RESUMO
Mammalian orthoreoviruses (reoviruses) are nonenveloped viruses that replicate in cytoplasmic membranous organelles called viral inclusions (VIs) where progeny virions are assembled. To better understand cellular routes of nonlytic reovirus exit, we imaged sites of virus egress in infected, nonpolarized human brain microvascular endothelial cells (HBMECs) and observed one or two distinct egress zones per cell at the basal surface. Transmission electron microscopy and 3D electron tomography (ET) of the egress zones revealed clusters of virions within membrane-bound structures, which we term membranous carriers (MCs), approaching and fusing with the plasma membrane. These virion-containing MCs emerged from larger, LAMP-1-positive membranous organelles that are morphologically compatible with lysosomes. We call these structures sorting organelles (SOs). Reovirus infection induces an increase in the number and size of lysosomes and modifies the pH of these organelles from â¼4.5-5 to â¼6.1 after recruitment to VIs and before incorporation of virions. ET of VI-SO-MC interfaces demonstrated that these compartments are connected by membrane-fusion points, through which mature virions are transported. Collectively, our results show that reovirus uses a previously undescribed, membrane-engaged, nonlytic egress mechanism and highlights a potential new target for therapeutic intervention.
Assuntos
Células Endoteliais/virologia , Lisossomos/virologia , Reoviridae/metabolismo , Vesículas Transportadoras/virologia , Liberação de Vírus/fisiologia , Cloreto de Amônio/farmacologia , Transporte Biológico , Biomarcadores/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/virologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Microscopia Eletrônica de Transmissão , Reoviridae/ultraestrutura , Vesículas Transportadoras/efeitos dos fármacos , Vesículas Transportadoras/metabolismo , Vírion/metabolismo , Vírion/ultraestrutura , Liberação de Vírus/efeitos dos fármacosRESUMO
Receptor-interacting protein (RIP) kinase 3 (RIPK3)-dependent necroptosis directs inflammation and tissue injury, as well as anti-viral host defense. In human cells, herpes simplex virus 1 (HSV1) UL39-encoded ICP6 blocks RIP homotypic interacting motif (RHIM) signal transduction, preventing this leakage form of cell death and sustaining viral infection. TNF receptor 1 (TNFR1)-induced necroptosis is known to require the formation of a RIPK1-RIPK3-mixed lineage kinase domain-like pseudokinase (MLKL) signaling complex (necrosome) that we find compartmentalizes exclusively to caveolin-1-associated detergent-resistant membrane (DRM) vesicles in HT-29 cells. Translocation proceeds in the presence of RIPK3 kinase inhibitor GSK'840 or MLKL inhibitor necrosulfonomide but requires the kinase activity, as well as RHIM signaling of RIPK1. ICP6 impedes the translocation of RIPK1, RIPK3, and MLKL to caveolin-1-containing DRM vesicles without fully blocking the activation of RIPK3 or phosphorylation of MLKL. Consistent with the important contribution of RIPK1 RHIM-dependent recruitment of RIPK3, overexpression of RHIM-deficient RIPK3 results in phosphorylation of MLKL, but this does not lead to either translocation or necroptosis. Combined, these data reveal a critical role of RHIM signaling in the recruitment of the MLKL-containing necrosome to membrane vesicle-associated sites of aggregation. A similar mechanism is predicted for other RHIM-containing signaling adaptors, Z-nucleic acid-binding protein 1 (ZBP1) (also called DAI and DLM1), and TIR domain-containing adapter-inducing interferon-ß (TRIF).
