DISRUPTION OF THE 5'-SPLICE AND 3'-SPLICE SITES FLANKING THE MAJOR LATENCY-ASSOCIATED TRANSCRIPTS OF HERPES-SIMPLEX VIRUS TYPE-1 - EVIDENCEFOR ALTERNATE SPLICING IN LYTIC AND LATENT INFECTIONS

The herpes simplex virus type 1 (HSV-1) latency-associated transcripts (LATs) are the only viral gene products expressed within latently inf ected neurones. The most abundant (major) LATs consist of two collinea r nuclear polyA(-) RNAs of 2 kb and 1.5 kb which it has been suggested represent stable introns derived from a less abundant primary transcr ipt (minor LAT). Consistent with this proposition is the identificatio n of consensus splice donor and acceptor sites flanking major LATs whi ch are conserved between HSV types 1 and 2. Here we test the functiona lity of the predicted splice sites within the context of the virus gen ome during productive infection in vitro and latent infection in vivo. To this end viruses in which the LAT splicing signals were disrupted by site-directed mutagenesis were constructed. We report that mutation of the splice acceptor site abrogates 2 kb major LAT generation durin g productive infection but does not significantly influence major LAT synthesis during neuronal latency. Similarly, mutation of the splice d onor site significantly reduces levels of 2 kb major LAT during produc tive infection but has no detectable effect on the generation of 2 kb major LAT during neuronal latency as assessed by Northern and in site hybridization analyses of latently infected neuronal tissue. From thes e data it can be concluded that the proposed splice sites Ranking the major LAT region are dispensable for 2 kb major LAT production in neur ones latently infected with HSV-1 but constitute functional splicing s ignals in productively infected non-neuronal cells.