RESUMEN
In order to initiate successful infection, viruses have to transmit and deliver their genome from one host cell or organism to another. To achieve this, enveloped viruses must first fuse their membrane with those of the target host cell. Here, we describe the sequence of events leading to the entry of representative enveloped viruses, highlighting the strategies they use to gain access to the host cell cytosol.
Asunto(s)
Endocitosis , Endosomas/virología , Fusión de Membrana , Internalización del Virus , Virus/metabolismo , Animales , Endosomas/metabolismo , Humanos , Virosis/enzimología , Virosis/metabolismo , Virus/genéticaRESUMEN
Cellular attachment of viruses determines their cell tropism and species specificity. For entry, vaccinia, the prototypic poxvirus, relies on four binding proteins and an eleven-protein entry fusion complex. The contribution of the individual virus binding proteins to virion binding orientation and membrane fusion is unclear. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards the virus fusion machinery to facilitate fusion. Using a membrane-bleb model system together with super-resolution and electron microscopy we find that side-bound vaccinia virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins reveals that three of four contribute to binding orientation, amongst which the chondroitin sulfate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection. Our results show that viral binding proteins are active participants in successful virus membrane fusion and illustrate the importance of virus protein architecture for successful infection.
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Poxviridae , Vaccinia , Humanos , Sulfatos de Condroitina , Virus Vaccinia/metabolismo , Poxviridae/metabolismo , Proteínas Virales/metabolismo , Fusión de Membrana , Proteínas PortadorasRESUMEN
Although viruses are simple in structure and composition, their interactions with host cells are complex. Merely to gain entry, animal viruses make use of a repertoire of cellular processes that involve hundreds of cellular proteins. Although some viruses have the capacity to penetrate into the cytosol directly through the plasma membrane, most depend on endocytic uptake, vesicular transport through the cytoplasm, and delivery to endosomes and other intracellular organelles. The internalization may involve clathrin-mediated endocytosis (CME), macropinocytosis, caveolar/lipid raft-mediated endocytosis, or a variety of other still poorly characterized mechanisms. This review focuses on the cell biology of virus entry and the different strategies and endocytic mechanisms used by animal viruses.
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Endocitosis , Internalización del Virus , Animales , Caveolas/metabolismo , Clatrina/metabolismo , Microdominios de Membrana/metabolismo , Fagocitosis , PinocitosisRESUMEN
Cardiac glycosides (CGs) are natural steroid glycosides, which act as inhibitors of the cellular sodium-potassium ATPase pump. Although traditionally considered toxic to human cells, CGs are widely used as drugs for the treatment of cardiovascular-related medical conditions. More recently, CGs have been explored as potential anti-viral drugs and inhibit replication of a range of RNA and DNA viruses. Previously, a compound screen identified CGs that inhibited vaccinia virus (VACV) infection. However, no further investigation of the inhibitory potential of these compounds was performed, nor was there investigation of the stage(s) of the poxvirus lifecycle they impacted. Here, we investigated the anti-poxvirus activity of a broad panel of CGs. We found that all CGs tested were potent inhibitors of VACV replication. Our virological experiments showed that CGs did not impact virus infectivity, binding, or entry. Rather, experiments using recombinant viruses expressing reporter proteins controlled by VACV promoters and arabinoside release assays demonstrated that CGs inhibited early and late VACV protein expression at different concentrations. Lack of virus assembly in the presence of CGs was confirmed using electron microscopy. Thus, we expand our understanding of compounds with anti-poxvirus activity and highlight a yet unrecognized mechanism by which poxvirus replication can be inhibited.