Assuntos
Herpesvirus Humano 1/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Vesículas Transportadoras/metabolismo , Proteínas Virais/metabolismo , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 1/genética , Humanos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas de Ligação a RNA , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Vesículas Transportadoras/genética , Vesículas Transportadoras/virologia , Células Vero , Proteínas Virais/genéticaRESUMO
The envelope glycoprotein (Env) plays a crucial role in the retroviral life cycle by mediating primary interactions with the host cell. As described previously and expanded on in this paper, Env mediates the trafficking of immature Mason-Pfizer monkey virus (M-PMV) particles to the plasma membrane (PM). Using a panel of labeled RabGTPases as endosomal markers, we identified Env mostly in Rab7a- and Rab9a-positive endosomes. Based on an analysis of the transport of recombinant fluorescently labeled M-PMV Gag and Env proteins, we propose a putative mechanism of the intracellular trafficking of M-PMV Env and immature particles. According to this model, a portion of Env is targeted from the trans-Golgi network (TGN) to Rab7a-positive endosomes. It is then transported to Rab9a-positive endosomes and back to the TGN. It is at the Rab9a vesicles where the immature particles may anchor to the membranes of the Env-containing vesicles, preventing Env recycling to the TGN. These Gag-associated vesicles are then transported to the plasma membrane.
Assuntos
Produtos do Gene env/metabolismo , Vírus dos Macacos de Mason-Pfizer/fisiologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vesículas Transportadoras/virologia , Animais , Membrana Celular/metabolismo , Membrana Celular/virologia , Endossomos/metabolismo , Endossomos/virologia , Produtos do Gene env/genética , Vírus dos Macacos de Mason-Pfizer/genética , Transporte Proteico , Vesículas Transportadoras/metabolismo , Montagem de VírusRESUMO
Adoptive transfer of genetically-modified T cells is a promising approach for treatment of both human malignancies and viral infections. Due to its ability to efficiently infect lymphocytes, the chimeric adenovirus Ad5F35 is potentially useful as an immunotherapeutic for the genetic modification of T cells. In previous studies, it was found that the infection efficiency of Ad5F35 was significantly increased without enhanced expression of the viral receptor after T cell stimulation; however, little is known about the underlying mechanism. Nonetheless, cell physiology has long been thought to affect viral infection. Therefore, we aimed to uncover the physiologic changes responsible for the increased infection efficiency of Ad5F35 following T cell stimulation. Given the complexity of intracellular transport we analyzed viral binding, entry, and escape using a Jurkat T cell model and found that both cell membrane fluidity and endosomal escape of Ad5F35 were altered under different physiological states. This, in turn, resulted in differences in the amount of virus entering cells and reaching the cytoplasm. These results provide additional insight into the molecular mechanisms underlying Ad5F35 infection of T cells and consequently, will help further the clinical application of genetically-modified T cells for immunotherapy.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Proteínas Recombinantes de Fusão/genética , Linfócitos T/metabolismo , Proteínas do Capsídeo/metabolismo , Ciclo Celular/fisiologia , Proliferação de Células , Células Cultivadas , Endossomos/ultraestrutura , Endossomos/virologia , Recuperação de Fluorescência Após Fotodegradação , Vetores Genéticos/genética , Células HEK293 , Humanos , Células Jurkat , Fluidez de Membrana , Microscopia Confocal , Microscopia Eletrônica , Receptores de Superfície Celular/metabolismo , Linfócitos T/fisiologia , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestrutura , Vesículas Transportadoras/virologia , Internalização do VírusRESUMO
West Nile virus (WNV) particles assemble at and bud into the endoplasmic reticulum (ER) and are secreted from infected cells through the secretory pathway. However, the host factor related to these steps is not fully understood. Rab proteins, belonging to the Ras superfamily, play essential roles in regulating many aspects of vesicular trafficking. In this study, we sought to determine which Rab proteins are involved in intracellular trafficking of nascent WNV particles. RNAi analysis revealed that Rab8b plays a role in WNV particle release. We found that Rab8 and WNV antigen were colocalized in WNV-infected human neuroblastoma cells, and that WNV infection enhanced Rab8 expression in the cells. In addition, the amount of WNV particles in the supernatant of Rab8b-deficient cells was significantly decreased compared with that of wild-type cells. We also demonstrated that WNV particles accumulated in the recycling endosomes in WNV-infected cells. In summary, these results suggest that Rab8b is involved in trafficking of WNV particles from recycling endosomes to the plasma membrane.
