RESUMEN
Herpes simplex virus type 1 (HSV-1) has evolved mechanisms to exploit the host cytoskeleton during entry, replication and exit from cells. In this study, we determined the role of actin and the molecular motor proteins, myosin II and myosin V, in the transport and release of HSV-1 from axon termini, or growth cones. Using compartmentalized neuronal devices, we showed that inhibition of actin polymerization, but not actin branching, significantly reduced the release of HSV-1 from axons. Furthermore, we showed that inhibition of myosin V, but not myosin II, also significantly reduced the release of HSV-1 from axons. Using confocal and electron microscopy, we determined that viral components are transported along axons to growth cones, despite actin or myosin inhibition. Overall, our study supports the role of actin in virus release from axonal growth cones and suggests myosin V as a likely candidate involved in this process.
Asunto(s)
Citoesqueleto de Actina/virología , Conos de Crecimiento/virología , Herpes Simple/virología , Liberación del Virus/fisiología , Animales , Transporte Axonal/fisiología , Conos de Crecimiento/ultraestructura , Herpesvirus Humano 1 , Ratas , Ratas WistarRESUMEN
The emerging coronavirus (CoV) pandemic is threatening the public health all over the world. Cytoskeleton is an intricate network involved in controlling cell shape, cargo transport, signal transduction, and cell division. Infection biology studies have illuminated essential roles for cytoskeleton in mediating the outcome of hostâvirus interactions. In this review, we discuss the dynamic interactions between actin filaments, microtubules, intermediate filaments, and CoVs. In one round of viral life cycle, CoVs surf along filopodia on the host membrane to the entry sites, utilize specific intermediate filament protein as co-receptor to enter target cells, hijack microtubules for transportation to replication and assembly sites, and promote actin filaments polymerization to provide forces for egress. During CoV infection, disruption of host cytoskeleton homeostasis and modification state is tightly connected to pathological processes, such as defective cytokinesis, demyelinating, cilia loss, and neuron necrosis. There are increasing mechanistic studies on cytoskeleton upon CoV infection, such as viral proteinâcytoskeleton interaction, changes in the expression and post-translation modification, related signaling pathways, and incorporation with other host factors. Collectively, these insights provide new concepts for fundamental virology and the control of CoV infection.
Asunto(s)
Infecciones por Coronavirus/virología , Coronavirus/patogenicidad , Citoesqueleto/virología , Interacciones Microbiota-Huesped/fisiología , Citoesqueleto de Actina/fisiología , Citoesqueleto de Actina/virología , Animales , Transporte Biológico Activo , Encéfalo/patología , Cilios/patología , Coronavirus/clasificación , Coronavirus/fisiología , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/fisiopatología , Citoesqueleto/patología , Citoesqueleto/fisiología , Humanos , Filamentos Intermedios/fisiología , Filamentos Intermedios/virología , Microtúbulos/fisiología , Microtúbulos/virología , Modelos Biológicos , Filogenia , Receptores Virales/fisiología , Transducción de Señal , Ensamble de Virus , Internalización del Virus , Replicación ViralRESUMEN
The actin cytoskeleton and Rho GTPase signaling to actin assembly are prime targets of bacterial and viral pathogens, simply because actin is involved in all motile and membrane remodeling processes, such as phagocytosis, macropinocytosis, endocytosis, exocytosis, vesicular trafficking and membrane fusion events, motility, and last but not least, autophagy. This article aims at providing an overview of the most prominent pathogen-induced or -hijacked actin structures, and an outlook on how future research might uncover additional, equally sophisticated interactions.
