Differentiation of PC12 cells by infection with an HSV-1 vector expressing nerve growth factor.
We have developed an HSV-1 vector with mutations in the viral IE 3 and VP16 genes that expresses mouse beta nerve growth factor (NGF) from a latency associated transcript (LAT) promoter modified by insertion of a Rous sarcoma virus (RSV) enhancer. The backbone double mutant vector has reduced cytotoxicity compared with a single mutant deleted for IE 3 and is able to express the reporter luciferase gene in rat pheochromocytoma (PC12) cells at low but relatively stable levels in vitro. Intracellular NGF levels of approximately 3 pg per mg of cellular protein were detected in infected PC12 cells for at least 7 days after infection. Furthermore, infected PC12 cells exhibited differentiated morphology marked by neurite outgrowth similar to that found after exposure of PC12 cells to purified 2.5S beta NGF. A persistent level of 0.2 to 0.3 pM of the expressed NGF was detected in the culture media. The NGF-expressing vector also showed reduced cytotoxicity compared with that of the parental virus in infected PC12 cells. In PC12 cells that overexpress the human proto-oncogene bcl-2, the cytotoxicity of both viruses was significantly reduced. These studies demonstrate that the reduced cytotoxicity of the IE 3-VP16 double mutant virus and the increased duration of transgene expression from the RSV-modified LAT promoter permit terminal differentiation of PC12 cells after infection with an NGF-expressing HSV-1 vector.
Wang
M J
MJ
Department of Pediatrics, University of California at San Diego, La Jolla 92093-0634, USA.
Friedmann
T
T
Johnson
P A
PA
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D001483:Base Sequence; D002454:Cell Differentiation / Q000235:genetics*; D002470:Cell Survival / Q000235:genetics; D018076:DNA, Complementary; D004742:Enhancer Elements, Genetic; D017930:Genes, Reporter; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000235:genetics*; D016716:PC12 Cells; D011401:Promoter Regions, Genetic; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2; D051381:Rats
D018076:DNA, Complementary; C000715470:MAS1 protein, human; D009414:Nerve Growth Factors; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D008156:Luciferases