach, we demonstrated that VP3 domain PATCH 2 (P2) mediates the association of VP3 with the endosomal membranes. To determine the role of VP3 P2 in the context of the virus replication cycle, we used avian cells stably overexpressing VP3 P2 for IBDV infection. Importantly, the intra- and extracellular virus yields, as well as the intracellular levels of VP2 viral capsid protein, were significantly diminished in cells stably overexpressing VP3 P2. Together, our results indicate that the association of VP3 with endosomes has a relevant role in the IBDV replication cycle. This report provides direct experimental evidence for membranous compartments such as endosomes being required by a dsRNA virus for its replication. The results also support the previously proposed role of birnaviruses as an evolutionary link between +ssRNA and dsRNA viruses.IMPORTANCE Infectious bursal disease (IBD; also called Gumboro disease) is an acute, highly contagious immunosuppressive disease that affects young chickens and spreads worldwide. The etiological agent of IBD is infectious bursal disease virus (IBDV). This virus destroys the central immune organ (bursa of Fabricius), resulting in immunosuppression and reduced responses of chickens to vaccines, which increase their susceptibility to other pathogens. IBDV is a member of Birnaviridae family, which comprises unconventional members of dsRNA viruses, whose replication strategy has been scarcely studied. In this report we show that IBDV hijacks the endosomes of the infected cells for establishing viral replication complexes via the association of the ribonucleoprotein complex component VP3 with the phospholipids in the cytosolic leaflet of endosomal membranes. We show that this interaction is mediated by the VP3 PATCH 2 domain and demonstrate its relevant role in the context of viral infection.

Asunto(s)

Endosomas/virología , Virus de la Enfermedad Infecciosa de la Bolsa/fisiología , Fosfolípidos/metabolismo , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismo , Animales , Línea Celular , Células HeLa , Humanos , Virus de la Enfermedad Infecciosa de la Bolsa/patogenicidad , Mutagénesis , Dominios Proteicos , Codorniz , Proteínas Estructurales Virales/química , Replicación Viral

RESUMEN

Cathepsins, endosomal acid proteases, are transported from the trans-Golgi network to late endosomes by the mannose-6-phosphate receptor (M6PR). We have previously demonstrated that some rotavirus strains, like UK, Wa, WI61, DS-1, and YM, require the cation-dependent (CD-) M6PR and cathepsins to enter from late endosomes to the cytoplasm in MA104 cells, while other strains, like the simian strain RRV, which enter cells from maturing endosomes, do not. However, the role of other trans-Golgi network-late endosome transporters, such as the cation-independent (CI-) M6PR and sortillin-1, has not been evaluated. In this work, we found that several rotavirus strains that require the CD-M6PR for cell entry are also dependent on CI-M6PR and sortilin-1. Furthermore, we showed that the infectivity of all these rotavirus strains also requires cathepsins to enter not only MA104 cells, but also human intestinal Caco-2 cells. This study identifies sortilin-1 as a novel cell factor necessary for the infectivity of a virus; in addition, our results strongly suggest that cathepsins could be common cell factors needed for the infectivity of most rotavirus strains.

Asunto(s)

Proteínas Adaptadoras del Transporte Vesicular/genética , Catepsinas/genética , Interacciones Huésped-Patógeno , Receptor IGF Tipo 2/genética , Rotavirus/metabolismo , Internalización del Virus , Proteínas Adaptadoras del Transporte Vesicular/antagonistas & inhibidores , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Transporte Biológico , Células CACO-2 , Catepsinas/antagonistas & inhibidores , Catepsinas/metabolismo , Línea Celular , Endosomas/metabolismo , Endosomas/virología , Fibroblastos/metabolismo , Fibroblastos/virología , Regulación de la Expresión Génica , Genotipo , Humanos , Macaca mulatta , Ratones , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor IGF Tipo 2/antagonistas & inhibidores , Receptor IGF Tipo 2/metabolismo , Rotavirus/clasificación , Rotavirus/genética , Rotavirus/crecimiento & desarrollo , Especificidad de la Especie , Red trans-Golgi/metabolismo , Red trans-Golgi/virología