Asunto(s)
Citoesqueleto de Actina/microbiología , Citoesqueleto de Actina/virología , Membrana Celular/microbiología , Membrana Celular/virología , Interacciones Huésped-Patógeno , Citoesqueleto de Actina/metabolismo , Autofagia , Bacterias/patogenicidad , Membrana Celular/metabolismo , Humanos , Transducción de Señal , Virulencia , Virus/patogenicidadRESUMEN
When infecting host cells, influenza virus must move on microfilaments (MFs) at the cell periphery and then move along microtubules (MTs) through the cytosol to reach the perinuclear region for genome release. But how viruses switch from the actin roadway to the microtubule highway remains obscure. To settle this issue, we systematically dissected the role of related motor proteins in the transport of influenza virus between cytoskeletal filaments in situ and in real-time using quantum dot (QD)-based single-virus tracking (SVT) and multicolor imaging. We found that the switch between MF- and MT-based retrograde motor proteins, myosin VI (myoVI) and dynein, was responsible for the seamless transport of viruses from MFs to MTs during their infection. After virus entry by endocytosis, both the two types of motor proteins are attached to virus-carrying vesicles. MyoVI drives the viruses on MFs with dynein on the virus-carrying vesicle hitchhiking. After role exchanges at actin-microtubule intersections, dynein drives the virus along MTs toward the perinuclear region with myoVI remaining on the vesicle moving together. Such a "driver switchover" mechanism has answered the long-pending question of how viruses switch from MFs to MTs for their infection. It will also facilitate in-depth understanding of endocytosis.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Interacciones Huésped-Patógeno , Subtipo H9N2 del Virus de la Influenza A/fisiología , Microtúbulos/metabolismo , Infecciones por Orthomyxoviridae/metabolismo , Citoesqueleto de Actina/patología , Citoesqueleto de Actina/virología , Animales , Perros , Dineínas/metabolismo , Endocitosis , Células de Riñón Canino Madin Darby , Microscopía Confocal , Microtúbulos/patología , Microtúbulos/virología , Cadenas Pesadas de Miosina/metabolismo , Imagen Óptica , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Internalización del VirusRESUMEN
AIM: To investigate the detailed mechanism of Japanese encephalitis virus (JEV) cell entry. MATERIALS & METHODS: Utilize a siRNA library targeting cellular membrane trafficking genes to identify key molecules that mediate JEV entry into human neuronal cells. RESULTS: JEV enters human neuronal cells by caveolin-1-mediated endocytosis, which depends on a two-step regulation of actin cytoskeleton remodeling triggered by RhoA and Rac1: RhoA activation promoted the phosphorylation of caveolin-1, and then Rac1 activation facilitated caveolin-associated viral internalization. Specifically, virus attachment activates the EGFR-PI3K signaling pathway, thereby leading to RhoA activation. CONCLUSION: This work provides a detailed picture of the entry route and intricate cellular events following the entry of JEV into human neuronal cells, and promotes a better understanding of JEV entry.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/virología , Caveolina 1/metabolismo , Virus de la Encefalitis Japonesa (Especie)/metabolismo , Virus de la Encefalitis Japonesa (Especie)/fisiología , Internalización del Virus/efectos de los fármacos , Citoesqueleto de Actina/efectos de los fármacos , Factores Despolimerizantes de la Actina/metabolismo , Factores Despolimerizantes de la Actina/farmacología , Animales , Caveolina 1/efectos de los fármacos , Caveolina 1/genética , Línea Celular , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/virología , Colesterol/metabolismo , Cricetinae , Dinamina II/genética , Dinamina II/metabolismo , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis Japonesa (Especie)/patogenicidad , Encefalitis Japonesa/virología , Endocitosis/fisiología , Receptores ErbB/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno/fisiología , Humanos , Estadios del Ciclo de Vida/fisiología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Neuronas/virología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/farmacología , ARN Interferente Pequeño/genética , Transducción de Señal , Transfección , Acoplamiento Viral , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/farmacología , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/farmacologíaAsunto(s)
Citoesqueleto de Actina/virología , Corriente Citoplasmática , Retículo Endoplásmico/virología , Proteínas de Movimiento Viral en Plantas/metabolismo , Virus de Plantas/fisiología , Plantas/virología , Citoesqueleto de Actina/metabolismo , Retículo Endoplásmico/metabolismo , Interacciones Huésped-Patógeno , Virus de Plantas/metabolismo , Plantas/metabolismoRESUMEN
AIMS: Human cytomegalovirus (HCMV) infection has been linked to the pathogenesis of vasculopathies; however, its pathogenic relevance remains to be established. A prerequisite for vascular repair is endothelial cell migration. We evaluated the influence of HCMV on chemokinesis and chemotactic response of human coronary artery endothelial cells (HCAEC) towards vascular endothelial growth factor (VEGF). METHODS AND RESULTS: A virus dose-dependent reduction in chemokinesis and VEGF-dependent chemotaxis was observed (P < 0.05). UV-inactivated virus did not inhibit chemotaxis or chemokinesis, indicating that viral gene expression is mandatory. We identified two HCMV-induced mechanisms explaining the reduction of chemotaxis: first, a non-ambiguous reduction of VEGFR-2 protein was observed, due to decreased transcription. This protein down-modulation could not be inhibited by Ganciclovir. The remaining VEGFR-2 expressed on infected HCAEC was able to stimulate cell activation. Second, HCMV infection influences actin polymerization in HCAEC as shown by FACS analysis: actin polymerization was significantly reduced to 53 and 51% (P < 0.05) compared with non-infected HCAEC at 24 and 72 h p.i., respectively. Genetically and pharmacologically eliminated VEGFR-2 function resulted in a significant (P < 0.05) reduction of VEGF-induced activation of actin polymerization. CONCLUSION: We demonstrated a significant reduction of the chemotactic mobility of HCMV-infected HCAEC mediated by down-modulation of the VEGFR-2 and by inhibition of actin polymerization. This VEGF resistance of HCMV-infected endothelial cells is likely to promote atherogenesis.