Assuntos
Endossomos/enzimologia , Vírus do Nilo Ocidental/fisiologia , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Transporte Biológico , Chlorocebus aethiops , Endossomos/virologia , Fibroblastos/enzimologia , Fibroblastos/virologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Knockout , Transporte Proteico , Vesículas Transportadoras/virologia , Células Vero , Proteínas Virais , Liberação de Vírus , Replicação ViralRESUMO
The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Nef interacts with a multitude of cellular proteins, manipulating the host membrane trafficking machinery to evade immune surveillance. Nef interactions have been analyzed using various in vitro assays, co-immunoprecipitation studies, and more recently mass spectrometry. However, these methods do not evaluate Nef interactions in the context of viral infection nor do they define the sub-cellular location of these interactions. In this report, we describe a novel bimolecular fluorescence complementation (BiFC) lentiviral expression tool, termed viral BiFC, to study Nef interactions with host cellular proteins in the context of viral infection. Using the F2A cleavage site from the foot and mouth disease virus we generated a viral BiFC expression vector capable of concurrent expression of Nef and host cellular proteins; PACS-1, MHC-I and SNX18. Our studies confirmed the interaction between Nef and PACS-1, a host membrane trafficking protein involved in Nef-mediated immune evasion, and demonstrated co-localization of this complex with LAMP-1 positive endolysosomal vesicles. Furthermore, we utilized viral BiFC to localize the Nef/MHC-I interaction to an AP-1 positive endosomal compartment. Finally, viral BiFC was observed between Nef and the membrane trafficking regulator SNX18. This novel demonstration of an association between Nef and SNX18 was localized to AP-1 positive vesicles. In summary, viral BiFC is a unique tool designed to analyze the interaction between Nef and host cellular proteins by mapping the sub-cellular locations of their interactions during viral infection.
Assuntos
Fluorescência , Vesículas Transportadoras/fisiologia , Integração Viral/fisiologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/fisiologia , Western Blotting , Citometria de Fluxo , Genes MHC Classe I/fisiologia , Células HEK293 , HIV-1/fisiologia , Células HeLa , Humanos , Células Jurkat , Lentivirus , Transporte Proteico/fisiologia , Nexinas de Classificação/fisiologia , Fator de Transcrição AP-1/fisiologia , Vesículas Transportadoras/virologia , Proteínas de Transporte Vesicular/fisiologia , Replicação Viral/fisiologiaRESUMO
Extracellular vesicles (EVs) being released from two adjacent adeno-associated virus serotype 1 (AAV1)-producing 293T cells are shown by electron microscopy. We have shown that AAV vectors can associate with EVs and enter the media. Furthermore, we have recently reported that EV-associated AAV has robust gene delivery and antibody evasion properties in vivo.
Assuntos
Dependovirus/ultraestrutura , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Microscopia Eletrônica/métodos , Vesículas Transportadoras/genética , Vesículas Transportadoras/virologiaRESUMO
The endocytic pathway is the principal cell entry pathway for large cargos and pathogens. Among the wide variety of specialized lipid structures within endosomes, the intraluminal vesicles (ILVs) formed in early endosomes (EEs) and transferred to late endosomal compartments are emerging as critical effectors of viral infection and immune recognition. Various viruses deliver their genomes into these ILVs, which serve as vehicles to transport the genome to the nuclear periphery for replication. When secreted as exosomes, ILVs containing viral genomes can infect permissive cells or activate immune responses in myeloid cells. We therefore propose that endosomal ILVs and exosomes are key effectors of viral pathogenesis.
