Splicing regulatory elements within tat exon 2 of human immunodeficiency virus type 1 (HIV-1) are characteristic of group M but not group O HIV-1 strains.

Human immunodeficiency virus type 1 (HIV-1) is a complex retrovirus that transcribes its RNA from an integrated proviral genome and uses the host splicing machinery to produce its mRNAs. To generate the more than 30 different singly and multiply spliced mRNAs, the HIV-1 9.2-kb primary transcript undergoes splicing by a complex pathway. The regulatory proteins Tat, Rev, and Nef are encoded by multiply spliced ∼2-kb mRNAs, whereas the Env, Vif, Vpr, and Vpu proteins are encoded by singly spliced mRNAs. In addition, approximately half of the HIV-1 RNA remains unspliced and is used as message for gag and pol gene products. The unspliced RNA is also packaged into progeny virions. The HIV-1 Rev protein, which binds to the Rev-responsive element located in the env gene, facilitates the stabilization and nuclear transport of unspliced and partially spliced mRNAs. However, prior to the action of Rev, the efficiency of viral RNA splicing at the different splice sites is regulated by a number of cis elements within the viral genome. Several splicing elements have been localized within the first tat coding exon of the NL4-3 HIV-1 strain (tat exon 2). One of these elements, has been termed exon splicing silencer 2 (ESS2), is located 60 to 70 nucleotides (nt) downstream from the 3′ splice site (3′ splice site A3) used to generate Tat mRNA (Fig. ​(Fig.1A)1A) (3, 4). It maps to the 10-nt core sequence CUAGACUAGA and acts specifically to inhibit splicing at this 3′ splice site (18). Mutations within ESS2 result in a selective increase in tat splicing when tested in an in vitro splicing system or in cell culture after transfection of infectious proviral DNA or virus infection (3, 7, 18). Inspection of HIV-1 sequences in the database (9a) indicates that the region of ESS2 in many strains within the M (major) group of HIV-1 strains contain only one copy of the CUAGA sequence, which is repeated in the NL4-3 ESS2. The corresponding sequences of the O (outlier) group of HIV-1 strains are more divergent and do not contain even a single copy of the CUAGA sequence, which suggests that there may be significant differences in the regulation of tat splicing in different HIV strains. FIG. 1 RT-PCR analysis of multiply spliced HIV-mRNAs in cells transfected with wild-type and minigene NL4-3 constructs. (A) Structure of the NL4-3 HIV-1 genome. Locations of the known 5′ (D) and 3′ (A) splice sites (ss) and the ESS2 are shown. ... A second splicing element in tat exon 2 is the most 3′-terminal of the three AG dinucleotides used to generate rev mRNAs (3′ splice site A4b [Fig. 1A]). We have shown that the A4b AG serves as both a splice site for rev mRNA and a branchpoint sequence (BPS) for splicing of single-spliced and double-spliced mRNAs encoding the Env and Nef proteins, respectively (22). Mutagenesis of the A4b AG results in significantly increased splicing at the env/nef 3′ splice site (A5) and a concomitant replication defect (16, 22). We have hypothesized that the increase in splicing at A5 results from relief of competition for AG binding factors. The consequence of this is facilitated binding of a factor(s) to the branchpoint. The A4b 3′ splice site is conserved in all group M HIV-1 strains but not in any group O strains. However, in a fraction of group M and all group O strains, some of the other rev AG dinucleotides are not present and different AG dinucleotides are created that are potential new rev splice sites. These sequence differences among the HIV-1 strains suggest that regulation of splicing may be different in these strains. To test for this, we have compared alternative splice site usage of NL4-3 in the region of the first tat coding exon to two other virus strains: (i) a diverged group M, clade B virus (SF2 strain) with changes in the ESS2 sequence as well as in the region of the rev 3′ splice sites and (ii) a highly divergent group O virus strain (ANT70C). Our results indicate that regulatory elements in the region of tat exon 2 are characteristic of group M but not group O viruses.