POLY(A) SITE SELECTION IN THE HIV-1 PROVIRUS - INHIBITION OF PROMOTER-PROXIMAL POLYADENYLATION BY THE DOWNSTREAM MAJOR SPLICE DONOR SITE

In common with all retroviruses, the human immunodeficiency virus type 1 (HIV-1) contains duplicated long terminal repeat (LTR) sequences fl anking the proviral genome. These LTRs contain identical poly(A) signa ls, which are both transcribed into RNA. Therefore, to allow efficient viral expression, a mechanism must exist to either restrict promoter- proximal poly(A) site use or enhance the activity of the promoter-dist al poly(A) site. We have examined the use of both poly(A) sites using proviral clones. Mutation of the previously defined upstream activator y sequences of the 3' LTR poly(A) site decreases the efficiency of pol yadenylation when placed in competition with an efficient downstream p rocessing signal. However, in the absence of competition, these mutati ons have no effect on HIV-1 polyadenylation. In addition, the 5' LTR p oly(A) site is inactive, whereas a heterologous poly(A) site positione d in its place is utilized efficiently. Furthermore, transcription ini tiating from the 3' LTR promoter utilizes the 3' LTR poly(A) signal ef ficiently. Therefore, the main determinant of the differential poly(A) site use appears to be neither proximity to a promoter element in the 5' LTR nor the presence of upstream activating sequences at the 3' LT R. Instead, we show that the major splice donor site that is immediate ly downstream of the 5' LTR inhibits cleavage and polyadenylation at t he promoter-proximal site. The fact that this poly(A) site is active i n a proviral clone when the major splice donor site is mutated suggest s that the selective use of poly(A) signals in HIV-1 is mediated by a direct inhibition of the HIV-1 poly(A) site by downstream splicing eve nts or factors involved in splicing.