Determination of the 5' end of the lactate dehydrogenase-elevating virus genome by two independent approaches.

Abstract

We have determined the 5' end of the lactate dehydrogenase-elevating virus (LDV) genome (strain LDV-P) using two independent approaches. In one approach, methylmercuric hydroxide-denatured genomic RNA was reverse-transcribed using as primer an oligonucleotide complementary to the 5' end of open reading frame (ORF) 1a. The first-strand cDNA was ligated with T4 RNA ligase to an oligonucleotide of which the 3' end was blocked. The ligated product was amplified by PCR, cloned and sequenced. In the second approach, untreated or decapped genomic RNA was ligated between the 3' and 5' ends, reverse-transcribed across the ligation junction and the product was amplified by PCR, cloned and sequenced. Both approaches yielded the same results, indicating that the 5' leader of LDV-P is 156 nucleotides long, inclusive of the 5' UAUAACC 3' sequence involved in the linkage of the 5' leader to the bodies of the seven subgenomic mRNAs of LDV. The 5' leader of LDV is about 50 nucleotides shorter than those of the related viruses, equine arteritis virus and Lelystad virus, but at least twice as long as the leaders of the coronaviruses. The finding that untreated LDV RNA was ligated 5' to 3' end as efficiently as RNA treated with decapping enzyme suggests that genomic LDV RNA may not possess a 5' cap but terminates with 5' phosphoryl-A.