RESUMO
Interleukin 6 (IL-6) acts as a pro and anti-inflammatory cytokine, has an intense correlation with exercise intensity, and activates various pathways such as autophagy and mitochondrial unfolded protein response. Also, IL-6 is interconnected to circadian clock-related inflammation and can be suppressed by the nuclear receptor subfamily 1, group D, member 1 (Nr1d1, protein product REV-ERBα). Since IL-6 is linked to physical exercise-modulated metabolic pathways such as autophagy and mitochondrial metabolism, we investigated the relationship of IL-6 with REV-ERBα in the adaptations of these molecular pathways in response to acute intense physical exercise in skeletal muscle. The present study was divided into three experiments. In the first one, wild-type (WT) and IL-6 knockout (IL-6 KO) mice were divided into three groups: Basal time (Basal; sacrificed before the acute exercise), 1 hour (1hr post-Ex; sacrificed 1 hour after the acute exercise), and 3 hours (3hr post-Ex; sacrificed 3 hours after the acute exercise). In the second experiment, C2C12 cells received IL-6 physiological concentrations or REV-ERBα agonist, SR9009. In the last experiment, WT mice received SR9009 injections. After the protocols, the gastrocnemius muscle or the cells were collected for reverse transcription-quantitative polymerase chain reaction (RTq-PCR) and immunoblotting techniques. In summary, the downregulation of REV-ERBα, autophagic flux, and most mitochondrial genes was verified in the IL-6 KO mice independent of exercise. The WT and IL-6 KO treated with SR9009 showed an upregulation of autophagic genes. C2C12 cells receiving IL-6 did not modulate the Nr1d1 mRNA levels but upregulated the expression of some mitochondrial genes. However, when treated with SR9009, IL-6 and mitochondrial gene expression were upregulated in C2C12 cells. The autophagic flux in C2C12 suggest the participation of REV-ERBα protein in the IL-6-induced autophagy. In conclusion, the present study verified that the adaptations required through physical exercise (increases in mitochondrial content and improvement of autophagy machinery) might be intermediated by an interaction between IL-6 and REVERBα.
Assuntos
Interleucina-6 , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares , Animais , Camundongos , Autofagia/genética , Biomarcadores , Produtos do Gene rev , Interleucina-6/genética , Interleucina-6/metabolismo , Músculo Esquelético/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismoRESUMO
BACKGROUND: The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv) has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. RESULTS: Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. CONCLUSION: The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.
Assuntos
Produtos do Gene rev/antagonistas & inibidores , HIV-1/fisiologia , Proteínas do Fator Nuclear 90/fisiologia , RNA Viral/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Produtos do Gene rev/metabolismo , Genes Reporter , HIV-1/metabolismo , Humanos , Proteínas do Fator Nuclear 90/química , Proteínas do Fator Nuclear 90/metabolismo , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Replicação Viral/fisiologia , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
Expression of fusion proteins in the plasma membrane enables cells to bind and fuse with surrounding cells to form syncytia. Cell fusion can have important functional outcomes for the interacting cells, as syncytia formation does in AIDS pathogenesis. Studies on cell fusion would be facilitated by a quantitative method able to discriminate between cellular aggregates and bona fide fused cells in a cell population. Flow cytometry with fluorescence resonance energy transfer is applied here for analyzing fusion of HIV-1 envelope-expressing cells with CD4+ Jurkat cells. Fusion partners were labeled with the vital lipophilic fluorescent probes DiO (green) and DiI (red) and FRET is manifested by an enhancement of the DiI red fluorescence intensity in double fluorescent cells, thus allowing discrimination between fused and aggregated cells. The inhibitory effect of anti-CD4 monoclonal antibodies and the inhibitory peptide T-20 upon cell fusion were readily quantified by this technique. This method allows the distinction of fused and aggregated cells even when they are at low frequencies.
