Open AccessEvaluation of VP22 Spread in Tissue Culture
Author(s) -
Neil Brewis,
Anne M. Phelan,
Jennifer A. Webb,
Jeff Drew,
Gill Elliott,
Peter O’Hare
Publication year - 2000
Publication title -
journal of virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.617
H-Index - 292
eISSN - 1070-6321
pISSN - 0022-538X
DOI - 10.1128/jvi.74.2.1051-1056.2000
Subject(s) - green fluorescent protein , biology , paraformaldehyde , immunofluorescence , herpes simplex virus , fluorescence , microbiology and biotechnology , fixation (population genetics) , cell culture , fusion protein , biophysics , virus , virology , gene , biochemistry , antibody , chemistry , recombinant dna , immunology , genetics , physics , organic chemistry , quantum mechanics
We compare methods of detection of intercellular transport of the herpes simplex virus protein VP22 and of a green fluorescent protein (GFP)-VP22 fusion protein. Spread of both proteins was observed by immunofluorescence (IF) using organic fixatives. Spread of both proteins was also detected by IF after paraformaldehyde (PFA) fixation and detergent permeabilization, albeit at reduced levels. However, while spread of GFP-VP22 was observed by examining intrinsic GFP fluorescence after methanol fixation, little spread was observed after PFA fixation, suggesting that the levels of the fusion protein in recipient cells were below the detection limits of intrinsic-fluorescence or that PFA fixation quenches the fluorescence of GFP-VP22. We further considered whether elution of VP22 from methanol-fixed cells and postfixation binding to surrounding cells contributed to the increased detection of spread observed after methanol fixation. The results show that while this could occur, it appeared to be a minor effect not accounting for the observed VP22 cell-to-cell spread in culture.