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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.

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.

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 approximately 60 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 U-to-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.

Using in vitro splicing assays with HeLa cell nuclear extracts, we showed that the HIV-1 pairs of splice sites D1-A2 and D2-A2 are efficiently used in vitro, as compared to the control D1-A2 pair of sites from the E3 transcription unit of human adenovirus-2. The strong efficiency of the two HIV-1 pairs of sites is surprising, as we also showed by primer extension analysis that the branch-site sequence used at the HIV-1 acceptor site A2 is UAGCAGA, with a dominant utilization of the ultimate G as the branched residue. No significant increase of the splicing efficiency was observed upon replacement of the wild-type branch-site sequence by a canonical sequence, in spite of the utilization of an A residue as the branched nucleotide. Results are discussed taking into account the present knowledge on branch-site selection.