- Open Access
Visualizing fusion of pseudotyped HIV-1 particles in real time by live cell microscopy
© Koch et al; licensee BioMed Central Ltd. 2009
- Received: 22 April 2009
- Accepted: 18 September 2009
- Published: 18 September 2009
Most retroviruses enter their host cells by fusing the viral envelope with the plasma membrane. Although the protein machinery promoting fusion has been characterized extensively, the dynamics of the process are largely unknown.
We generated human immunodeficiency virus-1 (HIV-1) particles pseudotyped with the envelope (Env) protein of ecotropic murine leukemia virus eMLV to study retrovirus entry at the plasma membrane using live-cell microscopy. This Env protein mediates highly efficient pH independent fusion at the cell surface and can be functionally tagged with a fluorescent protein. To detect fusion events, double labeled particles carrying one fluorophor in Env and the other in the matrix (MA) domain of Gag were generated and characterized. Fusion events were defined as loss of Env signal after virus-cell contact. Single particle tracking of >20,000 individual traces in two color channels recorded 28 events of color separation, where particles lost the Env protein, with the MA layer remaining stable at least for a short period. Fourty-five events were detected where both colors were lost simultaneously. Importantly, the first type of event was never observed when particles were pseudotyped with a non-fusogenic Env.
These results reveal rapid retroviral fusion at the plasma membrane and permit studies of the immediate post-fusion events.
- Fusion Event
- Fusion Pore
- Single Particle Tracking
- Color Separation
- Live Cell Microscopy
Enveloped viruses enter host cells by membrane fusion at the plasma membrane or at intracellular membranes. This process is mediated by the interaction of cellular receptors and Env glycoproteins. Numerous studies have revealed detailed information about the proteins involved in fusion for many viruses and have elucidated fundamental principles of viral fusion mechanisms [1, 2]. The dynamics of the fusion process, however, is still incompletely characterized. Furthermore, the early post-entry steps immediately following membrane fusion remain enigmatic for many viruses.
Previous investigations have employed bulk biochemical assays or cell-cell fusion to study the viral fusion process (for review see ). More recently, single particle tracking of fluorescently labeled viruses has become possible and has been successfully applied to characterize the entry of various viruses (for review see ). In most cases, the lipophilic dye DiD was used for labeling the membrane of enveloped virus particles [5–7]. As DiD is incorporated into the outer leaflet of the membrane its redistribution after virus-cell contact indicates primarily the lipid mixing of the contacting leaflets (termed hemifusion) and not the formation of the fusion pore .
HIV-1 entry, as well as entry of many other retroviruses, has long been believed to occur exclusively at the plasma membrane. More recently, however, productive infection by pH-independent, clathrin-dependent endocytosis of HIV-1 has also been reported  and was recently suggested to constitute the only route of productive entry . We have developed a system to study the dynamics of HIV-1 entry based on fluorescent live cell microscopy, in which the MA domain of the main structural protein Gag is labeled by fusion to a fluorescent protein . MA lines the inner surface of the viral membrane and is believed to separate from the core of the virion upon membrane fusion. The inner core is subsequently transformed into the reverse transcription complex, and after reverse transcription it is again transformed into the viral preintegration complex (PIC) (for review see ). These nucleoprotein complexes are poorly characterized, but are believed to contain no or only a small proportion of MA molecules . MA is believed to remain at the site of fusion from where it is redistributed within the membrane or into the cytosol . To allow for direct detection of fusion events, the fluorescent label at the MA domain was combined with a differently colored label at the core-associated viral protein R (Vpr), which remains associated with the PIC during cytoplasmic transport to the nucleus . Fusion should thus be accompanied by a rapid separation of the two labels in this system. However, tracking >10,000 individual interactions at high time resolution did not yield clear separation events . Since this may be due to the low fusogenicity of HIV, the possibility to pseudotype retroviruses was applied, and HIV-1 particles carrying the highly fusogenic glycoprotein of vesicular stomatitis virus (VSV-G) were analyzed. This approach resulted in readily detectable bulk color separation over time with the mRFP.Vpr that accumulated at the nuclear membrane and MA.eGFP exhibiting mostly cytoplasmic staining . Thus, efficient fusion must have occurred, but only sporadic events of color separation were observed for individual particles. This raised the question as to whether membrane fusion may not be accompanied by immediate separation of the bulk of MA from the viral core. Furthermore, pseudotyping with VSV-G diverted the entry route of the particles to a pH dependent endocytic pathway, thereby potentially influencing subsequent events.