Asunto(s)
Citoesqueleto de Actina/virología , Quimiotaxis/efectos de los fármacos , Infecciones por Citomegalovirus/virología , Citomegalovirus/patogenicidad , Células Endoteliales/virología , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Línea Celular , Citomegalovirus/genética , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/metabolismo , Regulación hacia Abajo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Regulación Viral de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Interferencia de ARN , Factores de Tiempo , Transcripción Genética , Transfección , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Tick-borne encephalitis virus (TBEV) causes one of the most dangerous human neuroinfections in Europe and Asia. To infect neurons it must cross the blood-brain-barrier (BBB), and presumably also cells adjacent to the BBB, such as astrocytes, the most abundant glial cell type. However, the knowledge about the viral infection of glial cells is fragmental. Here we studied whether TBEV infects rat astrocytes. Rats belong to an animal group serving as a TBEV amplifying host. We employed high resolution quantitative fluorescence microscopy to investigate cell entry and cytoplasmic mobility of TBEV particles along with the effect on the cell cytoskeleton and cell survival. We report that infection of astrocytes with TBEV increases with time of exposure to TBEV and that with post-infection time TBEV particles gained higher mobility. After several days of infection actin cytoskeleton was affected, but cell survival was unchanged, indicating that rat astrocytes resist TBEV-mediated cell death, as reported for other mammalian cells. Therefore, astrocytes may present an important pool of dormant TBEV infections and a new target for therapeutic intervention.
Asunto(s)
Astrocitos/patología , Astrocitos/virología , Virus de la Encefalitis Transmitidos por Garrapatas/fisiología , Encefalitis Transmitida por Garrapatas/patología , Encefalitis Transmitida por Garrapatas/veterinaria , Ratas/virología , Citoesqueleto de Actina/patología , Citoesqueleto de Actina/virología , Animales , Astrocitos/citología , Supervivencia Celular , Células Cultivadas , Humanos , Ratas Wistar , Internalización del VirusRESUMEN
The role of clathrin in pathogen entry has received much attention and has highlighted the adaptability of clathrin during internalization. Recent studies have now uncovered additional roles for clathrin and have put the spotlight on its role in pathogen spread. Here, we discuss the manipulation of clathrin by pathogens, with specific attention to the processes that occur at the plasma membrane. In the majority of cases, both clathrin and the actin cytoskeleton are hijacked, so we also examine the interplay between these two systems and their role during pathogen internalization, egress and spread.
