Methods for detecting the HSV-1 LAT anti-apoptosis activity in virus infected tissue culture cells

Plasmids expressing the herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) reduce apoptosis in transient transfection assays in tissue culture. LAT also reduces apoptosis in the context of the virus in trigeminal ganglia of rabbits and mice at approximately 6-7 days post-infection during the switch from acute to latent HSV-1 infection, a time at which LAT is the only abundantly transcribed viral gene. Analysis of LAT's anti-apoptosis function is complicated in tissue culture by the expression of at least five additional viral gene products that can block apoptosis, and by the fact that apoptosis usually occurs in only a fraction of the cells. Here, we present two approaches for detecting LAT's anti-apoptosis activity in the context of the whole virus in tissue culture. Using a combination of serum starvation to both partially synchronize the cells and induce apoptosis, and Hoechst staining to detect chromatin condensation, we found that there was a small window of time post-infection during which Schwann cells infected with the LAT(-) mutant dLAT2903 reproducibly had more apoptotic nuclei than identically treated cells infected with the LAT(+) parental virus HSV-1 strain McKrae. Using serum starvation and/or UV treatment and a method to isolate fragmented DNA away from large chromosomal DNA, we found a similar window of time post-infection during which Neuro2A cells infected with dLAT2903 had increased DNA fragmentation (as judged by a DNA laddering assay) compared to identically treated cells infected with wild type McKrae or the LAT(+) marker rescued dLAT2903R virus. These assays should permit the use of culture assays, rather than labor intensive animal models, to examine LAT's anti-apoptosis activity in the context of the virus in a large number of existing LAT mutant viruses.