For these reasons we developed a system where the fate of the viral membrane can be unequivocally determined. We made use of fluorescent HIV particles, pseudotyped with an Env protein from eMLV. This approach provides two main advantages: First, MLV Env carrying particles targeting DFJ-8 cells with a high surface density of murine cationic aminoacid transporter (mCAT-1, the receptor for eMLV) represent one of the most efficient systems for studying pH independent fusion at the plasma membrane . Second a well characterized fluorescent variant of eMLV Env is available which has been shown to mediate fusion with wild-type efficiency and remains associated with the host cell membrane after fusion . We have studied the dynamics of retroviral fusion and investigated immediate post fusion events by live cell imaging using double labeled pseudotypes carrying the fluorescent variant of eMLV Env and the MA domain of HIV-1 Gag fused to another fluorescent protein. Here, we report single particle tracking of >20,000 individual traces of double-fluorescent pseudotyped HIV recording 28 events of color separation and 45 additional events, where both colors were lost simultaneously.
Characterization of double labeled HIV-1 pseudotypes
Analysis of Env-dependent fusion by fluorescence microscopy
We compared the infection efficiency of immobilized particles with that of free particles to determine whether adherence to the cover slip affected the capacity of pseudotyped particles to infect DFJ-8 cells. Parallel infections were performed in which either particles or DFJ-8 cells were pre-bound to fibronectin-coated cover slips and cells or viruses were seeded on top. Infected cells were subsequently quantified by staining for β-galactosidase activity and infectivity was normalized to the particle input determined by measuring the reverse transcriptase activity of immobilized and free particles, respectively. These experiments revealed that the infectivity of the immobilized particles was equal or slightly better than that of the free particles (data not shown).
Visualization of individual fusion events by single particle tracing
Quantification of the red and green signal intensities originating from MA.mCherry and Env.YFP, respectively, of at least 400 individual double labeled particles as a function of time revealed a significant loss of the Env-associated YFP signal relative to the MA-associated mCherry signal for particles bearing fusion-competent Env.YFP (approximately 50% decrease after 20 minutes) as depicted in Figure 4E. To determine whether loss of the Env-YFP signal could be due to quenching of the pH-sensitive fluorophore YFP upon exposure of endocytosed particles to the low pH of the endosome, experiments were performed in the presence of ammonium chloride which prevents endosomal acidification (Figure 4D). As indicated in Figure 4E, ammonium chloride treatment had no significant impact on the loss of the Env.YFP signal over time. Furthermore, specific loss of the Env-associated signal could also be observed when Env was labeled with the less pH-sensitive protein mCherry (data not shown). Immobilized particles which had no cell contact did not display a significant loss of the Env.YFP signal, which indicates that photobleaching also did not contribute significantly to the loss of YFP fluorescence (indicated as background in Figure 4E). As expected, fusion impaired particles (Env.YFP.PR(-) and Env.YFP.H8R bearing VLPs, respectively) showed only a minor reduction of the YFP signal (approximately 10% decrease in the first 20 minutes after cell contact).
The observation of a persistent MA signal after loss of the viral membrane was not expected considering current models of retroviral entry. To determine whether the MA shell could have been artificially stabilized by fusion of the fluorescent protein, we analyzed MA shell dissociation in vitro using two different approaches. First, the Env.YFP/MA.mCherry labeled particles were adhered to a glass cover slip, incubated with 0.05% Triton X-100 and the number of single and double labeled particles was recorded over time. These experiments showed a rapid and concomitant loss of both signals upon detergent addition (Additional file 4A). Second, we made use of a FRET based assay to monitor the time course of MA shell dissociation. Purified particles labeled with a mixture of MA.eCFP and MA.eYFP displayed a strong FRET signal which rapidly disappeared upon disruption of the particle membrane with 0.05% Triton X-100. As expected, stabilization of the Gag shell by prevention of Gag processing prevented the decay of this FRET signal. Dissociation of the mature MA.XFP shell (indicated by a fluorescence spectrum resembling that of free eCFP) was complete within ~10 seconds at 37°C (Additional file 4B).
Summary of the automated tracking results.
