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1.
Blood ; 113(15): 3443-52, 2009 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-19211937

RESUMEN

The viral infectivity factor (Vif) is essential for HIV-1 infectivity and hence is an ideal target for promising anti-HIV-1/AIDS gene therapy. We previously demonstrated that F12-Vif mutant inhibits HIV-1 replication in CD4(+) T lymphocytes. Despite macrophage relevance to HIV-1 pathogenesis, most gene therapy studies do not investigate macrophages because of their natural resistance to genetic manipulation. Here, we confirm the F12-Vif antiviral activity also in macrophages differentiated in vitro from transduced CD34(+) human stem cells (HSCs). Moreover, we identified the 126- to 170-amino-acid region in the C-terminal half of F12-Vif as responsible for its antiviral function. Indeed, Chim3 protein, containing this 45-amino-acid region embedded in a WT-Vif backbone, is as lethal as F12-Vif against HIV-1. Of major relevance, we demonstrated a dual mechanism of action for Chim3. First, Chim3 functions as a transdominant factor that preserves the antiviral function of the natural restriction factor APOBEC3G (hA3G). Second, Chim3 blocks the early HIV-1 retrotranscript accumulation and thereby HIV-1 DNA integration regardless of the presence of WT-Vif and hA3G. In conclusion, by impairing the early steps of HIV-1 life cycle, Chim3 conceivably endows engineered cells with survival advantage, which is required for the efficient immune reconstitution of patients living with HIV/AIDS.


Asunto(s)
Linfocitos T CD4-Positivos/virología , Terapia Genética/métodos , Infecciones por VIH/terapia , VIH-1/crecimiento & desarrollo , Macrófagos/virología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Antígenos CD34/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/fisiología , Diferenciación Celular/inmunología , Línea Celular , Sangre Fetal/citología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/inmunología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Riñón/citología , Macrófagos/citología , Macrófagos/fisiología , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Transducción Genética , Integración Viral , Replicación Viral , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/química
3.
Nucleic Acids Res ; 37(11): 3660-9, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19369217

RESUMEN

The HIV-1 accessory protein Vif plays a dual role: it counteracts the natural restriction factors APOBEC3G and 3F and ensures efficient retrotranscription of the HIV-1 RNA genome. We have previously shown that Vif can act as an auxiliary factor for HIV-1 reverse transcriptase (RT), increasing its rate of association to RNA or DNA templates. Here, by using seven different Vif mutants, we provide in vitro evidences that Vif stimulates HIV-1 RT through direct protein-protein interaction, which is mediated by its C-terminal domain. Physical interaction appears to require the proline-rich region comprised between amino acid (aa) 161 and 164 of Vif, whereas the RT stimulatory activity requires, in addition, the extreme C-terminal region (aa 169-192) of the Vif protein. Neither the RNA interaction domain, nor the Zn(++)-binding domain of Vif are required for its interaction with the viral RT. Pseudotyped HIV-1 lentiviral vectors bearing Vif mutants deleted in the RNA- or RT-binding domains show defects in retrotranscription/integration processes in both permissive and nonpermissive cells. Our results broaden our knowledge on how three important functions of Vif (RNA binding, RT binding and stimulation and Zn(++) binding), are coordinated by different domains.


Asunto(s)
Transcriptasa Inversa del VIH/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/química , Línea Celular , Humanos , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN/metabolismo , Transcripción Reversa , Integración Viral , Dedos de Zinc , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo
4.
Open Virol J ; 1: 26-32, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-19440456

RESUMEN

HIV-1 can be subdivided into distinct subtypes; the consequences of such a genomic variability remain largely speculative. The long terminal repeats (LTR) control HIV transcription and reflect the major differences of distinct viral subtypes. Three regions in the HIV-1 subtype B LTR are close matches to the Signal Transducer and Activator of Transcription (STAT) consensus sequence. Here, we show heterogeneity in these putative STAT binding sites among HIV-1 LTR subtypes A through G. Transfection of constitutively activated STAT5 lead to transcriptional activation of HIV-1 expression in 293T cells transfected with a reporter assay driven by HIV-1 LTR subtype B. Constitutively activated STAT5 transactivated the LTR of various subtypes in U937 cells with different potency. These findings support and expand the potential relevance of STAT5 activation in HIV infection and may bear relevance for a differential regulation of latency and expression of different subtypes of HIV-1.

