Introducing point mutations into the ATGs of the putative open reading frames of the HSV-1 gene encoding the latency associated transcript (LAT) reduces its anti-apoptosis activity

The herpes simplex virus type 1 (HSV-1) latency associated transcript (LAT) gene has anti-apoptosis activity that directly or indirectly enhances the virus's reactivation phenotype in small animal models. The first 1.5 kb of the primary 8.3 kb LAT is sufficient and some or all of it is necessary for LAT's anti-apoptosis in transient transfection assays and for LAT's ability to enhance the reactivation phenotype. Based on LAT's genomic sequence, the first 1.5 kb contains eight potential open reading frames (ORFs) defined as an ATG followed by an in frame termination codon. In this study, point mutations were introduced into the ATGs of ORFs present in the 1.5 kb fragment of LAT. Mutagenesis of all eight ATGs in LAT ORFs consistently reduced the anti-apoptotic activity of LAT in transiently transfected mouse neuroblastoma cells regardless of whether apoptosis was induced by caspase 8 or caspase 9. Mutation of the six ATGs located in the stable intron sequences within the 1.5 kb LAT had a dramatic effect on caspase 9, but not caspase 8, induced apoptosis. For both caspase 8 and caspase 9 induced apoptosis, mutating the two ATGs in the exon of the LAT 1.5 kb fragment reduced, but did not eliminate the anti-apoptotic activity of LAT. These studies suggest that altering the fine structure of regulatory RNA or expression of a putative LAT ORF regulates the anti-apoptosis activity of LAT. These studies also indicate that more than one function is present in the 1.5 kb LAT fragment.