Asunto(s)
Glicósidos Cardíacos , Poxviridae , Vaccinia , Humanos , Virus Vaccinia/genética , Glicósidos Cardíacos/farmacología , Glicósidos Cardíacos/metabolismo , Replicación ViralRESUMEN
All poxviruses contain a set of proteinaceous structures termed lateral bodies (LB) that deliver viral effector proteins into the host cytosol during virus entry. To date, the spatial proteotype of LBs remains unknown. Using the prototypic poxvirus, vaccinia virus (VACV), we employed a quantitative comparative mass spectrometry strategy to determine the poxvirus LB proteome. We identified a large population of candidate cellular proteins, the majority being mitochondrial, and 15 candidate viral LB proteins. Strikingly, one-third of these are VACV redox proteins whose LB residency could be confirmed using super-resolution microscopy. We show that VACV infection exerts an anti-oxidative effect on host cells and that artificial induction of oxidative stress impacts early and late gene expression as well as virion production. Using targeted repression and/or deletion viruses we found that deletion of individual LB-redox proteins was insufficient for host redox modulation suggesting there may be functional redundancy. In addition to defining the spatial proteotype of VACV LBs, these findings implicate poxvirus redox proteins as potential modulators of host oxidative anti-viral responses and provide a solid starting point for future investigations into the role of LB resident proteins in host immunomodulation.
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Poxviridae , Línea Celular , Oxidación-Reducción , Poxviridae/genética , Poxviridae/metabolismo , Virus Vaccinia/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
The regulation of size, volume and mass in living cells is physiologically important, and dysregulation of these parameters gives rise to many diseases. Cell mass is largely determined by the amount of water, proteins, lipids, carbohydrates and nucleic acids present in a cell, and is tightly linked to metabolism, proliferation and gene expression. Technologies have emerged in recent years that make it possible to track the masses of single suspended cells and adherent cells. However, it has not been possible to track individual adherent cells in physiological conditions at the mass and time resolutions required to observe fast cellular dynamics. Here we introduce a cell balance (a 'picobalance'), based on an optically excited microresonator, that measures the total mass of single or multiple adherent cells in culture conditions over days with millisecond time resolution and picogram mass sensitivity. Using our technique, we observe that the mass of living mammalian cells fluctuates intrinsically by around one to four per cent over timescales of seconds throughout the cell cycle. Perturbation experiments link these mass fluctuations to the basic cellular processes of ATP synthesis and water transport. Furthermore, we show that growth and cell cycle progression are arrested in cells infected with vaccinia virus, but mass fluctuations continue until cell death. Our measurements suggest that all living cells show fast and subtle mass fluctuations throughout the cell cycle. As our cell balance is easy to handle and compatible with fluorescence microscopy, we anticipate that our approach will contribute to the understanding of cell mass regulation in various cell states and across timescales, which is important in areas including physiology, cancer research, stem-cell differentiation and drug discovery.
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Ciclo Celular/fisiología , Tamaño de la Célula , Análisis de la Célula Individual/métodos , Adenosina Trifosfato/biosíntesis , Animales , Adhesión Celular , Muerte Celular , Supervivencia Celular , Fibroblastos/citología , Células HeLa , Humanos , Interfase , Ratones , Microscopía Fluorescente , Análisis de la Célula Individual/instrumentación , Virus Vaccinia/fisiología , Agua/metabolismoRESUMEN
The replication and assembly of vaccinia virus (VACV), the prototypic poxvirus, occurs exclusively in the cytoplasm of host cells. While the role of cellular cytoskeletal components in these processes remains poorly understood, vimentin-a type III intermediate filament-has been shown to associate with viral replication sites and to be incorporated into mature VACV virions. Here, we employed chemical and genetic approaches to further investigate the role of vimentin during the VACV lifecycle. The collapse of vimentin filaments, using acrylamide, was found to inhibit VACV infection at the level of genome replication, intermediate- and late-gene expression. However, we found that CRISPR-mediated knockout of vimentin did not impact VACV replication. Combining these tools, we demonstrate that acrylamide treatment results in the formation of anti-viral granules (AVGs) known to mediate translational inhibition of many viruses. We conclude that vimentin is dispensable for poxvirus replication and assembly and that acrylamide, as a potent inducer of AVGs during VACV infection, serves to bolster cell's anti-viral response to poxvirus infection.
