Evaluation of VP22 spread in tissue culture.
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.
Brewis
N
N
Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, United Kingdom.
Phelan
A
A
Webb
J
J
Drew
J
J
Elliott
G
G
O'Hare
P
P
D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D005404:Fixatives; D005453:Fluorescence; D005557:Formaldehyde; D049452:Green Fluorescent Proteins; D018259:Herpesvirus 1, Human / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011108:Polymers; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*
D005404:Fixatives; D008164:Luminescent Proteins; D011108:Polymers; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; C073413:herpes simplex virus type 1 protein VP22; D049452:Green Fluorescent Proteins; D005557:Formaldehyde; C003043:paraform