RESUMEN
OBJECTIVE: We measure the transcript levels of the proapoptotic GALIG, antiapoptotic MCL1 genes and those of the autophagy genes BECN1, MAP1LC3B, ATG9a, P62/SQSTM1, GABARAP, GABARAPL1 and GABARAPL2 to define if mRNA alteration can characterize HIV-infected patients effectively treated with combined antiretroviral therapy (cART). DESIGN: Monocentric pilot study conducted on peripheral blood mononuclear cell (PBMC) of 40 uninfected donors and 27 HIV-positive patients effectively treated by cART for at least 8.4 years. METHODS: Transcripts of the various genes were quantified by reverse transcription (RT)-quantitative PCR (qPCR) and RT-droplet digital PCR and compared using the standard statistical Mann-Whitney U test and machine learning algorithms. RESULTS: A concomitant overexpression of GALIG and MCL1 is detected in PBMC of effectively cART-treated patients. Overexpression of MAP1LC3B and GABARAPL1 is also measured, whereas BECN1 is underexpressed. Finally, accurate classification (94.5%) of our PBMC samples as HIV-negative donors or HIV-positive cART-treated is obtained in three separate machine-learning algorithms with GABARAPL1 and ATG9a as input variables. CONCLUSION: cART-treated HIV patients display altered transcript levels for three genes of basal autophagy. Some of these alterations may appear contradictory: BECN1 and ATG9a, both key actors in the formation of mammalian autophagosome, exhibit decreased amount of transcripts, whereas mRNA from the ATG8 family increase. Given the known role of impaired basal autophagy in immune senescence and chronic inflammation, the functional significance of our findings should be explored in larger studies.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Apoptosis , Autofagia , Expresión Génica , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/patología , Leucocitos Mononucleares/patología , Terapia Antirretroviral Altamente Activa , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
GALIG gene expression induces apoptosis in cultured cells through a pathway still under investigation. It is highly expressed in leukocytes but weakly detectable in bone marrow, suggesting a role in the myeloid lineage homeostasis. We show here that GALIG-induced cell death is counteracted by the overexpression of MCL-1, a pro-survival member of the Bcl2 family. Moreover, during spontaneous neutrophil apoptosis, a substantial increase in GALIG gene expression is observed: GALIG still opposes MCL-1. Finally, in bone marrow and peripheral blood cells from patients with Acute Myeloid Leukemia type 2, the level of GALIG transcripts is massively down-regulated when compared to their normal counterparts, while MCL-1 is expressed to the same extent. These data suggest that GALIG could be a key player in the cell death pathway involved in leukocytes homeostasis and myeloid malignancies.
Asunto(s)
Apoptosis/genética , Proteínas Sanguíneas/genética , Galectinas/genética , Leucemia Mieloide Aguda/genética , Neutrófilos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Sanguíneas/metabolismo , Células de la Médula Ósea/metabolismo , Supervivencia Celular/genética , Galectinas/metabolismo , Regulación Leucémica de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Leucocitos Mononucleares/metabolismo , Microscopía Fluorescente , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Mitogaligin, a protein encoded by galig, an internal cytotoxic gene of the galectin-3 locus, is mostly a mitochondrial protein. Mitochondrial targeting is due to an already identified mitochondrial localization signal. Interaction of mitogaligin with mitochondria leads to cytochrome c cytosolic leakage and ultimately to cell death. We have previously pointed out that mitogaligin can also be directed to the nucleus when the mitochondrial addressing signal is inactivated, indicating a possible dual intracellular localization of the protein. When expressed in the nucleus, mitogaligin exhibits also apoptotic properties leading to cell death. In this report, we show that nuclear addressing of mitogaligin depends on a sequence differing from classical signals containing basic, lysine or proline-tyrosine rich residues. The signal consists of a long sequence of amino acids residues based on a series of a short repetitive degenerated sequence.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Núcleo Celular/metabolismo , Galectinas/metabolismo , Señales de Localización Nuclear/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Galectinas/química , Galectinas/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Señales de Localización Nuclear/química , Señales de Localización Nuclear/genética , Estructura Terciaria de Proteína , Eliminación de SecuenciaRESUMEN
In this study, we demonstrated the efficiency and feasibility of a gene therapy protocol against HIV infection using the antiviral effects of IFN-beta expression. Lentiviral vectors containing the human or the simian IFN-beta sequences under the influence of the murine moderate H2-kb promoter were constructed. To examine the capacity of IFN-beta to inhibit the replication of HIV in human CD4(+) cells, a transduction protocol permitting to efficiently transduce CD4(+) cells or PBMC (85+/-12% of CD4(+)-transduced cells) with a moderate expression of IFN-beta was developed. Results indicate that enforced expression of IFN-beta has no negative effects in terms of apoptosis and proliferation. In human CD4(+) cells, it drastically inhibits (up to 99.9%) replication after challenging with different strains of HIV-1. The expression of exogenous IFN-beta leads to an amplification of the CD4(+) cells (11-fold) and to a drastic decrease of the p24 protein. Micro-array analyses indicated that antiviral effect of IFN-beta could be due to a major regulation of the inflammatory response. These results are encouraging for the development of a clinical study of gene therapy against AIDS using IFN-beta.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Terapia Genética/métodos , Infecciones por VIH/terapia , VIH-1/efectos de los fármacos , Interferón beta/administración & dosificación , Replicación Viral/efectos de los fármacos , Animales , Antivirales/administración & dosificación , Antivirales/farmacología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/trasplante , Proliferación Celular/efectos de los fármacos , Vectores Genéticos , Haplorrinos , Humanos , Inflamación/tratamiento farmacológico , Interferón beta/farmacología , Ratones , Transducción GenéticaRESUMEN
Dimerization of genomic RNA is directly related with the event of encapsidation and maturation of the virion. The initiating sequence of the dimerization is a short autocomplementary region in the hairpin loop SL1. We describe here a new solution structure of the RNA dimerization initiation site (DIS) of HIV-1(Lai). NMR pulsed field-gradient spin-echo techniques and multidimensional heteronuclear NMR spectroscopy indicate that this structure is formed by two hairpins linked by six Watson-Crick GC base pairs. Hinges between the stems and the loops are stabilized by intra and intermolecular interactions involving the A8, A9 and A16 adenines. The coaxial alignment of the three A-type helices present in the structure is supported by previous crystallography analysis but the A8 and A9 adenines are found in a bulged in position. These data suggest the existence of an equilibrium between bulged in and bulged out conformations in solution.