RESUMEN

The budded phenotype (BV) of the baculovirus AcMNPV has been demonstrated to have strong immunostimulatory properties that are relevant for the development of vaccines and antiviral therapies. Although the occluded phenotype (ODV) shares the main structural proteins and its genome with BV, it has been poorly studied in mammals. In this study, we assessed the capacity of ODV to induce immune responses in mice. In contrast to BVs, ODVs failed to promote the secretion of IFN-gamma, IL-6 and Il-12 and to induce antiviral activity against VSV in the short term. Furthermore, ODVs were unable to induce cellular immunity against a coadministered antigen 7 days after inoculation. By analyzing the interaction of ODVs with BMDCs, we observed that although ODVs entered the cells reaching late and acidic endosomes, they did not induce their maturation. Finally, we also analyzed if BVs and ODVs followed different routes in the cell during the infection. BVs, but not ODVs, colocalized with the protein ovalbumin in compartments with the presence of proteases. The results suggest that structural differences could be responsible for their different destinies in the dendritic cell and this could lead to a different impact on the immune response.

Asunto(s)

Espacio Intracelular/virología , Nucleopoliedrovirus/fisiología , Fenotipo , Animales , Células de la Médula Ósea/citología , Células Dendríticas/citología , Células Dendríticas/inmunología , Endosomas/virología , Femenino , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos C57BL , Células Sf9 , Spodoptera

RESUMEN

The hantavirus membrane fusion process is mediated by the Gc envelope glycoprotein from within endosomes. However, little is known about the specific mechanism that triggers Gc fusion activation, and its pre- and post-fusion conformations. We established cell-free in vitro systems to characterize hantavirus fusion activation. Low pH was sufficient to trigger the interaction of virus-like particles with liposomes. This interaction was dependent on a pre-fusion glycoprotein arrangement. Further, low pH induced Gc multimerization changes leading to non-reversible Gc homotrimers. These trimers were resistant to detergent, heat and protease digestion, suggesting characteristics of a stable post-fusion structure. No acid-dependent oligomerization rearrangement was detected for the trypsin-sensitive Gn envelope glycoprotein. Finally, acidification induced fusion of glycoprotein-expressing effector cells with non-susceptible CHO cells. Together, the data provide novel information on the Gc fusion trigger and its non-reversible activation involving lipid interaction, multimerization changes and membrane fusion which ultimately allow hantavirus entry into cells.

Asunto(s)

Infecciones por Hantavirus/virología , Orthohantavirus/fisiología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Endosomas/química , Endosomas/virología , Orthohantavirus/química , Orthohantavirus/genética , Humanos , Concentración de Iones de Hidrógeno , Multimerización de Proteína , Proteínas del Envoltorio Viral/genética

RESUMEN

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55(Gag) is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4(+) T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55(Gag) membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55(Gag) with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication.

Asunto(s)

Membrana Celular/metabolismo , VIH-1/fisiología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ensamble de Virus/fisiología , Replicación Viral/fisiología , Proteínas de Unión al GTP rab/metabolismo , Transporte Biológico Activo/genética , Membrana Celular/genética , Membrana Celular/virología , Endosomas/genética , Endosomas/metabolismo , Endosomas/virología , Humanos , Células Jurkat , Macrófagos/metabolismo , Macrófagos/virología , Proteínas de la Membrana/metabolismo , Antígenos de Histocompatibilidad Menor , Fosfatidilinositol 4,5-Difosfato/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTP

