Molecular mechanisms regulating the expression of the HSV-1 LAT ORF and analysis of potential in vivo function

During latency, HSV-1 gene expression is limited to the latency-associated transcripts (LATs), which encode at least three potential open reading frames (ORFs) that are conserved between HSV-1 strains. This suggests that proteins that function at some point in the HSV lifecycle might be encoded, even though such an activity has not been detected previously, although work suggesting a function of the largest LAT ORF has previously been published (Thomas et al. 1999). This thesis concentrates on studying the regulation of the expression of this potentially functional ORF. In order to study the regulation of expression of the largest LAT ORF, viruses with green fluorescent protein (GFP) or lacZ replacing the ORF were constructed, with and without a polyA sequence. PolyA(-) viruses should express GFP/lacZ under conditions where the LAT ORF is usually expressed. Expression was not detected in vitro or at early time points in dorsal root ganglia after footpad inoculation of mice, but was detected at significant levels during latency. Similar expression was detected from polyA(+) and polyA(-) viruses indicating that differential polyadenylation is probably not important in regulating LAT ORF expression, at least in mice. Adding the woodchuck posttranscriptional regulatory element, after GFP, to potentially enhance the export of RNA from the nucleus gave similar results. Hence, differential polyadenylation or RNA export do not appear to play a key role in the regulation of LAT ORF expression. Marker gene expression decreased during reactivation. Studies aiming at determining if marker gene expression from these recombinant viruses during latency correlates with spontaneous reactivation were performed. Dual recombinant viruses containing both GFP within the LAT ORF and the IE1 promoter driving lacZ expression from within the UL43 gene were constructed and tested in vivo, either alone or together with a virus containing an intact LAT ORF. In both experiments, GFP expression did not correlate with lacZ expressing cells (i.e. cells in which spontaneous reactivation was probably occurring). Therefore, unless reactivation was aborted before ICP0 expression, LAT ORF expression does not appear to correlate with reactivation. Thus, it seems likely that the LAT ORF is expressed in vivo during latency, which was not expected from previous work (Thomas et al. 1999). Finally, work aimed at investigating an in vivo activity of the LAT ORF showed that CMV driven LAT ORF expression from an otherwise wild type virus could activate the ICP0 promoter in vivo from an already latent virus. This is the first preliminary evidence of a direct LAT protein mediated effect in vivo