Computational protocol: Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imaging

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Protocol publication

[…] CV-1/TVA950 or CV-1/TVA800 cells transfected with either CFP-Rab5 and YFP-Rab7 or mKO-Rab5 were placed on ice, washed with cold Hank’s buffer (HBSS), and centrifuged with ~1.5 · 104 IU of single labeled with Gag-mKate2 pseudoviruses or particles co-labeled with EcpH-ICAM and Gag-mKate2, respectively, at 2,100 × g (4°C) for 20 min. Unbound viruses were removed by washing, cells were mounted onto a microscope stage maintained at 37°C. Once a suitable image field was chosen, virus internalization and fusion were initiated by adding 1 ml of warm HBSS and imaged using a Zeiss LSM 780 confocal microscope (Zeiss Microsystems, Jena, Germany) with a 63×/1.4 NA oil immersion objective. Images were acquired every 8–12 sec for ~60 min. The axial position of a specimen during acquisition was stabilized using the Definite Focus module. Cells overexpressing the endosomal markers and/or containing aberrant swollen endosomes were excluded from analysis.EcpH and mKate2 were excited with the 488 and 561 nm laser lines, respectively. CFP-Rab5 and YFP-Rab7 were excited with 458 nm and 514 nm lines, respectively, whereas the 543 nm line was used for mKO-Rab5. Fluorescence emission was detected with the 32-channel GaAsP spectral detector. In experiments with the single labeled virus (Gag-mKate2) entering cells co-transfected with CFP-Rab5 and YFP-Rab7, the first 16 channels recorded the CFP signal and the remaining 16-channels acquired the YFP emission signal, the mKate2 fluorescence was detected using a cooled PMT. When using double-labeled viruses (EcpH-ICAM and Gag-mKate2) and cells expressing mKO-Rab5, the first 16 channels of the GaAsP detector were allocated for the EcpH signal and the remaining 16 channels for the mKO signal, while the mKate2 signal was acquired with the cooled PMT. The emission windows for the fluorescent proteins utilized where selected as follows: CFP (465–500 nm), EcpH (500–540 nm), YFP (520–560 nm), mKO (550–585 nm) and mKate2 (600–650 nm). Spectral unmixing was applied, as necessary, to correct for bleed-through between the CFP and YFP channels and between the EcpH and mKO channels. The above imaging conditions ensured negligible bleed through between CFP and YFP channels (e.g., Figures C and B). [...] Single virus tracking was performed with Imaris (BitPlane, Switzerland) or Volocity (Perkin Elmer, MA) software. Both single- (Gag-mKate2) and double-labeled (EcpH-ICAM and Gag-mKate2) viruses that entered the cell and colocalized with early endosomes (decorated with CFP-Rab5 or mKO-Rab5, depending on the experiment) were tracked using the red channel. The acquisition of Rab5 or Rab7 by virus-carrying endosomes was defined as the point when the endosomal marker signal exceeded the background level by 30%. Virus-endosome colocalization analysis was carried out with ImageJ (http://imagej.nih.gov/ij/). A line histogram that showed normalized intensity profiles of the viral and endosomal markers. The percentage of overlap between the profiles was obtained by calculating the area under the curve for each channel. Viruses showing at least 80% overlap with endosomal markers during >5 consecutive frames and traveled at least 1 μm were considered as co-trafficking with endosomes. After viruses colocalized with endosomal markers for 5 consecutive frames additional tracking was performed, using the CFP-Rab5 signal or the mKO-Rab5 signal as a reference, as indicated. Fusion was detected as disappearance of the red signal (Gag-mKate2 release) whilst the Rab5 signal remained steady. In some cases, quickly traveling endosomes co-localized with the virus were tracked manually using the Volocity software. These analyses yielded the mean fluorescence intensity of viral and endosomal markers, as well as their coordinates, as a function of time. Statistical analysis of the kinetics data was performed using a two-tailed t-test or Rank Sum test, and fraction of viruses fused in cells expressing TVA800 and TVA950 cells was analysed using a χ2 test (SigmaPlot, San Jose, CA). […]

Pipeline specifications

Software tools Spectral Unmixing Plugins, Imaris, ImageJ, SigmaPlot
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Diseases HIV Seropositivity, Infection