Cell lines and antibodies
The 293T/CD20 cell line was generated previously.10
293T and 293T/CD20 cells were maintained in a 5% CO2
environment in Dulbecco’s modified Eagle medium (Mediatech, Inc.) with 10% FBS (Sigma), and 2 mM L-glutamine (Hyclone). Mouse monoclonal antibody specific to the human CD20 antigen were purchased from Caltag Laboratories. Anti-HA-biotin to stain SINmu was obtained from Miltenyi Biotec Inc. Mouse monoclonal antibody against the early endosome antigen 1 (EEA1), rabbit polyclonal antibody specific to the Mannose 6 phosphate receptor (CI-MPR), and Cy5-conjugated goat anti-rabbit IgG antibody were purchased from Abcam. Cy5-conjugated streptavidin was purchased from Zymed Laboratories. Texas red-labeled goat anti-mouse IgG and AlexaFluor 594-labeled goat anti-human IgG antibodies were obtained from Molecular Probes. Taxol, nocodazole, cytochalasin D, and bafilomycin A1 were purchased from Sigma.
Assembly PCR was employed to fuse the GFP to the N-terminus of viral protein R (Vpr). The PCR product was then inserted into pcDNA3 (Invitrogen) to form pcDNA3-GFPVpr. The cDNAs for Rab5 and Rab7 were PCR-amplified and cloned into the pDsRed-monomer-C1 (Clontech) to form DsRed-Rab5 and DsRed-Rab7, respectively.
GFP-Vpr-labeled lentiviruses were produced by transfecting 293T cells by a calcium phosphate precipitation method. 293T cells at 80% confluence in 6 cm culture dishes were transfected with 5 μg of the lentiviral plasmid FUW, together with 2.5 μg each of pcDNA3-GFPVpr, pαCD20 (encodes a mouse/human chimeric anti-CD20 antibody), pIgαβ (encodes human Igα and Igβ, two immunoglobulin associated proteins that are required for surface expression of antibodies), pSINmu, and the packaging vector plasmids.10
Cells were washed at 4 h posttransfection, and then medium was replaced. The viral supernatant was collected after 48 h posttransfection and filtered through a 0.45-μm pore size filter. For high-titer lentivectors, the viral supernatant was concentrated by ultracentrifugation (Optima L-90 K ultracentrifuge, Beckman Coulter) for 90 min at 82,700 × g and resuspended in an appropriate volume of Hank’s balanced salt solution (HBSS, Hyclone).
For viral transduction, 293T/CD20 cells (0.2 × 106 per well) were plated in a 24-well culture dish and spin-infected with viral supernatants of FUGW/αCD20+SINmu (2 ml per well) at 2,500 rpm and 30°C for 90 min by using a Sorval Legend centrifuge. Then, the medium was removed and replaced with fresh medium and cultured for 3 days further before FACS analysis of GFP+ cells. For viral transduction with drug- or siRNA-treated cells, 293T/CD20 cells were preincubated with drugs (nocodazole (60 μM), cytochalasin D (20 μM), and bafilomycin A1 (25 nM)) or transfected with siRNAs, and then the cells (0.2 × 106 per well) were spin-infected with 2 ml of viral supernatants in a 24-well culture dish.
Time course study of fusion inhibition
293T/CD20 cells (0.4 × 105 per well) were seeded in a 96-well culture dish overnight. Viruses were added to cells and incubated at 4°C for 1 h and the resulting cells were washed with cold PBS to remove unbound viruses. The viral entry was initiated by shifting cells to 37°C. At the indicated time points, D10 media containing 25 nM of bafilomycin A1 was added to inhibit the endosome acidification. The drug was removed 3 h later and replaced with fresh D10 media. Cells were further incubated for 72 h, and the percentage of GFP-positive cells was analyzed by FACS.
For the detection of individual viral particles, fresh viral supernatant was overlaid upon poly-lysine-coated NO.1 coverslips in a six-well culture dish and centrifuged at 3,700 × g and 4°C for 2 h in a Sorval Legend RT centrifuge. The coverslips were rinsed with cold PBS twice, the adhered viruses were immunostained by Alexa 594 anti-human IgG and anti-HA-biotin antibodies, and they were then incubated with Cy5-streptavidin. The coverslips were mounted in Vectashield (Vector Laboratories), which is an antifade mounting medium. For imaging virus-cell binding, 5 × 105 cells were seeded onto a 35 mm glass-bottom culture dish (MatTek Corporation) and grown at 37°C overnight. The seeded cells were rinsed with cold PBS twice and incubated with the concentrated viruses for 1 h at 4°C to allow for binding. Cells were washed with cold PBS to remove unbound viruses and then fixed with 4% formaldehyde on ice for 10 min. To co-label the viral particles bound to the cell surfaces, Alexa594 anti-human IgG antibody was used to stain against the αCD20 heavy chain. Fluorescent images were taken by a Zeiss LSM 510 laser scanning confocal microscope equipped with filter sets for fluorescein, rhodamine or Texas red, and Cy5. A plan-apochromat 63×/1.4 oil immersion objective was used for imaging. Images were analyzed with the use of the Zeiss LSM 510 software version 3.2 SP2.
