Expression of the growth-associated protein B-50/GAP43 via a defective herpes-simplex virus vector results in profound morphological changes in non-neuronal cells.
This study describes the creation and application of a defective herpes simplex viral (HSV) vector for B-50/GAP-43, a neural growth-associated phosphoprotein. We demonstrate abundant expression of B-50/GAP-43 in cultured non-neuronal cells (African green monkey kidney cells [vero cells] and Rabbit skin cells) via this HSV vector. When B-50/GAP-43 was expressed in non-neuronal cells major morphological changes occurred that included extensive membrane ruffling, the formation of filopodia and long thin extensions reminiscent of neurites. These extensions often terminated in growth cone-like structures. Quantitation of these morphological changes at different times following infection demonstrates that the surface area of the B-50/GAP-43-expressing cells started to increase between 6 and 10 h post-infection. At 72 h, B-50/GAP-43-positive cells were 3.0 times larger in size and one third of the cells expressed long processes with a mean length of 165 +/- 14.5 microns. Ultrastructural studies of cells 48 h after infection revealed that B-50/GAP-43 is predominantly localized at the plasma membrane of the elaborated processes. Some immunoreactivity was associated with vesicular structures that appear to be in-transit in the processes. These observations suggest that B-50/GAP-43 acts at the plasmamembrane to induce a neuron-like morphology in non-neuronal cells persisting for several days in culture. In the future the defective viral vector will enable gene transfer to express B-50/GAP-43 in neurons in vivo in order to study its involvement in regenerative sprouting and neuroplasticity.
Rudolf Magnus Institute, Department of Medical Pharmacology, Utrecht, The Netherlands.
J
J
Verhaagen
W T
WT
Hermens
A B
AB
Oestreicher
W H
WH
Gispen
S D
SD
Rabkin
D W
DW
Pfaff
M G
MG
Kaplitt
D000818:Animals; D002522:Chlorocebus aethiops; D003587:Cytomegalovirus; D003673:Defective Viruses; D019922:GAP-43 Protein; D005822:Genetic Vectors; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D016900:Neurofilament Proteins / Q000096:biosynthesis; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis; D018139:Simplexvirus; D012867:Skin / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D014162:Transfection; D014709:Vero Cells
D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; D011994:Recombinant Proteins