Rapid formation of human immunodeficiency virus-like particles

Significance Currently, our knowledge of individual virus particle assembly kinetics is based solely on studies of the dynamics of fluorescent puncta corresponding to the viral structural protein called Gag labeled with fluorescent proteins (e.g., green fluorescent protein [GFP]) observed by fluorescence microscopy in membranes of cells. However, GFP tagging affects virus particle assembly and release. We directly measured topological changes during HIV-like particle assembly and found that they can reach full size in 20 s and release in 0.5 to 3 min. Compared to previous estimates of ∼8-min assembly time and 30- to 60-min release time, this is more than 10 times faster. In our opinion this is a highly important discovery that challenges current views on virus replication mechanisms.


Materials and Methods
Plasmids.
Gag-GFP was expressed from the plasmid pGag_eGFP, a Rev-independent full-length HIV-1 Gag gene fused to green fluorescent protein (1), kindly provided by Prof. Wesley Sundquist, University of Utah. The plasmid piGag-SNAP was generated from pGag_eGFP by Dr. David Nkwe and Ms. Carina Bannach (LMCB, UCL) by deleting the GFP sequence and inserting an internal SNAP tag (New England Biolabs (UK) Ltd.) between the MA and CA domains of Gag. The codon-optimised synthetic Gag-Pol construct pSynGP (2) was kindly provided by Prof. Stuart Neil, King's College London. Vpr containing an N-terminal GFP tag (3) was kindly provided by Prof. Greg Towers, UCL. The H2B mPlum/Gag plasmid (4) was purchased from Addgene (#102255).
Cell culture and transfection.
Jurkat-TAg cells (kindly provided by Prof. Stuart Neil, King's College London), are a derivative of CD4 + cells that have no detectable cell surface tetherin (5,6), so that fully assembled VLPs should be released. Cells were cultured in RPMI 1640 (Gibco) supplemented with 10% heat inactivated FBS (Sigma) and 1% glutamax (Gibco) in a 37°C incubator with an atmosphere of 5% CO 2 in air. For microscopic analysis 1.8×10 6 cells/well were seeded in 4-well plates and placed back in the incubator for 5 hours. The cells were then transfected with mix of either Gag-GFP or Vpr-GFP and SynGP or pH2B-mPlum/Gag plasmid DNA using Lipofectamine LTX (Invitrogen), according to the manufacturer's protocol. The total amount of plasmid was always 1.8 µg/well. After transfection, cells were placed in a 37°C incubator with an atmosphere of 16.5% CO 2 in air; this increase in CO 2 concentration in the incubator atmosphere causes decrease of culture medium pH from 7.4 in 5% CO 2 to 7.0 in 16.5% CO 2 . This pH has been reported to be optimal for lymphocyte proliferation and reduces apoptosis in cell culture (7,8). Prior to microscopic analysis, cells were seeded onto poly-l-lysinecoated glass bottom dishes (P35G-0-14-C, MatTek Corporation, USA) and allowed to adhere for 30 min.
HeLa, Cos7 or HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% heat inactivated FCS (Sigma) and 1% glutamax (Gibco) in a 37°C incubator with an atmosphere of 5% CO 2 in air. For microscopic analysis, cells were seeded in glass bottom dishes (P35G-0-14-C, MatTek Corporation, USA) at 50% confluency and incubated for 24 hours. The cells were then transfected with mix of either Gag-GFP or Vpr-GFP and SynGP or with piGag-SNAP only, using either Lipofectamine 2000 (Invitrogen) for Cos7 cells or FuGene 6 (Promega) for HEK293T and HeLa cells, according to the manufacturer's protocols. The total amount of plasmid was always 1.8 µg/dish. Live imaging was performed between 16 and 26 hours after transfection in phenol red free L-15 Medium (Leibovitz) or DMEM (Gibco).

Subtilisin A protease stripping
Bacillus licheniformis Subtilisin A (Sigma-Aldrich), in lyophilized powder form, was dissolved in HBSS and used at 100μg/mL final concentration. SICM imaging was performed in RPMI supplemented with 1% glutamax in room temperature with an atmosphere of 5% CO 2 in air.

