Zy. Chen et al., DETECTION OF NEGATIVE-STRANDED SUBGENOMIC RNAS BUT NOT OF FREE LEADERIN LDV-INFECTED MACROPHAGES, Virus research, 34(2), 1994, pp. 167-177
The mechanism of synthesis of the seven subgenomic mRNAs of lactate de
hydrogenase-elevating virus (LDV) was explored. One proposed mechanism
, leader-primed transcription, predicts the formation of free 5'-leade
r 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 vi
rions. Full-length antigenomic RNA was detected in LDV-infected macrop
hages 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 oligonucleo
tide complementary to the antileader followed by polymerase chain reac
tion 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 f
unction in the replication of the subgenomic mRNAs. They could also be
by-products of the RNA replication process. Finally, no subgenomic mR
NAs were detected in LDV virions.