Simultaneous loss of both colors
Additional file 5: Movie displaying an individual fusion event indicated by color separation, corresponding to still images in Figure 5A. Env.YFP (green) and MA.mCherry (red) labeled particles were coated onto a glass coverslip, and DFJ-8 cells were allowed to settle onto the virus particles. Image acquisition with a time resolution of 0.76 frames/sec was started at the time point of cell attachment to the coverslip. The video shows a section of the movie covering 38 sec and is displayed at a speed of 10 frames per second. The particle of interest is indicated by a white circle. While the Env.YFP signal vanished within 15 sec after virus-cell contact, the label of the MA domain remained punctated during the remaining period of observation. Still images of the video are shown in Figure 5A. (MOV 179 KB)
This study aimed at monitoring individual fusion events of eMLV Env pseudotyped HIV-1 particles and at analyzing the subsequent fate of the sub-membrane MA layer. So far, the dynamics of virus-cell fusion has been predominantly studied using cell-cell fusion assays in which cells expressing a viral Env protein fuse with cells expressing the cellular receptor for the virus [19–21]. However, the stoichiometry of Env and receptor as well as the geometry of the fusion area between two similarly sized cells do not accurately reflect the events occurring in the fusion between a small virion and a much larger cell. Analysis of cell-cell fusion events revealed an average half-time of 10 to 20 minutes [22, 23]. Scoring for loss of fluorescent Env molecules from double labeled HIV/eMLV pseudotypes, 28 fusion events were identified in the present study; and individual fusion events were already observed within seconds after the first virus-cell contact. This result is in agreement with a previous study, in which fusion of individual HIV-1 Env pseudotyped viruses labeled with the lipophilic dye DiD and GFP attached to the NC domain of Gag was monitored after binding to target cells at low temperature. These authors also observed initial fusion events within the first minute after shifting the temperature to 37°C , and they concluded that virus-cell fusion proceeds without significant delay during rising temperature. Thus, virus-cell fusion appears to be kinetically different from cell-cell fusion.
Our approach involved pseudotyping of fluorescent HIV-1 particles carrying a fluorophor in the MA domain of Gag with fluorescent eMLV Env. Both modifications have been shown to be compatible with particle formation and infectivity . Env is a membrane-embedded glycoprotein that is expected to remain attached to the plasma membrane after fusion. Accordingly, progressive plasma membrane labeling was observed upon incubation of DFJ-8 target cells with particles carrying wild-type Env, but not with particles carrying fusion-impaired or -defective variants. MA is associated with the inner leaflet of the virion membrane and is generally believed to remain at the plasma membrane after fusion before dissociating into the cytosol. Thus, the combination chosen in this report would not appear to be optimal for detecting color separation upon fusion. However, previous studies had shown bulk separation of labeled MA and inner core proteins over time when double labeled particles were incubated with permissive cells, while individual events of color separation were not detected . These observations raised the possibility that HIV-1 MA may remain attached with the entering viral core for at least a short period after membrane fusion. Consistent with this hypothesis, particulate MA signals were largely retained upon incubation of target cells with immobilized double labeled particles, while the Env.YFP signal was gradually lost over time. Tracking individual double labeled particles identified 28 events of color separation, indicating that the MA layer can dissociate from the surface glycoproteins upon membrane fusion. It may then remain associated with the entering viral core, at least for a short time. 10 of the 15 particles underwent a color separation event in the live cell experiments and could subsequently be followed until the end of the data acquisition. Consistent with our hypothesis, the 10 particles displayed a punctate MA.mCherry signal over the remaining observation period (corresponding to up to 4 minutes after color separation). While this does not clearly exclude a dissociation of the punctate MA.mCherry signal at later time points, it suggests that the MA shell may at least be transiently stable after the envelope is lost. Preliminary results on triple labeled particles carrying different fluorophors in Env, MA and the viral core also support this conclusion, revealing transient co-localization of MA and the entering core after fusion-dependent loss of the Env layer (unpublished observation). These events were rare, and it is currently not clear whether they give rise to productive entry. MLV pseudotypes efficiently fuse with DFJ-8 cells, however; and they exhibit a high infectivity on these cells, making it likely that at least some of the observed events represent productive fusion. Conceivably, the observed color separation events may constitute only a minority of all fusion events with the majority not being scored because of concomitant loss of MA together with Env fluorescence. This appears unlikely, however, because only 45 further events of particles losing the fluorescent signal were detected. In these cases both colors were lost simultaneously. Concomitant disappearance of both colors could be due to loss of the particle from the focus plane (e.g. during endosomal uptake), which may explain why such events were also seen for particles pseudotyped with fusion-defective Env. The number of events was much lower in this case (12 versus 45), indicating that at least some of the observed events of simultaneous loss of both colors also represent membrane fusion. Based on this study, such events do not appear to be more common than separation of Env and MA, however.
