THE HERPES-SIMPLEX VIRUS TYPE-1 2.0-KILOBASE LATENCY-ASSOCIATED TRANSCRIPT IS A STABLE INTRON WHICH BRANCHES AT A GUANOSINE

We have used a minigene construct of the herpes simplex virus type 1 ( HSV-1) latency-associated transcript (LAT) gene to analyze its transcr ipts in transient transfection assays. A 2.8-kb fragment of the approx imately 8.5-kb LAT gene encompassing the 2.0-Wb LAT,vas cloned into a eukaryotic expression vector downstream of the cytomegalovirus immedia te-early gene promoter. Northern hybridization of RNA isolated from tr ansfected COS-I cells identified three LAT-specific transcripts, 3.4, 2.0, and 1.4 kb in size. Mapping of these transcripts by Northern hybr idization indicated that the 1.4- and 2.0-kb RNAs are nonoverlapping, while the 3.4-kb RNA overlaps both smaller RNAs. Reverse transcription -PCR (RT-PCR) and partial sequencing of the 1.4-kb RNA revealed that t his RNA is the spliced exons of the 3.4-kb primary transcript. The 2.0 -kb LAT appears to be an intron accumulating after splicing of the min or LAT (mLAT) pre-mRNA. The splice donor and acceptor sites for the 2. 0-kb LAT identified in transfected and HSV-l-infected cells are identi cal. Mapping of the branch point of this intron by RT-PCR in transfect ed and HSV-l-infected cells, as well as in latently infected murine tr igemial ganglia, shows that it is a guanosine. This branch site does n ot bear homology to consensus mammalian branch site sequences. These d ata provide evidence that the 2.0-kb LAT is an intron of the mLAT pre- mRNA with a unique branch point.