An equilibrium between spliced and unspliced primary transcripts is essential for retrovirus multiplication. This equilibrium is maintained by the presence of inefficient splice sites. The A3 3-splice site of human immunodeficiency virus type I (HIV-1) is required for Tat mRNA production. The infrequent utilization of this splice site has been attributed to the presence of a suboptimal polypyrimidine tract and an exonic splicing silencer (ESS2) in tat exon 2 ϳ60 nucleotides downstream of 3-splice site A3. Here, using site-directed mutagenesis followed by analysis of splicing in vitro and in HeLa cells, we show that the 5 extremity of tat exon 2 contains a second exonic splicing silencer (ESS2p), which acts to repress splice site A3. The inhibitory property of this exonic silencer was active when inserted downstream of another HIV-1 3-splice site (A2). Protein hnRNP H binds to this inhibitory element, and two Uto-C substitutions within the ESS2p element cause a decreased hnRNP H affinity with a concomitant increase in splicing efficiency at 3-splice site A3. This suggests that hnRNP H is directly involved in splicing inhibition. We propose that hnRNP H binds to the HIV-1 ESS2p element and competes with U2AF 35 for binding to the exon sequence flanking 3-splice site A3. This binding results in the inhibition of splicing at 3-splice site A3.Because the unique transcript produced from the integrated proviral cDNA of retroviruses serves as the genome for newly synthesized virions and also for the production of mRNAs by alternative splicing, retrovirus multiplication depends upon an equilibrium between spliced and unspliced primary transcripts. To ensure this equilibrium, retroviral RNAs generally have splice sites that are used with low efficiencies. In human immunodeficiency virus type I (HIV-1), 3Ј-splice sites (3Јss) 1 and several central 3Јss (A3, A4a, A4b, A4c, and A5) compete with each other (Fig. 1A). Site A3 is required for production of tat mRNAs, sites A4a, b, and c for production of rev and env mRNAs and site A5 for production of nef and env mRNAs (Fig. 1A) (1). Metazoan 3Јss consist of three critical elements: the branchpoint sequence (2, 3), a polypyrimidine tract (PPT) sequence (4, 5) and an AG dinucleotide at the 3Ј-end of the intron (for reviews, see Refs. 6 -9). HIV-1 branchpoint sequences are highly divergent in comparison to the metazoan consensus sequence (10 -12), and HIV-1 PPTs are suboptimal (short and interspersed by purines) (13-15). The affinity of factor U2AF for the PPT depends upon the presence of a long stretch of U residues (9, 16). Factor U2AF consists of two proteins, U2AF 65 and U2AF35 , with molecular weight of 65 and 35, respectively (17). Introns with suboptimal PPTs, like those in HIV-1 RNA, require binding of U2AF 35 at the intron-exon junction for stable interaction of U2AF 65 with the PPT (9, 18, 19). Furthermore, suboptimal 3Јss are often the subject of positive or negative regulation by cis-regulatory elements, which are frequently located in the 3Ј exon. Exonic splicing en...

