THE TAT REV INTRON OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 IS INEFFICIENTLY SPLICED BECAUSE OF SUBOPTIMAL SIGNALS IN THE 3' SPLICE-SITE/

Proportional expression of retroviral genes requires that splicing of the viral primary transcript be an inefficient process. Much of our cu rrent knowledge about retroviral suboptimal splicing comes from studie s with Rous sarcoma virus. In this report, we describe the use of chim eric introns composed of human beta-globin and human immunodeficiency virus type 1 (HIV-1) splice sites to establish the basis for inefficie nt splicing of the intron which comprises most of the HIV-1 env coding sequences (referred to as the tat/rev intron). S1 RNA analysis of tra nsfected COS-7 cells revealed that the 3' splice site (3' ss) of this region was significantly less efficient than the 3' ss of the first in tron of beta-globin. Deletion of sequences flanking the tat/rev intron 3' ss demonstrated that the requirements for its inefficiency reside within the region that is expected to comprise the essential signals f or splicing (i.e., the branchpoint region, the polypyrimidine tract, a nd the AG dinucleotide). Introduction of an exact copy of the efficien t beta-globin branchpoint sequence within a highly conserved region re ndered the tat/rev intron 3' ss highly efficient. Improvement of the p olypyrimidine tract also increased the splicing efficiency, but to a d egree slightly less than that obtained with the branchpoint mutation. Subsequent examination of the tat/rev intron 5' splice site in a heter ologous context revealed that it is efficiently utilized. These result s indicate that both a poor branchpoint region and a poor polypyrimidi ne tract are responsible for the low splicing efficiency of the HIV-1 tat/rev intron. It is of fundamental interest to establish the basis f or inefficient splicing of the HIV-1 tat/rev intron since it may provi de the key to understanding why nuclear export of mRNAs encoding HIV-1 structural proteins is Rev dependent.