The exon splicing silencer in human immunodeficiency virus type 1 Tat exon 3 is bipartite and acts early in spliceosome assembly.

Human immunodeficiency virus type 1 (HIV-1) is a complex retrovirus whose RNA splicing is dependent on the host cell splicing machinery (12, 50). HIV-1 pre-mRNA contains five 5′ splice sites and nine 3′ splice sites which are used to generate more than 30 different mRNA species by alternative splicing. In addition, a pool of unspliced RNA must be maintained to serve as genomic RNA and as mRNA for the gag and pol gene products (18, 21, 32, 34, 35, 38). To achieve the balance between spliced and unspliced RNAs, splicing of HIV-1 RNA is inefficient, a feature which is shared with all other retroviruses (4, 7, 11, 20, 22, 45–47, 52). In contrast to the major HIV-1 5′ splice sites, which are strong, the 3′ splice sites are weak (33). These are characterized by the presence of nonconsensus polypyrimidine tracts and branch point sites other than the eukaryotic consensus adenosine residue (2, 13, 17, 33, 43). In addition, exon sequences downstream of the 3′ splice sites enhance or inhibit splicing at the 3′ splice sites (2, 3, 39, 42, 51). Furthermore, the virus-encoded protein Rev plays an essential role in facilitating the transport of unspliced and singly spliced mRNAs, thus preventing the viral RNA from splicing to completion (19, 37; for a review, see reference 12). HIV-1 tat mRNA is formed by the splicing of two coding exons (exon 2 and exon 3) and one or more upstream noncoding exons. Exon 3 is also joined to rev exon 2 and is therefore referred to as tat/rev exon 3 (34). The 3′ splice sites flanking exon 2 and exon 3 (3′ splice sites 3 and 7, respectively [Fig. 1]) contain suboptimal polypyrimidine tracts, and the 3′ splice site flanking exon 3 has been reported to contain a nonconsensus branch point site (17, 33, 39, 43). In addition, we and others have demonstrated that tat/rev exon 3 splicing is regulated by an exon splicing enhancer (ESE) and a juxtaposed exon splicing silencer (ESS) within this exon (ESS3) (Fig. ​(Fig.1)1) (3, 42). An ESS is also present within tat exon 2 (ESS2), which has been localized to the RNA sequence CUAGACUAGA in the region of vpr encoding the C terminus and in the region of tat encoding the N terminus. It acts at an early step in the splicing pathway to selectively inhibit splicing at the upstream 3′ splice site flanking this tat exon (3). Addition of competitor RNA containing ESS2 to splicing reaction mixtures with HIV-1 RNA substrates containing ESS2 causes an increase in splicing at the upstream 3′ splice site (3′ splice site 3). Based on these data, we hypothesized that the inhibition by ESS2 is mediated by a negative cellular factor(s) which binds to the ESS (3). In this study, we have delineated the ESS3 element in tat/rev exon 3 and have investigated the mechanism by which it inhibits splicing. The data suggest that the inhibition by ESS2 and ESS3 involves binding of a common factor(s). FIG. 1 (A) Schematic diagram of HIV-1 splice sites and structure of tat mRNA. ESS2 and ESS3 are ESSs located within tat exon 2 and tat/rev exon 3, respectively. 5′ splice sites are indicated above the line by arrows; 3′ splice sites are denoted ...