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Antivirales , Virus Vaccinia , Acrilamida , Filamentos Intermedios , Vimentina/genética , Replicación ViralRESUMEN
Super-resolution microscopy depends on steps that can contribute to the formation of image artifacts, leading to misinterpretation of biological information. We present NanoJ-SQUIRREL, an ImageJ-based analytical approach that provides quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same acquisition volume, this approach generates a quantitative map of super-resolution defects and can guide researchers in optimizing imaging parameters.
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Artefactos , Procesamiento de Imagen Asistido por Computador/métodos , Imagen Óptica/métodos , AlgoritmosRESUMEN
Super-resolution microscopy (SRM) has become essential for the study of nanoscale biological processes. This type of imaging often requires the use of specialised image analysis tools to process a large volume of recorded data and extract quantitative information. In recent years, our team has built an open-source image analysis framework for SRM designed to combine high performance and ease of use. We named it NanoJ-a reference to the popular ImageJ software it was developed for. In this paper, we highlight the current capabilities of NanoJ for several essential processing steps: spatio-temporal alignment of raw data (NanoJ-Core), super-resolution image reconstruction (NanoJ-SRRF), image quality assessment (NanoJ-SQUIRREL), structural modelling (NanoJ-VirusMapper) and control of the sample environment (NanoJ-Fluidics). We expect to expand NanoJ in the future through the development of new tools designed to improve quantitative data analysis and measure the reliability of fluorescent microscopy studies.
RESUMEN
Endocytosis plays a crucial role in receptor signalling. VEGFR2 (also known as KDR) and its ligand VEGFA are fundamental in neovascularisation. However, our understanding of the role of endocytosis in VEGFR2 signalling remains limited. Despite the existence of diverse internalisation routes, the only known endocytic pathway for VEGFR2 is the clathrin-mediated pathway. Here, we show that this pathway is the predominant internalisation route for VEGFR2 only in the absence of ligand. Intriguingly, VEGFA induces a new internalisation itinerary for VEGFR2, the pathway of macropinocytosis, which becomes the prevalent endocytic route for the receptor in the presence of ligand, whereas the contribution of the clathrin-mediated route becomes minor. Macropinocytic internalisation of VEGFR2, which mechanistically is mediated through the small GTPase CDC42, takes place through macropinosomes generated at ruffling areas of the membrane. Interestingly, macropinocytosis plays a crucial role in VEGFA-induced signalling, endothelial cell functions in vitro and angiogenesis in vivo, whereas clathrin-mediated endocytosis is not essential for VEGFA signalling. These findings expand our knowledge on the endocytic pathways of VEGFR2 and suggest that VEGFA-driven internalisation of VEGFR2 through macropinocytosis is essential for endothelial cell signalling and angiogenesis.
Asunto(s)
Neovascularización Fisiológica , Pinocitosis , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Clatrina/metabolismo , Dinaminas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/ultraestructura , Humanos , Modelos Biológicos , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Virus infection of humans and livestock can be devastating for individuals and populations, sometimes resulting in large economic and societal impact. Prevention of virus disease by vaccination or antiviral agents is difficult to achieve. A notable exception was the eradication of human smallpox by vaccination over 30 years ago. Today, humans and animals remain susceptible to poxvirus infections, including zoonotic poxvirus transmission. Here we identified a small molecule, bisbenzimide (bisbenzimidazole), and its derivatives as potent agents against prototypic poxvirus infection in cell culture. We show that bisbenzimide derivatives, which preferentially bind the minor groove of double-stranded DNA, inhibit vaccinia virus infection by blocking viral DNA replication and abrogating postreplicative intermediate and late gene transcription. The bisbenzimide derivatives are potent against vaccinia virus and other poxviruses but ineffective against a range of other DNA and RNA viruses. The bisbenzimide derivatives are the first inhibitors of their class, which appear to directly target the viral genome without affecting cell viability.IMPORTANCE Smallpox was one of the most devastating diseases in human history until it was eradicated by a worldwide vaccination campaign. Due to discontinuation of routine vaccination more than 30 years ago, the majority of today's human population remains susceptible to infection with poxviruses. Here we present a family of bisbenzimide (bisbenzimidazole) derivatives, known as Hoechst nuclear stains, with high potency against poxvirus infection. Results from a variety of assays used to dissect the poxvirus life cycle demonstrate that bisbenzimides inhibit viral gene expression and genome replication. These findings can lead to the development of novel antiviral drugs that target viral genomes and block viral replication.
