Analysis of HSV-1 Transcripts by RNA-PCR

Since the herpes simplex virus type 1 (HSV-1) genome has been sequenced and most HSV-1 RNAs are not spliced (1 ), detailed information about the structure of many HSV-1 RNAs can be obtained without the considerable time and effort that is required to construct and analyze cDNA libraries. Once the 5′ and 3′ ends of an RNA have been mapped precisely, the RNA nucleotide sequence can be deduced simply from the genomic DNA sequence. However, there are certain situations, such as the analysis of spliced RNAs or of chimeric RNAs expressed from foreign genes inserted into HSV-1 vectors, where cDNA cloning of HSV-1 transcripts may be informative. There are families of transcripts that arise by alternate splicing in several human herpesviruses: HSV-1, cytomegalovirus (CMV), and Epstein-Barr virus (EBV). For example, HSV-1 encodes several overlapping latency-associated transcripts or LATs (2 –4 ). The splice junctions of the intron within the HSV-1 2.0-kb LAT have been determined by RNA-PCR with primers located on either side of the intron, followed by direct DNA sequence determination of the PCR product (5 ). The construction of partial cDNAs by PCR saves much time-consuming effort and expense compared with the analysis of cDNA libraries. In addition, by sequencing PCR products directly, the need to analyze several cDNA clones in order to be assured of obtaining the consensus sequence is eliminated.