Asunto(s)
Actinas/metabolismo , Clatrina/metabolismo , Endocitosis , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/microbiología , Citoesqueleto de Actina/virología , Animales , Adhesión Bacteriana , Membrana Celular/metabolismo , Membrana Celular/microbiología , Membrana Celular/virología , Interacciones Huésped-Patógeno , Transporte de Proteínas , Shigella flexneri/fisiología , Virus Vaccinia/fisiología , Internalización del Virus , Liberación del Virus , Replicación ViralRESUMEN
Plant viruses overcome the barrier of the plant cell wall by encoding cell-to-cell movement proteins (MPs), which direct newly replicated viral genomes to, and across, the wall. The paradigm for how a single MP regulates and coordinates these activities is the Tobacco mosaic virus (TMV) 30-kDa protein (MP(TMV)). Detailed studies demonstrate that TMV multiplies exclusively in the cytoplasm and have documented associations of MP(TMV) with endoplasmic reticulum (ER) membrane, microtubules, and plasmodesmata throughout the course of infection. As TMV poorly infects Arabidopsis thaliana, Turnip vein clearing virus (TVCV) is the tobamovirus of choice for studies in this model plant. A key problem, which has contributed to confusion in the field, is the unproven assumption that the TVCV and TMV life cycles are identical. We engineered an infectious TVCV replicon that expressed a functional fluorescence-tagged MP(TVCV) and report here the unexpected discovery that MP(TVCV), beyond localizing to ER membrane and plasmodesmata, targeted to the nucleus in a nuclear localization signal (NLS)-dependent manner, where it localized to novel F-actin-containing filaments that associated with chromatin. The MP(TVCV) NLS appeared to be conserved in the subgroup 3 tobamoviruses, and our mutational analyses showed that nuclear localization of MP(TVCV) was necessary for efficient TVCV cell-to-cell movement and systemic infection in Nicotiana benthamiana and Arabidopsis thaliana. Our studies identify a novel nuclear stage in TVCV infection and suggest that nuclear MP encoded by TVCV and other subgroup 3 tobamoviruses interacts with F-actin and chromatin to modulate host defenses or cellular physiology to favor virus movement and infection.
Asunto(s)
Citoesqueleto de Actina/virología , Arabidopsis/virología , Núcleo Celular/virología , Nicotiana/virología , Enfermedades de las Plantas/virología , Proteínas de Movimiento Viral en Plantas/metabolismo , Tobamovirus/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Señales de Localización Nuclear , Proteínas de Movimiento Viral en Plantas/química , Proteínas de Movimiento Viral en Plantas/genética , Transporte de Proteínas , Nicotiana/metabolismo , Tobamovirus/química , Tobamovirus/genéticaRESUMEN
The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a haemorrhagic fever with high mortality in human. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post-translational modifications of DG in the context of LASV cell entry. Using the tyrosine kinase inhibitor genistein, we found that tyrosine kinases are required for efficient internalization of virus particles, but not virus-receptor binding. Engagement of cellular DG by LASV envelope glycoprotein (LASV GP) in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. This virus-induced dissociation of utrophin was affected by genistein treatment, suggesting a role of receptor tyrosine phosphorylation in the process.
Asunto(s)
Distroglicanos/metabolismo , Matriz Extracelular/virología , Fiebre de Lassa/genética , Virus Lassa/patogenicidad , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/virología , Matriz Extracelular/metabolismo , Humanos , Fiebre de Lassa/virología , Virus Lassa/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Tirosina/genética , Tirosina/metabolismo , Utrofina/genética , Utrofina/metabolismoRESUMEN
For an infecting viral pathogen, the actin cortex inside the host cell is the first line of intracellular components that it encounters. Viruses devise various strategies to actively engage or circumvent the actin structure. In this regard, the human immunodeficiency virus-1 (HIV-1) exemplifies command of cellular processes to take control of actin dynamics for the initiation of infection. It has becomes increasingly evident that cortical actin presents itself both as a barrier to viral intracellular migration and as a necessary cofactor that the virus must actively engage, particularly, in the infection of resting CD4 blood T cells, the primary targets of HIV-1. The coercion of this most fundamental cellular component permits infection by facilitating entry, reverse transcription, and nuclear migration, three essential processes for the establishment of viral infection and latency in blood T cells. It is the purpose of this review to examine, in detail, the manifestation of viral dependence on the actin cytoskeleton, and present a model of how HIV utilizes actin dynamics to initiate infection.