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Antivirales/farmacología , Bisbenzimidazol/farmacología , Replicación del ADN/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Virus Vaccinia/efectos de los fármacos , Virus Vaccinia/fisiología , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , Colorantes Fluorescentes , HumanosRESUMEN
The prototypic poxvirus, vaccinia virus (VACV), occurs in two infectious forms, mature virions (MVs) and extracellular virions (EVs). Both enter HeLa cells by inducing macropinocytic uptake. Using confocal microscopy, live-cell imaging, targeted RNAi screening and perturbants of endosome maturation, we analyzed the properties and maturation pathway of the macropinocytic vacuoles containing VACV MVs in HeLa cells. The vacuoles first acquired markers of early endosomes [Rab5, early endosome antigen 1 and phosphatidylinositol(3)P]. Prior to release of virus cores into the cytoplasm, they contained markers of late endosomes and lysosomes (Rab7a, lysosome-associated membrane protein 1 and sorting nexin 3). RNAi screening of endocytic cell factors emphasized the importance of late compartments for VACV infection. Follow-up perturbation analysis showed that infection required Rab7a and PIKfyve, confirming that VACV is a late-penetrating virus dependent on macropinosome maturation. VACV EV infection was inhibited by depletion of many of the same factors, indicating that both infectious particle forms share the need for late vacuolar conditions for penetration.
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Fagocitosis , Fagosomas/metabolismo , Virus Vaccinia/patogenicidad , Endosomas/metabolismo , Endosomas/virología , Células HeLa , Humanos , Proteína 1 de la Membrana Asociada a los Lisosomas/genética , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Fagosomas/virología , Nexinas de Clasificación/genética , Nexinas de Clasificación/metabolismo , Virus Vaccinia/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
Vaccinia virus (VACV), the model poxvirus, produces two types of infectious particles: mature virions (MVs) and extracellular virions (EVs). EV particles possess two membranes and therefore require an unusual cellular entry mechanism. By a combination of fluorescence and electron microscopy as well as flow cytometry, we investigated the cellular processes that EVs required to infect HeLa cells. We found that EV particles were endocytosed, and that internalization and infection depended on actin rearrangements, activity of Na(+)/H(+) exchangers, and signalling events typical for the macropinocytic mechanism of endocytosis. To promote their internalization, EVs were capable of actively triggering macropinocytosis. EV infection also required vacuolar acidification, and acid exposure in endocytic vacuoles was needed to disrupt the outer EV membrane. Once exposed, the underlying MV-like particle presumably fused its single membrane with the limiting vacuolar membrane. Release of the viral core into the host cell cytosol allowed for productive infection.
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Pinocitosis , Virus Vaccinia/fisiología , Virión/fisiología , Internalización del Virus , Actinas/metabolismo , Células HeLa , Humanos , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno/metabolismo , Vacuolas/metabolismoRESUMEN
For entry and infection viruses have developed numerous strategies to subjugate indispensable cellular factors and functions. Host cell lipids and cellular lipid synthesis machinery are no exception. Not only do viruses exploit existing lipid signalling and modifications for virus entry and trafficking, they also reprogram lipid synthesis, metabolism, and compartmentalization for assembly and egress. Here we review these various concepts and highlight recent progress in understanding viral interactions with host cell lipids during entry and assembly.
