RESUMO
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.
Assuntos
Éxons , Produtos do Gene tat/genética , HIV-1/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Splicing de RNA , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Sequência de Bases , Sítios de Ligação , Núcleo Celular/metabolismo , Produtos do Gene tat/metabolismo , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Homologia de Sequência do Ácido Nucleico , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
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.
Assuntos
Regiões 3' não Traduzidas , Sequência Conservada , HIV-1/química , Conformação de Ácido Nucleico , Sítios de Splice de RNA , RNA Viral/química , Sequências Reguladoras de Ácido Nucleico , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/metabolismo , Processamento Alternativo/genética , Sequência de Bases , Sequência Conservada/genética , Produtos do Gene tat/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sítios de Splice de RNA/genética , RNA Viral/síntese química , RNA Viral/genética , Sequências Reguladoras de Ácido Nucleico/genética , Células Tumorais Cultivadas , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
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.
Assuntos
Ácido Glutâmico/metabolismo , Protease de HIV/metabolismo , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Clonagem Molecular , Dimerização , Escherichia coli , Expressão Gênica , Ácido Glutâmico/genética , Transcriptase Reversa do HIV/genética , HIV-1/fisiologia , Cinética , Dados de Sequência Molecular , Mutagênese , Peptídeos/genética , Polímeros , Estrutura Terciária de Proteína , Vírion/fisiologia , Replicação ViralRESUMO
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.