MA carries the plasma membrane trafficking moiety of Gag and is thus responsible for Gag membrane association in the assembly phase . This is mediated by N-terminal myristoylation, basic charges and a phosphatidylinositol 4,5-bisphosphate binding site in MA [25, 26]. Membrane binding affinity is much lower for the cleaved MA domain than for full-length Gag [27, 28]. This is due to a myristoyl switch regulating exposure of the acyl chain and due also to the lack of stable multimerisation of MA [29, 30]. Accordingly, MA is rapidly stripped from the viral core upon detergent treatment [31–33], and only small amounts of MA have been detected in HIV pre-integration complexes [11, 12]. The bulk of MA can thus be expected to dissociate from the membrane into the cytoplasm as monomers or small oligomers after fusion. Such redistribution of MA is in agreement with previous observations using MA.eGFP/Vpr.mCherry labeled particles. After prolonged incubation, a diffuse cytoplasmic distribution was observed for the MA.eGFP signal in this case . This redistribution does not always occur directly upon fusion, however, since particulate MA.mCherry signals could be tracked for up to several minutes after loss of the Env signal in the present study. The simplest explanation for this phenotype would be the retention of a stable MA lattice at the fusion site with concomitant dissipation of Env molecules within the plasma membrane. There is currently no evidence, however, for a stable MA lattice. This hypothesis cannot explain the occasionally observed rapid movement of MA clusters after loss of the Env signal. Nor would this hypothesis be compatible with the temporary co-localisation of MA and the core in triple labeled particles. Such co-localisation could be due to a delayed opening of the fusion pore that allows dissipation of Env proteins within the plasma membrane while the core is still retained in the particle neck. A delayed release of an aqueous marker was observed after hemifusion had occurred in a previous study , and this could also apply to the later stages of fusion pore opening. Alternatively, the MA layer may dissociate from the membrane and remain transiently associated with the viral core after fusion and separation from the membrane. Furthermore, interaction of MA with the cytoplasmic tail of its cognate Env protein may be important for regular uncoating. Future live cell microscopy studies using high time resolution and fluorophors in different viral proteins will shed light on these immediate post-fusion events which are largely unexplored for most viruses.
The plasmid Friend MLV Env-YFP  was provided by W. Mothes (Yale University School of Medicine). The plasmids pMMP-LTR-LacZ and pMDoldGag-Pol were provided by Richard Mulligan (Department of Genetics, Harvard University). The plasmid 1765-H8R  that expresses the MLV envelope protein bearing a histidine to arginine mutation at position 8 was a gift from L. Albritton (University of Tennessee). To introduce the H8R mutation into Env.YFP we performed site directed mutagenesis using the Stratagene quick exchange kit (forward primer: 5'-CTCAGTGGGCCGCCCGATTGGGGGCTAGAGTATC-3'; reverse primer: 5'-GATACTCTAGCCCCCAATCGGGCGGCCCACTGAG-3') resulting in the plasmid Env.YFP.H8R. Plasmid pCHIV and derivatives have been described previously . The plasmid pCHIV.Env(-).PR(-) carrying a point mutation in the PR active site and a frameshift mutation in the env gene was constructed by exchange of an AgeI-XhoI fragment of pCHIV.PR(-) with the corresponding fragment of pCHIV.Env(-).Env.YFP with an uncleaved R-peptide is referred to as Env.YFP.PR(-).
Tissue culture and production of fluorescently labeled virus particles
293T, DF-1 and DFJ-8 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen), supplemented with 10% fetal calf serum (FCS; Biochrom), penicillin (100 IU/mL) and streptomycin (100 μg/mL). Live cell imaging studies were performed in PBS supplemented with 1 mM CaCl2, 0.5 mM MgCl2 and 1% FCS. For production of double fluorescently labeled particles, 293T cells were co-transfected with a mixture of pCHIV.Env(-), pCHIV.mCherry.Env(-), or their protease deficient variants, respectively, and the plasmid Env.YFP in a molar ratio of 1:1:4 by calcium phosphate precipitation. Supernatants were harvested at 36 hours post transfection and filtered through a 0.45 μm filter. Particles were concentrated by ultracentrifugation through a 20% (w/w) sucrose cushion. Virions were resuspended at 3 μl/ml culture supernatant in phosphate-buffered saline (PBS) supplemented with 10% FCS and 10 mM HEPES pH 7.3, frozen in liquid nitrogen, and stored at -80°C. Particle yield was determined by ELISA quantitation of the p24 capsid protein using an in house ELISA. For Western blotting, samples were separated by SDS-PAGE (16% acrylamide gels) and transferred by semi-dry blotting to an activated PVDF membrane (Immobilon-FL, Millipore). Viral proteins were detected by using polyclonal rabbit antiserum raised against recombinant HIV-1 CA protein or goat anti-Rauscher murine leukemia virus gp70 with known cross-reactivity to MLV Env (provided by C. Buchholz, Paul Ehrlich Institute, Langen). Rat polyclonal antiserum raised against mCherry was provided by Heinrich Leonhardt, LMU Munich. YFP was detected using rabbit polyclonal antiserum against recombinant GFP. Detection and documentation were performed with the Li-Cor Odyssey system according to the manufacturer's instructions, using the appropriate secondary antibodies provided by the manufacturer. MLV vector particles transducing β-galactosidase were quantified either by immunoblotting against the p30 CA protein (antiserum kindly provided by C. Buchholz, Paul Ehrlich Institute, Langen) or by measuring their reverse transcriptase activity using the RETRO SYS, RT Activity Kit (Innovagen AB) as recommended by the manufacturer.