Assuntos
Endossomos/metabolismo , Genoma Viral/genética , Vesículas Transportadoras/virologia , Animais , Endocitose , Humanos , Evasão da Resposta Imune , Internalização do VírusRESUMO
Knowledge on the morphogenesis of pestiviruses is limited due to low virus production in infected cells. In order to localize virion morphogenesis and replication sites of pestiviruses and to examine intracellular virion transport, a cell culture model was established to facilitate ultrastructural studies. Based on results of virus growth kinetic analysis and quantification of viral RNA, pestivirus strain Giraffe-1 turned out to be a suitable candidate for studies on virion generation and export from culture cells. Using conventional transmission electron microscopy and single-tilt electron tomography, we found virions located predominately in the lumen of the endoplasmic reticulum (ER) in infected cells and were able to depict the budding process of virions at ER membranes. Colocalization of the viral core protein and the envelope glycoprotein E2 with the ER marker protein disulfide isomerase (PDI) was demonstrated by immunogold labeling of cryosections. Moreover, pestivirions could be shown in transport vesicles and the Golgi complex and during exocytosis. Interestingly, viral capsid protein and double-stranded RNA (dsRNA) were detected in multivesicular bodies (MVBs), which implies that the endosomal compartment plays a role in pestiviral replication. Significant cellular membrane alterations such as those described for members of the Flavivirus and Hepacivirus genera were not found. Based on the gained morphological data, we present a consistent model of pestivirus morphogenesis.
Assuntos
Pestivirus/fisiologia , Pestivirus/ultraestrutura , Animais , Linhagem Celular , Retículo Endoplasmático/ultraestrutura , Retículo Endoplasmático/virologia , Endossomos/ultraestrutura , Endossomos/virologia , Complexo de Golgi/ultraestrutura , Complexo de Golgi/virologia , Cinética , Pestivirus/classificação , RNA de Cadeia Dupla/metabolismo , RNA Viral , Vesículas Transportadoras/ultraestrutura , Vesículas Transportadoras/virologia , Proteínas Virais/metabolismo , Montagem de Vírus , Liberação de Vírus , Replicação ViralRESUMO
Enteroviruses invade their hosts by crossing the intestinal epithelium. We have examined the mechanism by which echovirus 1 (EV1) enters polarized intestinal epithelial cells (Caco-2). Virus binds to VLA-2 on the apical cell surface and moves rapidly to early endosomes. Using inhibitory drugs, dominant negative mutants, and small interfering RNAs (siRNAs) to block specific endocytic pathways, we found that virus entry requires dynamin GTPase and membrane cholesterol but is independent of both clathrin- and caveolin-mediated endocytosis. Instead, infection requires factors commonly associated with macropinocytosis, including amiloride-sensitive Na(+)/H(+) exchange, protein kinase C, and C-terminal-binding protein-1 (CtBP1); furthermore, EV1 accumulates rapidly in intracellular vesicles with dextran, a fluid-phase marker. These results suggest a role for macropinocytosis in the process by which EV1 enters polarized cells to initiate infection.
Assuntos
Colesterol/metabolismo , Dinaminas/metabolismo , Enterovirus Humano B/fisiologia , Células Epiteliais/fisiologia , Interações Hospedeiro-Patógeno , Pinocitose , Internalização do Vírus , Oxirredutases do Álcool/metabolismo , Células CACO-2 , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/virologia , Humanos , Proteína Quinase C/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Vesículas Transportadoras/virologiaRESUMO
Infectious bursal disease virus (IBDV), a double-stranded RNA virus belonging to the Birnaviridae family, causes immunosuppression in chickens. In this study, we defined the localization of IBDV replication complexes based on colocalization analysis of VP3, the major protein component of IBDV ribonucleoproteins (RNPs). Our results indicate that VP3 localizes to vesicular structures bearing features of early and late endocytic compartments located in the juxtanuclear region. Interfering with the endocytic pathway with a dominant negative version of Rab5 after the internalization step leads to a reduction in virus titer. Triple-immunostaining studies between VP3, the viral RNA-dependent RNA polymerase VP1, and viral double-stranded RNA (dsRNA) showed a well-defined colocalization, indicating that the three critical components of the RNPs colocalize in the same structure, likely representing replication complexes. Interestingly, recombinant expressed VP3 also localizes to endosomes. Employing Golgi markers, we found that VP3-containing vesicles were closely associated with this organelle. Depolymerization of microtubules with nocodazole caused a profound change in VP3 localization, showing a punctate distribution scattered throughout the cytoplasm. However, these VP3-positive structures remained associated with Golgi ministacks. Similarly, brefeldin A (BFA) treatment led to a punctate distribution of VP3, scattered throughout the cytoplasm of infected cells. In addition, analysis of intra- and extracellular viral infective particles after BFA treatment of avian cells suggested a role for the Golgi complex in viral assembly. These results constitute the first study elucidating the localization of IBDV replication complexes (i.e., in endocytic compartments) and establishing a role for the Golgi apparatus in the assembly step of a birnavirus.