Asunto(s)
VIH-1/genética , Modelos Moleculares , ARN Viral/química , Secuencia de Bases , Dimerización , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido NucleicoRESUMEN
Reverse transcription of HIV-1 RNA is initiated from the 3' end of a tRNA3Lys molecule annealed to the primer binding site (PBS). An additional interaction between the anticodon loop of tRNA3Lys and a viral A-rich loop is required for efficient initiation of reverse transcription of the HIV-1 MAL isolate. In the HIV-1 HXB2 isolate, simultaneous mutations of the PBS and the A-rich loop (mutant His-AC), but not of the PBS alone (mutant His) allows the virus to stably utilize tRNA(His) as primer. However, mutant His-AC selects additional mutations during cell culture, generating successively His-AC-GAC and His-AC-AT-GAC. Here, we wanted to establish direct relationships between the evolution of these mutants in cell culture, their efficiency in initiating reverse transcription and the structure of the primer/template complexes in vitro. The initiation of reverse transcription of His and His-AC RNAs was dramatically reduced. However, His-AC-GAC RNA, which incorporated three adaptative point mutations, was reverse transcribed more efficiently than the wild type RNA. Incorporation of two additional mutations decreased the efficiency of the initiation of reverse transcription, which remained at the wild type level. Structural probing showed that even though both His-AC and His-AC-GAC RNAs can potentially interact with the anticodon loop of tRNA(His), only the latter template formed a stable interaction. Thus, our results showed that the selection of adaptative mutations by HIV-1 mutants utilizing tRNA(His) as primer was initially dictated by the efficiency of the initiation of reverse transcription, which relied on the existence of a stable interaction between the mutated A-rich loop and the anticodon loop of tRNA(His).
Asunto(s)
Regulación Viral de la Expresión Génica , VIH-1/genética , ARN de Transferencia de Histidina/metabolismo , ARN Viral/biosíntesis , Sitio de Iniciación de la Transcripción , Transcripción Genética , Secuencia de Bases , ADN Viral/biosíntesis , Transcriptasa Inversa del VIH/metabolismo , VIH-1/metabolismo , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutación , Sondas ARN , Procesamiento Postranscripcional del ARN , ARN Viral/genética , Alineación de Secuencia , Relación Estructura-Actividad , Moldes GenéticosRESUMEN
The conversion of the single-stranded RNA genome into double-stranded DNA by virus-coded reverse transcriptase (RT) is an essential step of the retrovirus life cycle. In human immunodeficiency virus type 1 (HIV-1), RT uses the cellular tRNA(Lys)3 to initiate the (-) strand DNA synthesis. Placement of the primer tRNA(Lys)3 involves binding of its 3'-terminal 18 nt to a complementary region of genomic RNA termed PBS. However, the PBS sequence is not the unique determinant of primer usage and additional contacts are important. This placement is believed to be achieved in vivo by the nucleocapsid domain of Gag or by the mature protein NCp. Up to now, structural information essentially arose from heat-annealed primer-template complexes (Isel et al., J Mol Biol, 1995, 247:236-250; Isel et al., EMBO J, 1999, 18:1038-1048). Here, we investigated the formation of the primer-template complex mediated by NCp and compared structural and functional properties of heat- and NCp-annealed complexes. We showed that both heat- and NCp-mediated procedures allow comparable high yields of annealing. Then, we investigated structural features of both kinds of complexes by enzymatic probing, and we compared their relative efficiency in (-) strong stop DNA synthesis. We did not find any significant differences between these complexes, suggesting that information derived from the heat-annealed complex can be transposed to the NCp-mediated complex and most likely to complexes formed in vivo.