R region sequences in the long terminal repeat of a murine retrovirus specifically increase expression of unspliced RNAs

A stem-loop structure at the 5' end of the R region of the long terminal re peat (LTR) of the murine leukemia virus SL3 and other type C mammalian retr oviruses is important for maximum levels of expression of a reporter gene u nder the control of the viral LTR, This element, termed the R region stem-l oop (RSL), has a small effect on transcriptional initiation and no effect o n RNA polymerase processivity. Its major effect is on posttranscriptional p rocessing of LTR-driven transcripts. Here we tested whether the RSL affecte d the production of RNAs from a full-length SL3 genome. Mutation of the RSL in the 5' LTR of SW reduced the cytoplasmic levels of full-length viral tr anscripts but not those of spliced, env mRNA transcripts. Thus, the RSL spe cifically affected the cytoplasmic levels of the unspliced viral RNA. To te st further whether the effect was specific for unspliced transcripts, a sys tem was devised in which the expression of a reporter gene under the contro l of the viral LTR was tested in the presence or absence of an intron. Muta tion of the RSL resulted in only about a twofold decline in the level of re porter gene expression when the transcripts contained an intron. However, w hen the intron was removed, mutation of the RSL reduced expression of the r eporter gene about 10- to 60-fold in various cell lines. The secondary stru cture of the RSI, was essential for its activity on the intronless transcri pt. Thus, the RSL appears to be important for the cytoplasmic accumulation of unspliced viral RNA and unspliced RNA from chimeric transcription units under the control of the viral LTR.