Assuntos
Complexo de Golgi/metabolismo , Complexo de Golgi/virologia , Vírus da Doença Infecciosa da Bursa/fisiologia , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/virologia , Montagem de Vírus , Replicação Viral , Animais , Linhagem Celular , Galinhas , Humanos , Proteínas Virais/metabolismoRESUMO
Rotavirus (RV) is the major cause of childhood gastroenteritis worldwide. This study presents a functional genome-scale analysis of cellular proteins and pathways relevant for RV infection using RNAi. Among the 522 proteins selected in the screen for their ability to affect viral infectivity, an enriched group that participates in endocytic processes was identified. Within these proteins, subunits of the vacuolar ATPase, small GTPases, actinin 4, and, of special interest, components of the endosomal sorting complex required for transport (ESCRT) machinery were found. Here we provide evidence for a role of the ESCRT complex in the entry of simian and human RV strains in both monkey and human epithelial cells. In addition, the ESCRT-associated ATPase VPS4A and phospholipid lysobisphosphatidic acid, both crucial for the formation of intralumenal vesicles in multivesicular bodies, were also found to be required for cell entry. Interestingly, it seems that regardless of the molecules that rhesus RV and human RV strains use for cell-surface attachment and the distinct endocytic pathway used, all these viruses converge in early endosomes and use multivesicular bodies for cell entry. Furthermore, the small GTPases RHOA and CDC42, which regulate different types of clathrin-independent endocytosis, as well as early endosomal antigen 1 (EEA1), were found to be involved in this process. This work reports the direct involvement of the ESCRT machinery in the life cycle of a nonenveloped virus and highlights the complex mechanism that these viruses use to enter cells. It also illustrates the efficiency of high-throughput RNAi screenings as genetic tools for comprehensively studying the interaction between viruses and their host cells.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Infecções por Rotavirus/metabolismo , Rotavirus/metabolismo , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/virologia , ATPases Associadas a Diversas Atividades Celulares , Animais , Células CACO-2 , Chlorocebus aethiops , Estudo de Associação Genômica Ampla , Humanos , Transporte Proteico/fisiologia , Interferência de RNA , Infecções por Rotavirus/virologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Células Vero , Proteínas de Transporte Vesicular/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7 , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Although Epstein-Barr virus (EBV) is an orally transmitted virus, viral transmission through the oropharyngeal mucosal epithelium is not well understood. In this study, we investigated how EBV traverses polarized human oral epithelial cells without causing productive infection. We found that EBV may be transcytosed through oral epithelial cells bidirectionally, from both the apical to the basolateral membranes and the basolateral to the apical membranes. Apical to basolateral EBV transcytosis was substantially reduced by amiloride, an inhibitor of macropinocytosis. Electron microscopy showed that virions were surrounded by apical surface protrusions and that virus was present in subapical vesicles. Inactivation of signaling molecules critical for macropinocytosis, including phosphatidylinositol 3-kinases, myosin light-chain kinase, Ras-related C3 botulinum toxin substrate 1, p21-activated kinase 1, ADP-ribosylation factor 6, and cell division control protein 42 homolog, led to significant reduction in EBV apical to basolateral transcytosis. In contrast, basolateral to apical EBV transcytosis was substantially reduced by nystatin, an inhibitor of caveolin-mediated virus entry. Caveolae were detected in the basolateral membranes of polarized human oral epithelial cells, and virions were detected in caveosome-like endosomes. Methyl ß-cyclodextrin, an inhibitor of caveola formation, reduced EBV basolateral entry. EBV virions transcytosed in either direction were able to infect B lymphocytes. Together, these data show that EBV transmigrates across oral epithelial cells by (i) apical to basolateral transcytosis, potentially contributing to initial EBV penetration that leads to systemic infection, and (ii) basolateral to apical transcytosis, which may enable EBV secretion into saliva in EBV-infected individuals.