RESUMEN

Infectious bursal disease virus (IBDV) internalization is sparsely known in terms of molecular components of the pathway involved. To describe the cell biological features of IBDV endocytosis, we employed perturbants of endocytic pathways such as pharmacological inhibitors and overexpression of dominant-negative mutants. Internalization analysis was performed quantifying infected cells by immunofluorescence and Western blot detection of the viral protein VP3 at 12 h post-infection reinforced by the analysis of the capsid protein VP2 localization after virus uptake at 1 h post-infection. We compared IBDV infection to the internalization of well-established ligands with defined endocytic pathways: transferrin, cholera-toxin subunit B and dextran. To describe virus endocytosis at the morphological level, we performed ultrastructural studies of viral internalization kinetics in control and actin dynamics-blocked cells. Our results indicate that IBDV endocytic internalization was clathrin- and dynamin-independent, and that IBDV uses macropinocytosis as the primary entry mechanism. After uptake, virus traffics to early endosomes and requires exposure to the low endocytic pH as well as a functional endocytic pathway to complete its replication cycle. Moreover, our results indicate that the GTPase Rab5 is crucial for IBDV entry supporting the participation of the early endosomal pathway in IBDV internalization and infection of susceptible cells.

Asunto(s)

Endosomas/virología , Virus de la Enfermedad Infecciosa de la Bolsa/fisiología , Pinocitosis , Internalización del Virus , Proteínas de Unión al GTP rab5/metabolismo , Animales , Transporte Biológico , Aves , Western Blotting , Línea Celular , Microscopía Fluorescente , Factores de Tiempo , Proteínas Estructurales Virales/análisis

RESUMEN

UNLABELLED: Rotaviruses (RVs) enter cells through different endocytic pathways. Bovine rotavirus (BRV) UK uses clathrin-mediated endocytosis, while rhesus rotavirus (RRV) employs an endocytic process independent of clathrin and caveolin. Given the differences in the cell internalization pathway used by these viruses, we tested if the intracellular trafficking of BRV UK was the same as that of RRV, which is known to reach maturing endosomes (MEs) to infect the cell. We found that BRV UK also reaches MEs, since its infectivity depends on the function of Rab5, the endosomal sorting complex required for transport (ESCRT), and the formation of endosomal intraluminal vesicles (ILVs). However, unlike RRV, the infectivity of BRV UK was inhibited by knocking down the expression of Rab7, indicating that it has to traffic to late endosomes (LEs) to infect the cell. The requirement for Rab7 was also shared by other RV strains of human and porcine origin. Of interest, most RV strains that reach LEs were also found to depend on the activities of Rab9, the cation-dependent mannose-6-phosphate receptor (CD-M6PR), and cathepsins B, L, and S, suggesting that cellular factors from the trans-Golgi network (TGN) need to be transported by the CD-M6PR to LEs to facilitate RV cell infection. Furthermore, using a collection of UK × RRV reassortant viruses, we found that the dependence of BRV UK on Rab7, Rab9, and CD-M6PR is associated with the spike protein VP4. These findings illustrate the elaborate pathway of RV entry and reveal a new process (Rab9/CD-M6PR/cathepsins) that could be targeted for drug intervention. IMPORTANCE: Rotavirus is an important etiological agent of severe gastroenteritis in children. In most instances, viruses enter cells through an endocytic pathway that delivers the viral particle to vesicular organelles known as early endosomes (EEs). Some viruses reach the cytoplasm from EEs, where they start to replicate their genome. However, other viruses go deeper into the cell, trafficking from EEs to late endosomes (LEs) to disassemble and reach the cytoplasm. In this work, we show that most RV strains have to traffic to LEs, and the transport of endolysosomal proteases from the Golgi complex to LEs, mediated by the mannose-6-phosphate receptor, is necessary for the virus to exit the vesicular compartment and efficiently start viral replication. We also show that this deep journey into the cell is associated with the virus spike protein VP4. These findings illustrate the elaborate pathway of RV entry that could be used for drug intervention.