Imaging virus fusion and transport through endosomes
The concentrated viruses were incubated with 100 μM of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD) (Molecular Probes) for 1 h at room temperature. For imaging virus-endosome fusion, double labeled viruses were incubated with 293T/CD20 cells at 37°C for various time periods and then fixed with 4% formaldehyde. GFP-Vpr and DiD were excited simultaneously with a 488 nm Argon and a 633 nm HeNe laser, respectively, and the emitted light was separated through the corresponding emission filter sets. All samples were scanned under the same conditions for magnification, laser intensity, brightness, gain, and pinhole size. For live cell imaging of virus fusion, 293T/CD20 cells were preincubated on a glass bottom culture dish at 37°C overnight. Viruses were incubated with the cells at 37°C for 30 min to initiate virus internalization. Images were then collected using the confocal microscope. Fluorescence intensity vs. time within the regions of interest around the virus particles were measured by using the Zeiss LSM 510 software package.
For observation of the colocalization of the virus with different endosomal markers, GFP-Vpr-labeled viruses were incubated with 293T/CD20 cells at 37°C and fixed after different time periods. Cells were then permeabilized with 0.1% Triton X-100 and immunostained with EEA1 and CI-MPR for early endosome and late endosome markers, respectively. Texas red-conjugated anti-mouse IgG and Cy5-conjugated goat anti-rabbit IgG antibodies were used as the secondary antibodies. To remove viral aggregates, virus-containing media was filtered by a 0.45 μm pore size centrifuge tube filter (Costar, NY) just before the experiments were conducted.
For the viral trafficking studies using Rab5 and Rab7 constructs, 293T/CD20 cells were transfected with DsRed-Rab5 or DsRed-Ran7 plasmid. At 48 h posttransfection, cells were seeded onto the glass-bottom culture dish and grown at 37°C overnight. GFP-Vpr-labeled viruses at MOI ~30 were added and incubated at 37°C for the different time points and then fixed.
For the colocalization study with transferrin, GFP-Vpr-labeled viruses and 2 μM of Alexa647-conjugated transferrin (Molecular Probes) were mixed and then incubated with 293T/CD20 cells at 37°C for 60 or 120 min. Cells were fixed, permeabilized, and immunostained with EEA1 to compare trafficking of the engineered lentivirus with transferring through early to recycling endosomes.
For the real-time observation of the release of the virus from the endosome, GFP/DiD-labeled viruses were incubated with cells at 37°C for 1 h to induce virus-endosome fusion, and confocal time-lapse images were then recorded.
To observe viruses on microtubule networks, the cells were incubated with GFP-Vpr-labeled viruses at 37°C for 1 h, fixed, and then permeabilized with 0.1% Triton X-100 containing 20 μM of taxol. Microtubules were then immunostained with anti-α-tubulin mAb (Sigma) and Texas red-conjugated anti-mouse secondary antibody. For the microtubule-disrupting assay, cells were preincubated with D10 media containing 60 μM of nocodazole at 37°C for 30 min, and then viruses were added and incubated for further studies.
For the inhibition assay using small interfering RNA (siRNA), we purchased α-tubulin siRNA and the negative control siRNA from Santa Cruz Biotechnology, Inc. The transfection of siRNA was performed as described by the manufacture’s protocol. At 72 h posttransfection, equal numbers of α-tubulin siRNA and control siRNA treated 293T/CD20 cells were used for further studies.
To visualize viruses on actin filaments, the cells were incubated with GFP-Vpr-labeled viruses at 37°C for 1 h, fixed, and permeabilized. Actin filaments were labeled with rhodamine-conjugated phalloidin (Molecular Probes). For the actin-disrupting assay, cells were preincubated with D10 media containing 20 μM of cytochalasin-D at 37°C for 30 min, and then viruses were added and incubated for further studies.