CONTROL OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 RNA-METABOLISM - ROLE OF SPLICE SITES AND INTRON SEQUENCES IN UNSPLICED VIRAL-RNA SUBCELLULAR-DISTRIBUTION

In the course of examining the various factors which affect the metabo lism of human immunodeficiency virus type 1 (HIV-1) RNA, we examined t he role of intron sequences and splice sites in determining the subcel lular distribution of the RNA. Using in situ hybridization, we demonst rated that in the absence of Rev, unspliced RNA generated with an HIV- 1 env expression construct displayed discrete localization in the nucl eus, coincident with the location of the gene and not associated with SC35-containing nuclear speckles. Expression of Rev resulted in a disp erse signal for the unspliced RNA throughout both the nucleus and the cytoplasm. Subsequent fractionation of the nucleus revealed that the m ajority of unspliced viral RNA within the nucleus is associated with t he nuclear matrix and that upon expression of Rev, a small proportion of the unspliced RNA is found within the nucleoplasm. Mutations which altered splice site utilization did not alter the sequestration of uns pliced RNA into discrete nuclear regions. In contrast, a 2.2-kb deleti on of intron sequence resulted in a shift from discrete regions within the nucleus to a disperse signal throughout the cell, indicating that intron sequences, and not just splice sites, are required for the obs erved nuclear sequestration of unspliced viral RNA.