Assuntos
Herpesvirus Humano 4/fisiologia , Mucosa Bucal/virologia , Transcitose/fisiologia , Fator 6 de Ribosilação do ADP , Amilorida/farmacologia , Animais , Callithrix , Linhagem Celular , Primers do DNA/genética , Imunofluorescência , Herpesvirus Humano 4/patogenicidade , Humanos , Imunoglobulina G/metabolismo , Queratinócitos/virologia , Microscopia Eletrônica de Transmissão , Nistatina/farmacologia , Tonsila Palatina/citologia , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia , Transcitose/efeitos dos fármacos , Vesículas Transportadoras/ultraestrutura , Vesículas Transportadoras/virologia , Vírion/fisiologia , Vírion/ultraestruturaRESUMO
Although membrane perforation was suggested as the means of penetration mediated by IBDV, the cellular mechanism being hijacked to facilitate its entry is largely unknown. In this study, the entry pathway of cell culture adapted IBDV (caIBDV) was characterized in DF-1 chicken embryonic fibroblasts. We observed that the entry of caIBDV was inhibited by bafilomycin A1 and CaEGTA which interfere with the function of vacuolar H(+)-ATPase (V-ATPase) and retain endosomal Ca(2+). This result suggests that the intact caIBDV particle was transported to the V-ATPase positive vesicles for uncoating and implicates an essential role of endocytosis during the viral entry. The IBDV-mediated endocytosis was demonstrated to be clathrin-independent. Instead, the entry of caIBDV in DF-1 was reduced under the inhibitions or depletions of lipid raft, c-Src tyrosine kinase, dynamin and actin polymerization. In summary, this study confirmed the role of endocytosis in caIBDV entry and characterized the route of its endocytosis.
Assuntos
Infecções por Birnaviridae/veterinária , Endocitose , Fibroblastos/virologia , Vírus da Doença Infecciosa da Bursa/fisiologia , Doenças das Aves Domésticas/virologia , Vesículas Transportadoras/virologia , Internalização do Vírus , Animais , Infecções por Birnaviridae/fisiopatologia , Infecções por Birnaviridae/virologia , Técnicas de Cultura de Células , Galinhas , Fibroblastos/fisiologia , Vírus da Doença Infecciosa da Bursa/genética , Microdomínios da Membrana/virologia , Doenças das Aves Domésticas/fisiopatologiaRESUMO
During herpes simplex virus 1 (HSV1) egress in neurons, viral particles travel from the neuronal cell body along the axon towards the synapse. Whether HSV1 particles are transported as enveloped virions as proposed by the 'married' model or as non-enveloped capsids suggested by the 'separate' model is controversial. Specific viral proteins may form a recruitment platform for microtubule motors that catalyze such transport. However, their subviral location has remained elusive. Here we established a system to analyze herpesvirus egress by cryo electron tomography. At 16 h post infection, we observed intra-axonal transport of progeny HSV1 viral particles in dissociated hippocampal neurons by live-cell fluorescence microscopy. Cryo electron tomography of frozen-hydrated neurons revealed that most egressing capsids were transported independently of the viral envelope. Unexpectedly, we found not only DNA-containing capsids (cytosolic C-capsids), but also capsids lacking DNA (cytosolic A-/B-capsids) in mid-axon regions. Subvolume averaging revealed lower amounts of tegument on cytosolic A-/B-capsids than on C-capsids. Nevertheless, all capsid types underwent active axonal transport. Therefore, even few tegument proteins on the capsid vertices seemed to suffice for transport. Secondary envelopment of capsids was observed at axon terminals. On their luminal face, the enveloping vesicles were studded with typical glycoprotein-like spikes. Furthermore, we noted an accretion of tegument density at the concave cytosolic face of the vesicle membrane in close proximity to the capsids. Three-dimensional analysis revealed that these assembly sites lacked cytoskeletal elements, but that filamentous actin surrounded them and formed an assembly compartment. Our data support the 'separate model' for HSV1 egress, i.e. progeny herpes viruses being transported along axons as subassemblies and not as complete virions within transport vesicles.
