Computational protocol: Ubiquitin-Regulated Nuclear-Cytoplasmic Trafficking of the Nipah Virus Matrix Protein Is Important for Viral Budding

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Protocol publication

[…] For the live viral infection experiments, cells infected with NiV were fixed with 10% formalin for 24 hrs and removed from the BSL4. Cells were then permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 5 min at room temperature (RT), washed with PBS and then incubated with rabbit polyclonal anti-NiV-M antibody. After extensive washing with PBS, cells were incubated with Alexa488-conjugated goat anti-rabbit secondary antibody (Invitrogen Molecular Probes) for 30 min at RT. Cells were then counterstained with DAPI. The slides were imaged on a Zeiss LSM 510 confocal microscope in the UTMB optical imaging core. For the cell transfection experiments, HeLa cells transfected with the indicated constructs were fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton in PBS and stained with mouse anti-FLAG antibody followed by Alexa488 or 594-conjugated goat anti-mouse secondary antibodies. Cells were imaged on a Nikon Eclipse TE300 fluorescent microscope with MetaMorph software (Molecular Devices).Image analysis was performed with MetaXpress software from Molecular Devices using the Enhanced Translocation module. The algorithm identifies nuclei as compartments using DAPI stain. The nuclear region was defined as the central region 20 pixels inset from the nuclear/cytoplasm boundary. The cytoplasmic region was defined as a disc beginning at the nuclear/cytoplasmic boundary and extending 5 pixels into the cytoplasm. Cells were manually included or excluded by inspection to insure that all cells included in the final scoring had the cytoplasm and nuclear regions correctly defined. A minimum cutoff intensity level was applied to ensure NiV matrix expression was sufficient. This was to exclude aberrant cell morphology and non-transfected cells. The statistic evaluated was the ratio of the average cytoplasmic region intensity to the average nuclear region intensity for each cell. Since the cytoplasmic/nuclear fluorescent intensity (C∶N) ratio for wild-type M is close to 1, C∶N ratios greater than 1 implies increased cytoplasmic retention whereas C∶N ratios less than 1 indicates increased nuclear retention. Between 10-50 cells were counted for Mwt and all mutants analyzed. […]

Pipeline specifications

Software tools MetaMorph, MetaXpress
Application Laser scanning microscopy
Organisms Nipah henipavirus, Homo sapiens
Diseases Encephalitis, Multiple Myeloma, Scleroderma, Localized, HIV Infections