Expression of vhs and VP16 during HSV-1 helper virus-free amplicon packaging enhances titers.
Recently developed helper virus-free methods of herpes simplex virus (HSV) amplicon vector packaging provide stocks that are virtually devoid of the cytotoxic component normally associated with traditional helper virus-based packaging methods. These approaches involve cotransfection of amplicon plasmid DNA with either a five-cosmid set or a bacterial artificial chromosome (BAC) that contains the HSV genome without its cognate pac signals. Helper virus-free amplicon packaging produces low-titer stocks (<10(5) expressing particles/ml) that exhibit a high frequency of pseudotransduction. In an effort to enhance amplicon titers, we introduced in trans a genomic copy of the virion host shutoff (vhs) protein-encoding gene UL41 into both cosmid- and BAC-based packaging strategies. Cotransfection of this plasmid with the amplicon and packaging reagents results in a 10-fold higher amplicon titer, and stocks that do not exhibit the pseudotransduction phenomenon. To further enhance packaging efficiency, the HSV transcriptional activator VP16 was introduced into packaging cells 1 day before the packaging components. Pre-loading of packaging cells with VP16 led to an additional enhancement of amplicon titers, an effect that did not occur in the absence of vhs. Increased helper virus-free amplicon titers resulting from these modifications will make in vivo transduction experiments more feasible.
Bowers
W J
WJ
Department of Neurology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA.
Howard
D F
DF
Brooks
A I
AI
Halterman
M W
MW
Federoff
H J
HJ
D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics; D006224:Cricetinae; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006378:Helper Viruses / Q000235:genetics; D017405:Herpes Simplex Virus Protein Vmw65 / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000235:genetics*; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods; D012260:Ribonucleases; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism
D017405:Herpes Simplex Virus Protein Vmw65; D014764:Viral Proteins; C070722:virion host shutoff protein, Simplexvirus; D012260:Ribonucleases; D001616:beta-Galactosidase