SICM-FCM
Correlative SICM and confocal live imaging was performed between 6 and 26 hours after transfection in HBSS (Gibco) supplemented with 10mM HEPES (Sigma) and 1% glutamax, pH 6.9 or in culture medium. Where specified, cells were fixed in 0.5% formaldehyde in HBSS supplemented with 10mM HEPES and 1% glutamax.
All experiments were performed using a custom built SICM setup consisting of XY-stage (45 x 45 μm, Direct Drive, Capacitive Sensors, Parallel Metrology, IC-XY-4545-001) and Z-stage (25 μm, Direct Metrology, Capacitive Sensor, IC-Z-25-001) powered by Piezo Controller System (IC-PDC-001) and operated using ICAPPIC Controller (IC-UN-001, ICAPPIC Ltd., UK). To allow high speed imaging the setup was equipped with a fast Z piezo actuator (IC-HSZ-001, ICAPPIC Ltd. UK). Fine alignment of the scanning nanopipette tip with the laser beam in the XY plane was done using two N-470 PiezoMike Linear Actuators (Physik Instrumente, Germany) and was based on the optical image acquired using a 100×/1.3NA oil immersion objective. Therefore, partial offset of fluorescence images in relation to topography could be observed.
Nanopipettes were pulled from BF-100-50-7.5 borosilicate glass capillaries (Sutter Instrument Co., USA) using a P-2000 laser puller (Sutter Instrument Co., USA). Ion current was detected by Axopatch 200B amplifier (Molecular Devices, UK) using a gain of 1 mV/pA and a low-pass filter cutoff setting of 5 kHz. The internal holding voltage source of the Axopatch 200B was used to supply a direct current voltage of +200 mV to the pipette. The ion current and outputs of the capacitive sensors from all three piezo elements were monitored using Axon Digidata 1322A and Clampex 9.2 (Molecular Devices, UK).
The excitation was provided by a 488-nm wavelength diode-pumped solid-state laser (Laser 200; Protera). Fluorescence images were recorded using a D-104 Microscope Photometer (Photon Technology International, Inc. USA) through a 100×/1.3NA oil immersion objective.
SICM-FCM control, data acquisition and analysis were performed using custom modified HPICM scanner software and SICM Image Viewer (ICAPPIC Ltd, UK).

Electron microscopy
Correlative light and electron microscopy: Cells expressing mixtures of either Gag-GFP or Vpr-GFP and SynGP were fixed with 3% formaldehyde and 2% sucrose dissolved in HBSS, 10mM HEPES, 1% glutamax for 30 min. and subsequently in 2% formaldehyde and 1.5% glutaraldehyde in HBSS for another 30 min. Cells expressing pH2B-mPlum/Gag were fixed with a mix of 1.5% formaldehyde and 0.1% glutaraldehyde in HBSS, 10mM HEPES, 1% glutamax. Transfected cells were identified by fluorescence imaging and their location on gridded glass bottomed dishes (MatTek Corp, USA) was imaged and documented using an inverted fluorescence widefield microscope (Leica, UK). Aldehydefixed cells were then post-fixed for 1 hr at 4°C in 1% OsO4/1.5% K3[Fe(CN)6], and treated with 1.5% tannic acid. Samples were dehydrated in graded ethanol and embedded in Epon 812 (TAAB Laboratories, UK). The transfected cells were then relocated on the resin block surface and sectioned either in xy or xz orientation. Ultrathin sections (~70nm) were cut on an UC7 ultramicrotome (Leica Microsystems, UK), collected on formvar-coated slot grids and stained with lead citrate.
Immuno-electron microscopy: Transfected or untransfected control cells were fixed by adding an equal volume of pre-warmed double-strength fixative (8% PFA in 0.1 M NaPi buffer, pH 7.4) directly into the culture medium. After 10 min, the medium was replaced with 4% PFA and fixation continued for 90 min. Cells were rinsed in PBS containing 25 mM glycine, embedded in gelatine (12%), infiltrated