Assuntos
Linfócitos T CD4-Positivos/citologia , Agregação Celular , Fusão Celular , Citometria de Fluxo/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Carbocianinas/análise , Carbocianinas/farmacologia , Técnicas de Cocultura , Corantes Fluorescentes/análise , Produtos do Gene env/biossíntese , Produtos do Gene rev/biossíntese , Inibidores da Fusão de HIV/farmacologia , HIV-1/genética , Humanos , Células Jurkat , Coloração e Rotulagem , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
The findings that BF intersubtype recombinant human immunodeficiency type 1 viruses (HIV-1) with coincident breakpoints in pol are circulating widely in Argentina and that non-recombinant F subtype viruses have failed to be detected in this country were reported recently. To analyse the mosaic structures of these viruses and to determine their phylogenetic relationship, near full-length proviral genomes of eight of these recombinant viruses were amplified by PCR and sequenced. Intersubtype breakpoints were analysed by bootscanning and examining the signature nucleotides. Phylogenetic relationships were determined with neighbour-joining trees. Five viruses, each with predominantly subtype F genomes, exhibited mosaic structures that were highly similar. Two intersubtype breakpoints were shared by all viruses and seven by the majority. Of the consensus breakpoints, all nine were present in two viruses, which exhibited identical recombinant structures, and four to eight breakpoints were present in the remaining viruses. Phylogenetic analysis of partial sequences supported both a common ancestry, at least in part of their genomes, for all recombinant viruses and the phylogenetic relationship of F subtype segments with F subtype viruses from Brazil. A common ancestry of the recombinants was supported also by the presence of shared signature amino acids and nucleotides, either unreported or highly unusual in F and B subtype viruses. These results indicate that HIV-1 BF recombinant viruses with diverse mosaic structures, including a circulating recombinant form (which are widespread in Argentina) derive from a common recombinant ancestor and that F subtype segments of these recombinants are related phylogenetically to the F subtype viruses from Brazil.
Assuntos
Variação Genética , Genoma Viral , Infecções por HIV/virologia , HIV-1/genética , Mosaicismo , Recombinação Genética , Proteínas Virais , Argentina , Sequência de Bases , DNA Viral , Feminino , Produtos do Gene gag/genética , Produtos do Gene gag/fisiologia , Produtos do Gene rev/genética , Produtos do Gene rev/fisiologia , Antígenos HIV/genética , Antígenos HIV/fisiologia , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/fisiologia , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/fisiologia , HIV-1/classificação , Proteínas do Vírus da Imunodeficiência Humana , Humanos , Masculino , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Análise de Sequência de Proteína , Análise de Sequência de RNA , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/fisiologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
Among the major circulating HIV-1 subtypes, subtype C is the most prevalent. To generate full-length subtype C clones and sequences, we selected 13 primary (PBMC-derived) isolates from Zambia, India, Tanzania, South Africa, Brazil, and China, which were identified as subtype C by partial sequence analysis. Near full-length viral genomes were amplified by using a long PCR technique, sequenced in their entirety, and phylogenetically analyzed. Amino acid sequence analysis revealed 10.2, 6.3, and 17.3% diversity in predicted Gag, Pol, and Env protein sequences. Ten of 13 viruses were nonmosaic subtype C genomes, while all three isolates from China represented B/C recombinants. One of them was composed primarily of subtype C sequences with three small subtype B portions in gag, pol, and nef genes. Two others exhibited these same mosaic regions, but contained two additional subtype B portions at the gag/pol overlap and in the accessory gene region, suggesting ongoing B/C recombination in China. All subtype C genomes contained a prematurely truncated second exon of rev, but other previously proposed subtype C signatures, including three potential NF-kappa B-binding sites in the viral promoter-enhancer regions, were found in only a subset of these genomes.
Assuntos
Genoma Viral , Infecções por HIV/virologia , HIV-1/genética , Adulto , Sequência de Bases , Brasil , China , Feminino , Produtos do Gene env , Produtos do Gene gag/genética , Produtos do Gene pol/genética , Produtos do Gene rev , Repetição Terminal Longa de HIV/genética , HIV-1/classificação , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , África do Sul , Tanzânia , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
One of the most intriguing aspects concerning the pathogenesis of AIDS is the long period of latency of the HIV in human cells, not causing any cytopatic effect in some and, on the other hand, causing cell destruction, at short periods, in others. The various agents and the mechanisms they adopt to reactivate the latente HIV, were described. Also the frequent epidemiological observation on the presence of both such agents and the HIV in AIDS patients allowed the authors to speculate on the probable important role of a cohort of co-factors which determine the destiny of such individuals. Special considerations were made in respect to the hepatitis B virus, cytomegalovirus, herpesviruses (HHV-1, e and 6), EB virus, HTLV-1 and 2 retroviruses, group B arbovirus Maguary, malaria and other endemic infectious diseases which victimize millions of Brazilians. Accepting the importance of such co-factors acting on the viral gens that regulate the HIV expression in the host cell, it was speculated on the possible role of vaccines, such as the hepatitis B vaccine, and some antiviral drugs which could be useful in the indirect prevention of AIDS-disease in both HIV-carriers and those practising AIDS-high-risk-activities.