5.
Blood ; 109(12): 5380-9, 2007 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-17332243

RESUMEN

CD4(+) cells of most individuals infected with HIV-1 harbor a C-terminally truncated and constitutively activated form of signal transducer and activator of transcription-5 (STAT5 Delta). We report that the chronically HIV-infected U1 cell line expresses STAT5 Delta but not full-length STAT5. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of U1 cells promoted early activation of STAT5 Delta and of extracellular signal regulated kinases (ERKs), followed by later activation of activator protein 1 (AP-1) and HIV expression. Inhibition of ERK/AP-1 by PD98,059 abolished, whereas either tyrphostin AG490 or a STAT5 small interfering RNA (siRNA) enhanced, virion production in GM-CSF-stimulated U1 cells. Chromatin immunoprecipitation demonstrated the induction of STAT5 Delta binding to STAT consensus sequences in the HIV-1 promoter together with a decreased recruitment of RNA polymerase II after 1 hour of GM-CSF stimulation of U1 cells. Down-regulation of STAT5 Delta by siRNA resulted in the up-regulation of both HIV-1 gag-pol RNA and p24 Gag antigen expression in CD8-depleted leukocytes of several HIV-positive individuals cultivated ex vivo in the presence of interleukin-2 but not of interleukin-7. Thus, the constitutively activated STAT5 Delta present in the leukocytes of most HIV-positive individuals acts as a negative regulator of HIV expression.


Asunto(s)
VIH-1/genética , Factor de Transcripción STAT5/inmunología , Línea Celular , Células Cultivadas , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Infecciones por VIH , Humanos , Leucocitos/virología , Replicación Viral
6.
Mol Ther ; 12(4): 697-706, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16039909

RESUMEN

The viral infectivity factor (Vif) is an essential component of the HIV-1 infectious cycle. Vif counteracts the action of the cytidine deaminase APOBEC3G (AP3G), which confers nonimmune antiviral defense against HIV-1 to T lymphocytes. Disabling or interfering with the function of Vif could represent an alternative therapeutic approach to AIDS. We have expressed a natural mutant of Vif, F12-Vif, in a VSV-G-pseudotyped lentiviral vector under the Tat-inducible control of the HIV-1 LTR. Conditional expression of F12-Vif prevents replication and spreading of both CXCR4 and CCR5 strains of HIV-1 in human primary T lymphocyte and T cell lines. T cells transduced with F12-Vif release few HIV-1 virions and with reduced infectivity. Several lines of evidence indicate that HIV-1 interference requires the presence of both wild-type and F12-Vif proteins, suggesting a dominant-negative feature of the F12-Vif mutant. Surprisingly, however, the F12-Vif-mediated inhibition does not depend on the reestablishment of the AP3G function.


Asunto(s)
Productos del Gen vif/genética , Terapia Genética , Infecciones por VIH/terapia , VIH-1 , Linfocitos T/virología , Desaminasa APOBEC-3G , Secuencia de Aminoácidos , Línea Celular , Citidina Desaminasa , Productos del Gen vif/biosíntesis , Vectores Genéticos , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Duplicado del Terminal Largo de VIH , VIH-1/genética , VIH-1/metabolismo , Humanos , Lentivirus/genética , Datos de Secuencia Molecular , Nucleósido Desaminasas , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Proteínas Represoras , Linfocitos T/citología , Transducción Genética , Virión/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana
7.
Biochem Biophys Res Commun ; 302(2): 421-6, 2003 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-12604365

RESUMEN

HMGB1 is an architectural factor that enhances the DNA binding affinity of several proteins. We have investigated the influence of HMGB1 on DNA binding by members of the Rel family. HMGB1 enhances DNA binding by p65/p50 and p50/p50, but reduces binding by p65/p65, c-Rel/c-Rel, p65/c-Rel, and p50/c-Rel. In pull-down assays, HMGB1 interacts directly with the p50 subunit via its HMG boxes and this interaction is weakened by the presence of the acidic tail. Functionally, HMGB1 is required for the NF-kappaB-dependent expression of the adhesion molecule VCAM-1.


Asunto(s)
Fibroblastos/metabolismo , Proteína HMGB1/metabolismo , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Animales , Células Cultivadas , Dimerización , Expresión Génica/efectos de los fármacos , Ratones , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Molécula 1 de Adhesión Celular Vascular/genética
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