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 transcripts in transient transfection assays. A 2.8-kb fragment of the approximately 8.5-kb LAT gene encompassing the 2.0-kb LAT was cloned into a eukaryotic expression vector downstream of the cytomegalovirus immediate-early gene promoter. Northern hybridization of RNA isolated from transfected COS-1 cells identified three LAT-specific transcripts, 3.4, 2.0, and 1.4 kb in size. Mapping of these transcripts by Northern hybridization 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 this 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 minor LAT (mLAT) pre-mRNA. The splice donor and acceptor sites for the 2.0-kb LAT identified in transfected and HSV-1-infected cells are identical. Mapping of the branch point of this intron by RT-PCR in transfected and HSV-1-infected cells, as well as in latently infected murine trigemial ganglia, shows that it is a guanosine. This branch site does not bear homology to consensus mammalian branch site sequences. These data provide evidence that the 2.0-kb LAT is an intron of the mLAT pre-mRNA with a unique branch point.
Zabolotny
J M
JM
The Wistar Institute, Philadelphia, Pennsylvania 19104-4268, USA.
Krummenacher
C
C
Fraser
N W
NW
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D005814:Genes, Viral; D006151:Guanosine; D006860:Hydrogen Bonding; D007438:Introns; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D012326:RNA Splicing; D012367:RNA, Viral / Q000235:genetics*; D018139:Simplexvirus / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics*; D017735:Virus Latency
D012367:RNA, Viral; D015678:Viral Structural Proteins; D006151:Guanosine