Control of HIV-1 gene expression depends on two viral regulatory proteins, Tat and Rev. Tat stimulates transcription elongation by directing the cellular transcriptional elongation factor P-TEFb to nascent RNA polymerases. Rev is required for the transport from the nucleus to the cytoplasm of the unspliced and incompletely spliced mRNAs that encode the structural proteins of the virus. Molecular studies of both proteins have revealed how they interact with the cellular machinery to control transcription from the viral LTR and regulate the levels of spliced and unspliced mRNAs. The regulatory feedback mechanisms driven by HIV-1 Tat and Rev ensure that HIV-1 transcription proceeds through distinct phases. In cells that are not fully activated, limiting levels of Tat and Rev act as potent blocks to premature virus production.A fter integration into the host genome, the HIV-1 provirus acts as a transcription template that is regulated at the transcriptional and posttranscriptional levels. Immediately after infection, HIV-1 produces only short completely spliced mRNAs encoding the viral regulatory proteins Tat and Rev. As the infection proceeds, transcription increases sharply, and larger, incompletely spliced mRNAs are produced. These encode Env and the HIV-1 accessory genes Vif, Vpr, and Vpu. Also synthesized late are the full-length unspliced transcripts which act both as the virion genomic RNA and the mRNA for the Gag-Pol polyprotein (Kim et al. 1989;Pomerantz et al. 1990).This complex pattern of gene expression is controlled by the regulatory proteins Tat and Rev. Tat activates viral transcription by stimulating elongation from the viral long terminal repeat (LTR). Rev transports the unspliced and incompletely spliced mRNAs encoding the structural proteins from the nucleus to the cytoplasm. In this article, we review our current understanding of how these unique regulatory proteins orchestrate HIV-1 gene expression through their interactions with the cellular transcription, RNA splicing, and RNA transport machinery.

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In HIV-1 infected cells, over 40 different mRNA species are produced by alternative splicing of the single HIV-1 primary RNA transcript. In addition, approximately half of the HIV-1 primary RNA transcripts are not spliced and are exported to the cytoplasm where they serve as mRNA and as genomic RNA. In this article, we will review current knowledge of the mechanisms by which the HIV-1 alternative splicing is regulated. Several negatively and positively-acting cis-acting elements have been detected within the viral genome that repress or facilitate viral RNA splicing by binding to cellular proteins. These include exonic splicing silencers (ESS) and an intronic splicing silencer (ISS) that are selectively bound either by members of the hnRNP A/B family (hnRNPs A1, A1(B), A2, and B1) or by hnRNP H. Exonic splicing enhancers (ESE) are also present within the HIV-1 genome and are selectively bound by members of the SR protein family. ESS and ISS repression mediated by hnRNP A/B proteins occurs at early steps of splicing, prior to formation of pre-spliceosome complexes. Current models propose that ESS elements promote cooperative binding of hnRNP A/B proteins to the exon and prevent efficient binding of essential cellular splicing factors to the 3' splice site. SR proteins bound to ESE elements that are juxtaposed or overlapping ESS elements may counteract this inhibition. We will review data indicating the importance of the HIV-1 splicing elements and their cognate binding proteins for efficient virus replication. Differences in cis-acting splicing elements between the group M (major) and group O (outlier) HIV-1 strains will also be discussed. Finally we will review evidence suggesting the possibility that there may be changes in regulation of HIV-1 alternative splicing in infected human T cells, human macrophages and rodent cells.

Following ribonuclease digestion of methyl-3H-labeled B77 avian sarcoma virus RNA subunits, methylated oligonucleotides were isolated by diethylaminoethylcellulose chromotogrpahy. Partial nucleotide sequences were deduced from the known enzymatic specificities of the ribonucleases. In addition to methylated nucleosides in the 5'-terminal cap structure, m7G(5')GmpCp, N6-methyladenosine(m6A) was found to be present in only two internal sequences of the RNA molecule, Gpm6ApC and Apm6ApC. The average numbers of methylated nucleosides per RNA subunit are about 12-13 in Gpm6ApC, 1-2 in Apm6ApC, and 2 in m7GpppGmpCp. The sequences containing m6A in B77 sarcoma virus RNA are identical to m6A-containing sequences previously reported for the bulk mRNA from HeLa cells (Wei, C.M., Gershowitz, A., and Moss, B. (1976), Biochemistry 15, 397-401). Analysis of the oligonucleotides produced by RNase A digestion indicated that the sequence of bases on the 5' side of these trinucleotides is not specific. The oligonucleotide profile, however, was highly reproducible in different virus preparations. This suggests that the methylations occur at specific positions on the RNA molecule. Some of the methylated oligonucleotides produced by RNase A digestion appear to be present in less than molar amounts. Several hypotheses are proposed to explain this result.

Human immunodeficiency virus type 1 (HIV-1)pre-mRNA splicing is regulated in order to maintain pools of unspliced and partially spliced viral RNAs as well as the appropriate levels of multiply spliced mRNAs during virus infection. We have previously described an element in tat exon 2 that negatively regulates splicing at the upstream tat 3 splice site 3 (B. A. Amendt, D. Hesslein, L.-J. Chang, and C. M. Stoltzfus, Mol. Cell. Biol. 14:3960-3970, 1994). In this study, we further defined the element to a 20-nucleotide (nt) region which spans the C-terminal vpr and N-terminal tat coding sequences. By analogy with exon splicing enhancer (ESE) elements, we have termed this element an exon splicing silencer (ESS). We show evidence for another negative cis-acting region within tat-rev exon 3 of HIV-1 RNA that has sequence motifs in common with a 20-nt ESS element in tat exon 2. This sequence is juxtaposed to a purine-rich ESE element to form a bipartite element regulating splicing at the upstream tat-rev 3 splice site. Inhibition of the splicing of substrates containing the ESS element in tat exon 2 occurs at an early stage of spliceosome assembly. The inhibition of splicing mediated by the ESS can be specifically abrogated by the addition of competitor RNA. Our results suggest that HIV-1 RNA splicing is regulated by cellular factors that bind to positive and negative cis elements in tat exon 2 and tat-rev exon 3.Alternative splicing of mRNA precursors plays a critical role in the regulation of gene expression. In metazoan cells, splicing of pre-mRNA is mediated by cis-acting signals which include 5Ј and 3Ј splice sites, branchpoint sequences, and polypyrimidine tracts preceding 3Ј splice sites (for a review, see reference 19). However, the mechanisms by which alternative splice site selection is regulated are not well understood. There are numerous examples of sequences within introns that act to either enhance or inhibit splicing (3,7,10,16,21,25,35,40,43,65). Some of these intron sequences have been shown to bind cellular factors (21,35,40,43). Exon sequences have also been shown to play a role in alternative splicing. Positive-acting exon sequences and purine-rich regions or exon splicing enhancer (ESE) elements have been reported for a number of different cellular and viral genes (4,5,8,23,30,36,50,51,53,56,(58)(59)(60). Some of these positive-acting exon sequences are binding sites for cellular factors (5,23,30,50,58). A family of factors called SR proteins are required for splicing and, in some cases, have been shown to regulate alternative splice site selection in a concentration-dependent manner (14,17,28,33,61). Recent reports have shown that the SR proteins selectively bind to purine-rich splicing elements present in cellular exons (30,49,50). There are also several examples of negative-acting exon splicing elements (2,4,18,45,55). To date, factors interacting with negative-acting exon splicing elements affecting alternative 3Ј splice site usage in metazoan cells have not yet been reported.Human immunodeficie...