Detection of negative-stranded subgenomic RNAs but not of free leader in LDV-infected macrophages

The mechanism of synthesis of the seven subgenomic mRNAs of lactate dehydrogenase-elevating virus (LDV) was explored. One proposed mechanism, leader-primed transcription, predicts the formation of free 5'-leader in infected cells which then primes reinitiation of transcription at specific complementary sites on the antigenomic template. No free LDV 5'-leader of 156 nucleotides was detected in LDV-infected macrophages. Another mechanism, independent replication of the subgenomic mRNAs, predicts the presence of negative complements to all subgenomic mRNAs in infected cells which might be generated from subgenomic mRNAs in virions. Full-length antigenomic RNA was detected in LDV-infected macrophages by Northern hybridization at a level of < 1% of that of genomic RNA, but no negative polarity subgenomic RNAs. Negative complements to all subgenomic mRNAs, however, were detected by reverse transcription of total RNA from infected macrophages using as primer an oligonucleotide complementary to the antileader followed by polymerase chain reaction amplification using this sense primer in combination with various oligonucleotide primers complementary to a segment downstream of the junction between the 5' leader and the body of each subgenomic RNA. It is unclear whether these minute amounts of negative subgenomic RNAs function in the replication of the subgenomic mRNAs. They could also be by-products of the RNA replication process. Finally, no subgenomic mRNAs were detected in LDV virions.