Splicing is a crucial step for human immunodeficiency virus, type 1 (HIV-1) multiplication; eight acceptor sites are used in competition to produce the vif, vpu, vpr, nef, env, tat, and rev mRNAs. The effects of SR proteins have only been investigated on a limited number of HIV-1 splicing sites by using small HIV-1 RNA pieces. To understand how SR proteins influence the use of HIV-1 splicing sites, we tested the effects of overproduction of individual SR proteins in HeLa cells on the splicing pattern of an HIV-1 RNA that contained all the splicing sites. The steady state levels of the HIV-1 mRNAs produced were quantified by reverse transcriptase-PCR. For interpretation of the data, transcripts containing one or several of the HIV-1 acceptor sites were spliced in vitro in the presence or the absence of one of the tested SR proteins. Both in vivo and in vitro, acceptor sites A2 and A3 were found to be strongly and specifically regulated by SR proteins. ASF/SF2 strongly activates site A2 and to a lesser extent site A1. As a result, upon ASF/SF2 overexpression, the vpr mRNA steady state level is specifically increased. SC35 and SRp40, but not 9G8, strongly activate site A3, and their overexpression ex vivo induces a dramatic accumulation of the tat mRNA, to the detriment of most of the other viral mRNAs. Here we showed by Western blot analysis that the Nef protein synthesis is strongly decreased by overexpression of SC35, SRp40, and ASF/SF2. Finally, activation by ASF/ SF2 and 9G8 was found to be independent of the RS domain. This is the first investigation of the effects of variations of individual SR protein concentrations that is performed ex vivo on an RNA containing a complex set of splicing sites.Splicing plays a key role for production of the HIV-1 1 retroviral proteins. By using the integrated proviral genome as the template, RNA polymerase II of the infected cell produces long primary transcripts that are all identical. Some of these transcripts are transported to the cytoplasm in an intact form to serve as genomes for new virions or as messenger RNAs for the production of the Gag-Pol protein precursor. The other transcripts undergo alternative splicing to produce mRNAs for the auxiliary and regulatory proteins and the Env precursor protein. Production of mRNAs encoding HIV-1 proteins depends on the alternative utilization of four 5Ј-splice sites D1 to D4 (1) and eight 3Ј-splice sites (A1, A2, A3, A4a, -b, -c, A5, and A7) (1). An additional 3Ј-splice site (A6) was only found in one HIV-1 strain (2). Donor sites D1, D2, and D3 can be coupled to any of the A1 to A5 acceptor sites, whereas donor site D4 is exclusively coupled to site A7 and to site A6 when this site is present (1, 2). The combination of these various sites gives rise to at least 35 different mRNAs (1). Although the relative efficiencies of the HIV-1 donor sites seem to depend mainly upon their complementarity to the U1 snRNA 5Ј-terminal sequence (3, 4), efficiencies of HIV-1 acceptor sites depend upon the presence of cis-regulatory elements (5...

The HIV-1 transcript is alternatively spliced to over 30 different mRNAs. Whether RNA secondary structure can influence HIV-1 RNA alternative splicing has not previously been examined. Here we have determined the secondary structure of the HIV-1/BRU RNA segment, containing the alternative A3, A4a, A4b, A4c and A5 3' splice sites. Site A3, required for tat mRNA production, is contained in the terminal loop of a stem-loop structure (SLS2), which is highly conserved in HIV-1 and related SIVcpz strains. The exon splicing silencer (ESS2) acting on site A3 is located in a long irregular stem-loop structure (SLS3). Two SLS3 domains were protected by nuclear components under splicing condition assays. One contains the A4c branch points and a putative SR protein binding site. The other one is adjacent to ESS2. Unexpectedly, only the 3' A residue of ESS2 was protected. The suboptimal A3 polypyrimidine tract (PPT) is base paired. Using site-directed mutagenesis and transfection of a mini-HIV-1 cDNA into HeLa cells, we found that, in a wild-type PPT context, a mutation of the A3 downstream sequence that reinforced SLS2 stability decreased site A3 utilization. This was not the case with an optimized PPT. Hence, sequence and secondary structure of the PPT may cooperate in limiting site A3 utilization.

No abstract

The biological form of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer consisting of two polypeptides, p66 and p51, which have identical N-termini. The p51 polypeptide is generated by action of viral protease cleaving the p66 polypeptide between residues Phe440 and Tyr441. Dimerization has been mostly studied using bacterially purified RT bearing amino acid changes in either subunit, but not in the context of HIV-1 particles. We introduced changes of conserved amino acid residues 430-438 into the protease-sensitive subdomain of the p66 subunit and analyzed the reverse transcriptase processing and function using purified variants and their corresponding HIV-1 recombinant clones. Our mutational analysis shows that the conserved Glu438 residue is critical for proper heterodimerization and function of virion-associated RT, but not of bacterially expressed RT. In contrast, the conserved Glu430, Glu432, and Pro433 residues are not important for dimerization of virion-associated RT. The network of interactions made by the Glu438 carboxyl group with neighboring residues is critical to protect the Phe440-Tyr441 from cleavage in the context of the p66/p51 heterodimer and may explain why the p66/p51 is not processed further to p51/p51.