Asunto(s)
Citoesqueleto de Actina/virología , ADN Viral/biosíntesis , VIH-1/patogenicidad , Factores Despolimerizantes de la Actina/genética , Factores Despolimerizantes de la Actina/metabolismo , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/genética , VIH-1/fisiología , Humanos , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transcripción Reversa , Transducción de Señal , Internalización del Virus , Replicación ViralRESUMEN
Nef, a HIV-1 pathogenesis factor, elevates virus replication in vivo and thus progression to AIDS by incompletely defined mechanisms. As one of its biological properties, Nef enhances the infectivity of cell-free HIV-1 particles in single round infections, however it fails to provide a significant and amplifying growth advantage for HIV-1 on such virus producing cells. A major difference between HIV-1 cell-free single round infections and virus replication kinetics on T lymphocytes consists in the predominant role of cell-associated virus transmission rather than cell-free infection during multiple round virus replication. HIV-1 cell-to-cell transmission occurs across close cell contacts also referred to as virological synapse (VS) and involves polarization of the F-actin cytoskeleton, formation of F-actin rich membrane bridges as well as virus budding to cell-cell contacts. Since Nef potently interferes with triggered actin remodelling in several cell systems to reduce e.g. cell motility and signal transduction, we set out here to address whether Nef also affects organization and possibly function of the T lymphocyte VS. We find that in addition to increasing infectivity of cell-free virions, Nef can also moderately enhance single rounds of HIV-1 cell-cell transmission between Jurkat T lymphocytes. This occurs without affecting cell conjugation efficiencies or polarization of F-actin and HIV-1 p24Gag at the VS, identifying actin remodelling at the VS as an example of Nef-insensitive host cell actin rearrangements. However, Nef-mediated enhancement of single round cell-free infection or cell-to-cell transmission does not potentiate over multiple rounds of infection. These results suggest that Nef affects cell-free and cell-associated HIV-1 infection by the same mechanism acting on the intrinsic infectivity of HIV-1 particles. They further indicate that the high efficacy of cell-to-cell transmission can compensate such infectivity defects. Nef therefore selectively interferes with actin remodelling processes involved in antiviral host cell defense while actin driven processes that promote virus propagation remain unaltered.
Asunto(s)
Actinas/metabolismo , VIH-1/metabolismo , Linfocitos T/metabolismo , Linfocitos T/virología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Citoesqueleto de Actina/ultraestructura , Citoesqueleto de Actina/virología , Movimiento Celular , Humanos , Células Jurkat , Transducción de Señal , Linfocitos T/ultraestructura , Estructuras Virales/ultraestructura , Internalización del Virus , Replicación ViralRESUMEN
Many diverse viruses target a polarized epithelial monolayer during host invasion. The polarized epithelium is adept at restricting the movement of solutes, ions, macromolecules, and pathogens across the mucosa. This regulation can be attributed to the presence of a junctional complex between adjacent cells and to an intricate network of actin filaments that provides support to the subapical membrane and stabilizes intercellular junctions. It is therefore not surprising that many viruses have evolved highly varied strategies to dissolve or modulate the cortical actin meshwork to promote infection of polarized cells. In this review, we will discuss the cell biological properties of the actin cytoskeleton in polarized epithelial cells and review the known mechanisms utilized by viral pathogens to manipulate this system in order to facilitate their infection.
Asunto(s)
Citoesqueleto de Actina/virología , Células Epiteliales/virología , Citoesqueleto de Actina/fisiología , Adenoviridae/fisiología , Animales , Polaridad Celular/fisiología , Enterovirus Humano B/fisiología , Células Epiteliales/fisiología , Herpesviridae/fisiología , Humanos , Orthomyxoviridae/fisiología , Rotavirus/fisiología , Uniones Estrechas/fisiología , Uniones Estrechas/virología , Internalización del Virus , Proteínas de Unión al GTP rho/fisiologíaRESUMEN
To investigate the role of cytoskeletal components in canine distemper virus (CDV) replication, various agents were used that interfere with turnover of actin filaments and microtubules. Only inhibition of actin filaments significantly reduced viral infectivity. Analysis of the intracellular localization of the viral matrix (M) protein revealed that it aligned along actin filaments. Treatment with actin filament-disrupting drugs led to a marked intracellular redistribution of M protein during infection as well as transfection. In contrast, the localization of the CDV fusion (F) protein was not significantly changed during transfection. Thus, a M protein-actin filament interaction appears to be important for generation of infectious CDV.