Asunto(s)

Catepsinas/metabolismo , Enfermedades de los Bovinos/enzimología , Enfermedades de los Bovinos/virología , Endosomas/virología , Enfermedades de los Monos/enzimología , Receptor IGF Tipo 2/metabolismo , Infecciones por Rotavirus/veterinaria , Rotavirus/fisiología , Animales , Catepsinas/genética , Bovinos , Enfermedades de los Bovinos/genética , Enfermedades de los Bovinos/metabolismo , Endosomas/enzimología , Endosomas/metabolismo , Macaca mulatta , Ratones , Enfermedades de los Monos/genética , Enfermedades de los Monos/metabolismo , Enfermedades de los Monos/virología , Receptor IGF Tipo 2/genética , Rotavirus/genética , Infecciones por Rotavirus/enzimología , Infecciones por Rotavirus/metabolismo , Infecciones por Rotavirus/virología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus

RESUMEN

Oropouche virus (ORO), family Bunyaviridae, is the second most frequent cause of arboviral febrile illness in Brazil. Studies were conducted to understand ORO entry in HeLa cells. Chlorpromazine inhibited early steps of ORO replication cycle, consistent with entry/uncoating. The data indicate that ORO enters HeLa cells by clathrin-coated vesicles, by a mechanism susceptible to endosomal acidification inhibitors. Transmission electron microscopy and immunofluorescence indicated that ORO associates with clathrin-coated pits and can be found in association with late endosomes in a time shorter than 1h.

Asunto(s)

Ácidos/metabolismo , Infecciones por Bunyaviridae/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Endosomas/metabolismo , Orthobunyavirus/fisiología , Internalización del Virus , Brasil , Infecciones por Bunyaviridae/virología , Vesículas Cubiertas por Clatrina/ultraestructura , Vesículas Cubiertas por Clatrina/virología , Endosomas/ultraestructura , Endosomas/virología , Células HeLa , Humanos , Orthobunyavirus/ultraestructura

RESUMEN

The endocytic pathway followed by dengue virus to infect the mosquito cells C6/36 HT was analyzed. Using DIL-labeled virions and real-time imaging it was determined that viral entry into C6/36 HT takes approximately 5 to 7 min. Pretreatment of C6/36 HT cells with sucrose and bafilomycin A, but not filipin, inhibited dengue virus infection up to 80%. Furthermore, the overexpression of dominant-negative mutants of Eps15, a molecule required for the formation of clathrin-coated vesicles, reduced dengue infection up to 50%, indicating that dengue virus entry is through clathrin-mediated endocytosis and is pH-dependent. By double-immunofluorescence assays, DIL-labeled particles were colocalized with early endosomes at 5 min and with lysosomes mainly at 30 min post-infection. Finally, disruption of the microtubule and microfilaments by nocodazole and by cytochalasin D reduced viral infection by more than 80%. Taken together these results indicate that dengue virions enter into C6/36 HT cells by clathrin-mediated endocytosis, using the endosomal pathway from early endosomes to acidic lysosomes before viral RNA is released into the cytoplasm.

Asunto(s)

Aedes/citología , Aedes/virología , Clatrina/metabolismo , Virus del Dengue/patogenicidad , Endocitosis/fisiología , Aedes/clasificación , Animales , Línea Celular , Células Cultivadas , Cricetinae , Virus del Dengue/genética , Virus del Dengue/metabolismo , Endosomas/virología , Técnica del Anticuerpo Fluorescente , Lisosomas/virología , Microscopía Confocal , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Virión/patogenicidad , Internalización del Virus

RESUMEN

Entry of dengue virus 2 (DENV-2) into Aedes albopictus mosquito C6/36 cells was analysed using biochemical and molecular inhibitors, together with confocal and electron microscopy observations. Treatment with monodansylcadaverine, chlorpromazine, sucrose and ammonium chloride inhibited DENV-2 virus yield and protein expression, whereas nystatin, a blocker of caveolae-mediated endocytosis, did not have any effect. Using confocal microscopy, co-localization of DENV-2 E glycoprotein and the marker protein transferrin was observed at the periphery of the cytoplasm. To support the requirement of clathrin function for DENV-2 entry, overexpression of a dominant-negative mutant of Eps15 in C6/36 cells was shown to impair virus entry. The disruption of actin microfilaments by cytochalasin D also significantly affected DENV-2 replication. In contrast, microtubule disruption by colchicine treatment did not impair DENV-2 infectivity, suggesting that DENV-2 does not require transport from early to late endosomes for successful infection of mosquito cells. Furthermore, using transmission electron microscopy, DENV-2 particles of approximately 44-52 nm were found attached within electron-dense invaginations of the plasma membrane and in coated vesicles that resembled those of clathrin-coated pits and vesicles, respectively. Together, these results demonstrate for the first time that DENV-2 enters insect cells by receptor-mediated, clathrin-dependent endocytosis, requiring traffic through an acidic pH compartment for subsequent uncoating and completion of a productive infection.