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Membrana Celular/virología , Interacciones Huésped-Patógeno/fisiología , Metabolismo de los Lípidos/fisiología , Lípidos de la Membrana/metabolismo , Internalización del Virus , Humanos , Transducción de Señal , Virosis/metabolismo , Replicación ViralRESUMEN
BACKGROUND: Large-scale RNAi screening has become an important technology for identifying genes involved in biological processes of interest. However, the quality of large-scale RNAi screening is often deteriorated by off-targets effects. In order to find statistically significant effector genes for pathogen entry, we systematically analyzed entry pathways in human host cells for eight pathogens using image-based kinome-wide siRNA screens with siRNAs from three vendors. We propose a Parallel Mixed Model (PMM) approach that simultaneously analyzes several non-identical screens performed with the same RNAi libraries. RESULTS: We show that PMM gains statistical power for hit detection due to parallel screening. PMM allows incorporating siRNA weights that can be assigned according to available information on RNAi quality. Moreover, PMM is able to estimate a sharedness score that can be used to focus follow-up efforts on generic or specific gene regulators. By fitting a PMM model to our data, we found several novel hit genes for most of the pathogens studied. CONCLUSIONS: Our results show parallel RNAi screening can improve the results of individual screens. This is currently particularly interesting when large-scale parallel datasets are becoming more and more publicly available. Our comprehensive siRNA dataset provides a public, freely available resource for further statistical and biological analyses in the high-content, high-throughput siRNA screening field.
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Genómica/métodos , Interferencia de ARN , ARN Interferente Pequeño/genética , Línea Celular , Biblioteca de Genes , Genómica/normas , Ensayos Analíticos de Alto Rendimiento , Interacciones Huésped-Patógeno/genética , Humanos , Curva ROC , Reproducibilidad de los ResultadosRESUMEN
Vaccinia virus membrane biogenesis requires the A14 and A17 proteins. We show here that both proteins can associate with membranes co- but not posttranslationally, and we perform a structure function analysis of A14 and A17 using inducible recombinants. In the absence of A14, electron-dense virosomes and distinct clusters of small vesicles accumulate; in the absence of A17, small vesicles form a corona around the virosomes. When the proteins are induced at 12 h postinfection (hpi), crescents appear at the periphery of the electron-dense virosomes, with the accumulated vesicles likely contributing to their formation. A variety of mutant alleles of A14 and A17 were tested for their ability to support virion assembly. For A14, biologically important motifs within the N-terminal or central loop region affected crescent maturation and the immature virion (IV)âmature virion (MV) transition. For A17, truncation or mutation of the N terminus of A17 engendered a phenotype consistent with the N terminus of A17 recruiting the D13 scaffold protein to nascent membranes. When N-terminal processing was abrogated, virions attempted to undergo the IV-to-MV transition without removing the D13 scaffold and were therefore noninfectious and structurally aberrant. Finally, we show that A17 is phosphorylated exclusively within the C-terminal tail and that this region is a direct substrate of the viral F10 kinase. In vivo, the biological competency of A17 was reduced by mutations that prevented its serine-threonine phosphorylation and restored by phosphomimetic substitutions. Precleavage of the C terminus or abrogation of its phosphorylation diminished the IVâMV maturation; a block to cleavage spared virion maturation but compromised the yield of infectious virus.