Assuntos
Transporte Axonal , Axônios/virologia , Tomografia com Microscopia Eletrônica/métodos , Herpesvirus Humano 1/metabolismo , Neurônios/virologia , Animais , Axônios/ultraestrutura , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Células Cultivadas , Herpesvirus Humano 1/ultraestrutura , Microtúbulos/metabolismo , Neurônios/metabolismo , Ratos , Vesículas Transportadoras/virologia , Vírion/metabolismo , Vírion/ultraestruturaRESUMO
Pathogens of bacterial and viral origin hijack pathways operating in eukaryotic cells in many ways in order to gain access into the host, to establish themselves and to eventually produce their progeny. The detailed molecular characterization of the subversion mechanisms devised by pathogens to infect host cells is crucial to generate targets for therapeutic intervention. Here we review recent data indicating that coronaviruses probably co-opt membranous carriers derived from the endoplasmic reticulum, which contain proteins that regulate disposal of misfolded polypeptides, for their replication. In addition, we also present models describing potential mechanisms that coronaviruses could employ for this hijacking.
Assuntos
Coronaviridae/fisiologia , Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático/virologia , Mamíferos/metabolismo , Vesículas Transportadoras/virologia , Replicação Viral , Animais , Autofagia/fisiologia , Infecções por Coronaviridae/virologia , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Vesículas Transportadoras/metabolismoRESUMO
Viruses intricately interact with and modulate cellular membranes at several stages of their replication, but much less is known about the role of viral lipids compared to proteins and nucleic acids. All animal viruses have to cross membranes for cell entry and exit, which occurs by membrane fusion (in enveloped viruses), by transient local disruption of membrane integrity, or by cell lysis. Furthermore, many viruses interact with cellular membrane compartments during their replication and often induce cytoplasmic membrane structures, in which genome replication and assembly occurs. Recent studies revealed details of membrane interaction, membrane bending, fission, and fusion for a number of viruses and unraveled the lipid composition of raft-dependent and -independent viruses. Alterations of membrane lipid composition can block viral release and entry, and certain lipids act as fusion inhibitors, suggesting a potential as antiviral drugs. Here, we review viral interactions with cellular membranes important for virus entry, cytoplasmic genome replication, and virus egress.