Asunto(s)
Citoesqueleto de Actina/virología , Virus del Moquillo Canino/metabolismo , Moquillo/virología , Proteínas de la Matriz Viral/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Moquillo/metabolismo , Virus del Moquillo Canino/genética , Perros , Unión Proteica , Transporte de Proteínas , Proteínas de la Matriz Viral/genéticaRESUMEN
The actin cytoskeleton has been implicated in the intra- and intercellular movement of a growing number of plant and animal viruses. However, the range of viruses influenced by actin for movement and the mechanism of this transport are poorly understood. Here we determine the importance of microfilaments and myosins for the sustained intercellular movement of a group of RNA-based plant viruses. We demonstrate that the intercellular movement of viruses from different genera [tobacco mosaic virus (TMV), potato virus X (PVX), tomato bushy stunt virus (TBSV)], is inhibited by disruption of microfilaments. Surprisingly, turnip vein-clearing virus (TVCV), a virus from the same genus as TMV, did not require intact microfilaments for normal spread. To investigate the molecular basis for this difference we compared the subcellular location of GFP fusions to the 126-kDa protein and the homologous 125-kDa protein from TMV and TVCV, respectively. The 126-kDa protein formed numerous large cytoplasmic inclusions associated with microfilaments, whereas the 125-kDa protein formed few small possible inclusions, none associated with microfilaments. The dependence of TMV, PVX, and TBSV on intact microfilaments for intercellular movement led us to investigate the role of myosin motors in this process. Virus-induced gene silencing of the Nicotiana benthamiana myosin XI-2 gene, but not three other myosins, inhibited only TMV movement. These results indicate that RNA viruses have evolved differently in their requirements for microfilaments and the associated myosin motors, in a manner not correlated with predicted phylogeny.
Asunto(s)
Actinas/metabolismo , Miosinas/metabolismo , Virus de Plantas/fisiología , Virus ARN/fisiología , Citoesqueleto de Actina/virología , Arabidopsis/genética , Citoplasma/virología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Plantas/virología , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Enteropathogens are known to disrupt apical actin filaments and/or tight-junction barriers of intestinal epithelial cells to promote infection. In this study, we show that a controlled, cytochalasin-D (Cyto-D)-mediated disruption of actin filaments and tight junctions enhanced the apical delivery of the gene-therapy vector recombinant adeno-associated virus serotype 2 (rAAV2). This increase in transduction efficiency can be attributed to the enhanced delivery of rAAV2 across the Cyto-D disrupted tight junctions, allowing basolateral entry of rAAV2. Previously, we have shown that MG101 and doxorubicin are capable of overcoming proteasome-mediated transduction barriers of rAAV2 in enterocytes. In this study, when Cyto-D was combined with MG101 and doxorubicin in apical delivery of rAAV2 to transduce the differentiated Caco-2 enterocytes, a synergistic >2300-fold increase in transgene expression was achieved. We conclude that Cyto-D is capable of permeating the polarized enterocytes for rAAV2 transduction, which may potentially be a useful device to facilitate intestinal gene transfer via the gut lumen.
Asunto(s)
Citocalasina D/farmacología , Dependovirus/fisiología , Enterocitos , Vectores Genéticos , Recombinación Genética , Uniones Estrechas , Transducción Genética , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/virología , Células CACO-2 , Citocalasina D/metabolismo , Dependovirus/genética , Enterocitos/efectos de los fármacos , Enterocitos/virología , Terapia Genética/métodos , Humanos , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/virologíaRESUMEN
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 fármacosRESUMEN
Cytoskeletal components play a major role in the human immunodeficiency virus-1 (HIV-1) infection. A wide variety of molecules belonging to the microfilament system, including actin filaments and actin binding proteins, as well as microtubules have a key role in regulating both cell life and death. Cell shape maintenance, cell polarity and cell movements as well as cytoplasmic trafficking of molecules determining cell fate, including apoptosis, are in fact instructed by the cytoskeleton components. HIV infection and viral particle production seem to be controlled by cytoskeleton as well. Furthermore, HIV-associated apoptosis failure can also be regulated by the actin network function. In fact, HIV protein gp120 is able to induce cytoskeleton-driven polarization, thus sensitizing T cells to CD95/Fas-mediated apoptosis. The microfilament system seems thus to be a sort of cytoplasmic supervisor of the viral particle, the host cell and the bystander cell's very fate.