Asunto(s)

Aedes/virología , Clatrina/fisiología , Virus del Dengue/fisiología , Endocitosis/fisiología , Internalización del Virus , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/virología , Actinas/metabolismo , Animales , Transporte Biológico , Chlorocebus aethiops , Clatrina/genética , Citocalasina D/farmacología , Endocitosis/efectos de los fármacos , Endosomas/virología , Microscopía Confocal , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Transferrina/ultraestructura , Células Vero , Proteínas del Envoltorio Viral/ultraestructura , Internalización del Virus/efectos de los fármacos

RESUMEN

Venezuelan equine encephalitis virus (VEEV) is a New World alphavirus that can cause fatal encephalitis in humans. It remains a naturally emerging disease as well as a highly developed biological weapon. VEEV is transmitted to humans in nature by mosquito vectors. Little is known about VEEV entry, especially in mosquito cells. Here, a novel luciferase-based virus entry assay is used to show that the entry of VEEV into mosquito cells requires acidification. Furthermore, mosquito homologs of key human proteins (Rab5 and Rab7) involved in endocytosis were isolated and characterized. Rab5 is found on early endosomes and Rab7 on late endosomes and both are important for VEEV entry in mammalian cells. Each was shown to have analogous function in mosquito cells to that seen in mammalian cells. The wild-type, dominant negative and constitutively active mutants were then used to demonstrate that VEEV requires passage through early and late endosomes before infection can take place. This work indicates that the infection mechanism in mosquitoes and mammals is through a common and ancient evolutionarily conserved pathway.

Asunto(s)

Culicidae/virología , Virus de la Encefalitis Equina Venezolana/crecimiento & desarrollo , Endosomas/virología , Internalización del Virus , Proteínas de Unión al GTP rab/fisiología , Proteínas de Unión al GTP rab5/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Culicidae/citología , Endosomas/química , Genes Reporteros , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Microscopía Confocal , Datos de Secuencia Molecular , Alineación de Secuencia , Proteínas de Unión al GTP rab/análisis , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab5/análisis , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión a GTP rab7

RESUMEN

Virus envelope proteins determine receptor utilization and host range. The choice of receptor not only permits specific targeting of cells that express it, but also directs the virus into specific endosomal trafficking pathways. Disrupting trafficking can result in loss of virus infectivity due to redirection of virions to non-productive pathways. Identification of the pathway or pathways used by a virus is, thus, important in understanding virus pathogenesis mechanisms and for developing new treatment strategies. Most of our understanding of alphavirus entry has focused on the Old World alphaviruses, such as Sindbis and Semliki Forest virus. In comparison, very little is known about the entry route taken by more pathogenic New World alphaviruses. Here, we use a novel contents mixing assay to identify the cellular requirements for entry of a New World alphavirus, Venezuelan equine encephalitis virus (VEEV). Expression of dominant negative forms of key endosomal trafficking genes shows that VEEV must access clathrin-dependent endocytic vesicles for membrane fusion to occur. Unexpectedly, the exit point is different from Old World alphaviruses that leave from early endosomes. Instead, VEEV also requires functional late endosomes. Furthermore, unlike the Old World viruses, VEEV entry is insensitive to cholesterol sequestration from cell membranes and may reflect a need to access an endocytic compartment that lacks cholesterol. This indicates fundamental differences in the entry route taken by VEEV compared to Old World alphaviruses.

Asunto(s)

Colesterol/metabolismo , Virus de la Encefalitis Equina Venezolana/fisiología , Endosomas/virología , Línea Celular , Membrana Celular , Virus de la Encefalitis Equina Venezolana/genética , Endosomas/metabolismo , Vectores Genéticos/fisiología , Venezuela