DETERMINATION OF THE 5' END OF THE LACTATE DEHYDROGENASE-ELEVATING VIRUS GENOME BY 2 INDEPENDENT APPROACHES

We have determined the 5' end of the lactate dehydrogenase-elevating v irus (LDV) genome (strain LDV-P) using two independent approaches. In one approach, methylmercuric hydroxide-denatured genomic RNA was rever se-transcribed using as primer an oligonucleotide complementary to the 5' end of open reading frame (ORF) la. The first-strand cDNA was liga ted with T4 RNA ligase to an oligonucleotide of which the 3' end was b locked. The ligated product was amplified by PCR, cloned and sequenced . In the second approach, untreated or decapped genomic RNA was ligate d between the 3' and 5' ends, reverse-transcribed across the ligation junction and the product was amplified by PCR, cloned and sequenced. B oth approaches yielded the same results, indicating that the 5' leader of LDV-P is 156 nucleotides long, inclusive of the 5' UAUAACC 3' sequ ence involved in the linkage of the 5' leader to the bodies of the sev en subgenomic mRNAs of LDV. The 5' leader of LDV is about 50 nucleotid es shorter than those of the related viruses, equine arteritis virus a nd Lelystad virus, but at least twice as long as the leaders of the co ronavinrses. The finding that untreated LDV RNA was ligated 5' to 3' e nd as efficiently as RNA treated with decapping enzyme suggests that g enomic LDV RNA may not possess a 5' cap but terminates with 5' phospho ryl-A.