Analysis of viral infectivity
To determine viral infectivity, MLV vector particles carrying the lacZ gene and the indicated Env proteins were generated as described previously . Briefly, 293T cells were co-transfected with 5 μg of a plasmid encoding the vector RNA (pMMP-LTR-LacZ), 5 μg plasmid encoding wild-type Env or its labeled derivatives, respectively, and 5 μg plasmid encoding wild-type GagPol (pMDoldGag-Pol) in a 10 cm dish by calcium phosphate precipitation. The medium was changed 24 hours and 36 hours post transfection, the medium was harvested and particles were purified as described above. Comparable amounts of particles (as determined by p30 immunoblot) were adhered to fibronectin-coated coverslips and DFJ-8 cells were allowed to settle on top of the virus coated surface. After 48 hours cells were fixed with 4% PFA and β-galactosidase activity was determined by X-gal staining. The percentage of infected cells was determined by the ratio of stained cells to total cells.
Epifluorescence microscopy was performed on a Zeiss Axiovert 200 M microscope with a back illuminated EM-CCD camera (Cascade II, Roper Scientific). Images were acquired with Metamorph Software (Visitron). For live cell imaging, cells were incubated at 37°C in a microscope incubation chamber (EMBLEM, Heidelberg, Germany). The microscopic setup has been described previously . For experiments analyzing single particle fusion, eight-chambered cover glasses (LabTek, Nunc) were coated with fibronectin (Sigma) at a concentration of 100 μg/μl and incubated at 37°C for 1 h. Fibronectin was removed and the cover glasses were dried for 30 minutes and rinsed with PBS. Fluorescent virus particles in PBS were subsequently added to the chambers. To detect overall changes in the VLP population, we used a VLP amount corresponding to 500 ng p24. For single event tracing, a VLP amount corresponding to 100 ng p24 was used. VLPs were allowed to adhere to fibronectin for 30 minutes at room temperature before removal of the virus containing solution. Subsequently, a suspension containing approximately 5,000 DFJ-8 cells was added in pre-warmed PBS supplemented with 1 mM CaCl2 and 0.5 mM MgCl2 and 1% FCS. Image acquisition was started when cells attached to the bottom of the cover glasses. Cell positions were documented by bright field images recorded immediately before and after the time series. To block endosomal acidification, DFJ-8 cells were trypsinized and incubated in the presence of 30 mM NH4Cl for 3 h at 37°C. Afterwards the cells were added to prebound VLPs in PBS containing 30 mM NH4Cl and image acquisition was started.
Automated particle tracking
For automated analysis a 2D tracking approach was developed to track dual-colored particles with a low signal-to-noise ratio. Details of the particle localization and tracking algorithms are described elsewhere . Briefly, particles were localized using 2D Gaussian fitting and particle positions and fluorescence intensities in both YFP and RFP channels were recorded. The particles were tracked in consecutive frames using a probabilistic scheme based on the Kalman filter. To detect color separation or events where both labels vanished simultaneously the intensity profiles of each track in both green and red channel were analyzed. For this, the intensity profiles, which were derived from the automatically generated VLP traces of both channels, were compared to the corresponding background level. the mean intensity level of both signals had to differ by at least one standard deviation from the background signal to be considered as a double labeled particle. Color separation was defined as a drop of one signal to background intensity. Supplemental method information is available in Additional file 8.
We thank Walther Mothes, Rainer Pepperkok and Friedrich Frischknecht for inspiring and helpful discussions and Oliver Fackler, Christian Buchholz and Heinrich Leonhardt for their kind gifts of reagents. This study was supported in part by a grant from the DFG to BM and HGK (MU885/4-2) and by the BMBF-funded project Viroquant (0313923). PK was supported through the DFG Graduiertenkolleg GRK1188. HGK is a member of the excellence cluster CellNetworks (EXC81), whose support of the imaging facility is greatly acknowledged.
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