Assuntos
Lipídeos de Membrana/fisiologia , Replicação Viral/fisiologia , Genoma Viral , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/virologia , Modelos Biológicos , Vesículas Transportadoras/virologia , Internalização do VírusRESUMO
BACKGROUND: Proteins associated with the late endosome (LE) appear to play a central role in the envelopment of a number of taxonomically diverse viruses. How viral proteins interact with LE-associated proteins to facilitate envelopment is not well understood. LE-derived transport vesicles form through the interaction of Rab9 GTPase with cargo proteins, and TIP47, a Rab9-specific effector protein. Vaccinia virus (VV) induces a wrapping complex derived from intracellular host membranes to envelope intracellular mature virus particles producing egress-competent forms of virus. RESULTS: We show that VV p37 protein associates with TIP47-, Rab9-, and CI-MPR-containing membranes. Mutation of a di-aromatic motif in p37 blocks association with TIP47 and inhibits plaque formation. ST-246, a specific inhibitor of p37 function, inhibits these interactions and also blocks wrapped virus particle formation. Vaccinia virus expressing p37 variants with reduced ST-246 susceptibility associates with Rab9 and co-localizes with CI-MPR in the presence and absence of compound. CONCLUSION: These results suggest that p37 localizes to the LE and interacts with proteins associated with LE-derived transport vesicle biogenesis to facilitate assembly of extracellular forms of virus.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endossomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas da Gravidez/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Vesículas Transportadoras/metabolismo , Vaccinia virus/metabolismo , Vacínia/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Endossomos/virologia , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/virologia , Proteínas de Membrana/genética , Perilipina-3 , Ligação Proteica , Receptor IGF Tipo 2 , Vesículas Transportadoras/virologia , Vacínia/virologia , Vaccinia virus/genética , Proteínas de Transporte Vesicular , Proteínas do Envelope Viral/genéticaRESUMO
Chronic Helicobacter pylori infection is associated with an increased risk of gastric carcinogenesis. These non-invasive bacteria colonize the gastric mucosa and constitutively shed small outer membrane vesicles (OMV). In this study, we investigated the direct effect of H.pylori OMV on cellular events associated with carcinogenesis. We observed increased micronuclei formation in AGS human gastric epithelial cells treated with OMV isolated from a toxigenic H.pylori strain (60190). This effect was absent in OMV from strain 60190v:1 that has a mutant vacA, indicating VacA-dependent micronuclei formation. VacA induces intracellular vacuolation, and reduced acridine orange staining indicated disruption in the integrity of these vacuoles. This was accompanied by an alteration in iron metabolism and glutathione (GSH) loss, suggesting a role for oxidative stress in genomic damage. Increasing intracellular GSH levels with a GSH ester abrogated the VacA-mediated increase in micronuclei formation. In conclusion, OMV-mediated delivery of VacA to the gastric epithelium may constitute a new mechanism for H.pylori-induced gastric carcinogenesis.
Assuntos
Transformação Celular Neoplásica , Helicobacter pylori/patogenicidade , Neoplasias Gástricas/virologia , Vesículas Transportadoras/virologia , Proteínas de Bactérias , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/virologia , Imunofluorescência , Glutationa/metabolismo , Humanos , Ferro/metabolismo , Micronúcleos com Defeito Cromossômico , Estresse Oxidativo/fisiologia , Carbonilação Proteica/fisiologia , Neoplasias Gástricas/patologia , Vesículas Transportadoras/metabolismoRESUMO
Tandem affinity purification was used in Arabidopsis thaliana to identify cellular interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp). The heat shock cognate 70-3 (Hsc70-3) and poly(A)-binding (PABP) host proteins were recovered and shown to interact with the RdRp in vitro. As previously shown for PABP, Hsc70-3 was redistributed to nuclear and membranous fractions in infected plants and both RdRp interactors were co-immunoprecipitated from a membrane-enriched extract using RdRp-specific antibodies. Fluorescently tagged RdRp and Hsc70-3 localized to the cytoplasm and the nucleus when expressed alone or in combination in Nicotiana benthamiana. However, they were redistributed to large perinuclear ER-derived vesicles when co-expressed with the membrane binding 6K-VPg-Pro protein of TuMV. The association of Hsc70-3 with the RdRp could possibly take place in membrane-derived replication complexes. Thus, Hsc70-3 and PABP2 are potentially integral components of the replicase complex and could have important roles to play in the regulation of potyviral RdRp functions.