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Proteínas de la Membrana/metabolismo , Virus Vaccinia/fisiología , Proteínas del Envoltorio Viral/metabolismo , Ensamble de Virus , Animales , Línea Celular , Chlorocebus aethiops , Proteínas de la Membrana/genética , Microscopía Electrónica de Transmisión , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Virus Vaccinia/genética , Virus Vaccinia/metabolismo , Proteínas del Envoltorio Viral/genéticaRESUMEN
We previously identified the bisbenzimide Hoechst 33342 (H42) as a potent multi-stage inhibitor of the prototypic poxvirus, the vaccinia virus (VACV), and several parapoxviruses. A recent report showed that novel bisbenzimide compounds similar in structure to H42 could prevent human cytomegalovirus replication. Here, we assessed whether these compounds could also serve as poxvirus inhibitors. Using virological assays, we show that these bisbenzimide compounds inhibit VACV spread, plaque formation, and the production of infectious progeny VACV with relatively low cell toxicity. Further analysis of the VACV lifecycle indicated that the effective bisbenzimide compounds had little impact on VACV early gene expression but inhibited VACV late gene expression and truncated the formation of VACV replication sites. Additionally, we found that bisbenzimide compounds, including H42, can inhibit both monkeypox and a VACV mutant resistant to the widely used anti-poxvirus drug TPOXX (Tecovirimat). Therefore, the tested bisbenzimide compounds were inhibitors of both prototypic and pandemic potential poxviruses and could be developed for use in situations where anti-poxvirus drug resistance may occur. Additionally, these data suggest that bisbenzimide compounds may serve as broad-activity antiviral compounds, targeting diverse DNA viruses such as poxviruses and betaherpesviruses.IMPORTANCEThe 2022 mpox (monkeypox) outbreak served as a stark reminder that due to the cessation of smallpox vaccination over 40 years ago, most of the human population remains susceptible to poxvirus infection. With only two antivirals approved for the treatment of smallpox infection in humans, the need for additional anti-poxvirus compounds is evident. Having shown that the bisbenzimide H33342 is a potent inhibitor of poxvirus gene expression and DNA replication, here we extend these findings to include a set of novel bisbenzimide compounds that show anti-viral activity against mpox and a drug-resistant prototype poxvirus mutant. These results suggest that further development of bisbenzimides for the treatment of pandemic potential poxviruses is warranted.
Asunto(s)
Poxviridae , Viruela , Humanos , Bisbenzimidazol/metabolismo , Pandemias , Virus Vaccinia/genéticaRESUMEN
Autophagy is an essential degradation program required for cell homeostasis. Among its functions is the engulfment and destruction of cytosolic pathogens, termed xenophagy. Not surprisingly, many pathogens use various strategies to circumvent or co-opt autophagic degradation. For poxviruses, it is known that infection activates autophagy, which however is not required for successful replication. Even though these complex viruses replicate exclusively in the cytoplasm, autophagy-mediated control of poxvirus infection has not been extensively explored. Using the prototypic poxvirus, vaccinia virus (VACV), we show that overexpression of the xenophagy receptors p62, NDP52, and Tax1Bp1 restricts poxvirus infection. While NDP52 and Tax1Bp1 were degraded, p62 initially targeted cytoplasmic virions before being shunted to the nucleus. Nuclear translocation of p62 was dependent upon p62 NLS2 and correlated with VACV kinase mediated phosphorylation of p62 T269/S272. This suggests that VACV targets p62 during the early stages of infection to avoid destruction and further implies that poxviruses exhibit multi-layered control of autophagy to facilitate cytoplasmic replication.
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Autofagia , Núcleo Celular , Proteína Sequestosoma-1 , Virus Vaccinia , Humanos , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Núcleo Celular/virología , Células HEK293 , Células HeLa , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Fosforilación , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Vaccinia/metabolismo , Vaccinia/virología , Vaccinia/genética , Virus Vaccinia/metabolismo , Virus Vaccinia/genética , Replicación ViralRESUMEN
Recent studies have revealed new insights into the endocytosis of vaccinia virus (VACV). However, the mechanism of fusion between viral and cellular membranes remains unknown. We developed a microfluidic device with a cell-trap array for immobilization of individual cells, with which we analyzed the acid-dependent fusion of single virions. VACV particles incorporating enhanced green fluorescent protein (EGFP) and labeled with self-quenching concentrations of R18 membrane dye were used in combination with total internal reflection fluorescence microscopy to measure the kinetics of R18 dequenching and thus single hemifusion events initiated by a fast low-pH trigger. These studies revealed unexpectedly long lag phases between pH change and hemifusion. In addition, we found that EGFP fluorescence in the virus was quenched upon acidification, indicating that protons could access the virus core, possibly through a proton channel. In a fraction of virus particles, EGFP fluorescence was recovered, presumably after fusion-pore formation and exposure of the core to the physiological pH of the host-cell cytosol. Given that virus-encoded cation channels play a crucial role in the life cycle of many viruses and can serve as antiviral drug targets, further investigations into a potential VACV viroporin are justified. Our findings indicate that the microfluidic device described may be highly beneficial to similar studies requiring fast kinetic measurements.