RESUMEN
HIV-1 Tat is a key viral protein that stimulates several steps of viral gene expression. Tat is especially required for the transcription of viral genes. Nevertheless, it is still not clear if and how Tat is incorporated into HIV-1 virions. Cyclophilin A (CypA) is a prolyl isomerase that binds to HIV-1 capsid protein (CA) and is thereby encapsidated at the level of 200 to 250 copies of CypA/virion. Here, we found that a Tat-CypA-CA tripartite complex assembles in HIV-1-infected cells and allows Tat encapsidation into HIV virions (1 Tat/1 CypA). Biochemical and biophysical studies showed that high-affinity interactions drive the assembly of the Tat-CypA-CA complex that could be purified by size exclusion chromatography. We prepared different types of viruses devoid of transcriptionally active Tat. They showed a 5- to 10 fold decrease in HIV infectivity, and conversely, encapsidating Tat into ΔTat viruses greatly enhanced infectivity. The absence of encapsidated Tat decreased the efficiency of reverse transcription by ~50% and transcription by more than 90%. We thus identified a Tat-CypA-CA complex that enables Tat encapsidation and showed that encapsidated Tat is required to initiate robust viral transcription and thus viral production at the beginning of cell infection, before neosynthesized Tat becomes available. IMPORTANCE The viral transactivating protein Tat has been shown to stimulate several steps of HIV gene expression. It was found to facilitate reverse transcription. Moreover, Tat is strictly required for the transcription of viral genes. Although the presence of Tat within HIV virions would undoubtedly favor these steps and therefore enable the incoming virus to boost initial viral production, whether and how Tat is present within virions has been a matter a debate. We here described and characterized a tripartite complex between Tat, HIV capsid protein, and the cellular chaperone cyclophilin A that enables efficient and specific Tat encapsidation within HIV virions. We further showed that Tat encapsidation is required for the virus to efficiently initiate infection and viral production. This effect is mainly due to the transcriptional activity of Tat.
Asunto(s)
Proteínas de la Cápside , Ciclofilina A , Infecciones por VIH , VIH-1 , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Humanos , Proteínas de la Cápside/metabolismo , Ciclofilina A/metabolismo , Infecciones por VIH/virología , VIH-1/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Complejos Multiproteicos/metabolismo , Resonancia por Plasmón de Superficie , Citosol/metabolismo , Línea CelularRESUMEN
Human T cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and inflammatory diseases. The HTLV-1 bZIP factor (HBZ) gene is constantly expressed in HTLV-1 infected cells and ATL cells. HBZ protein suppresses transcription of the tax gene through blocking the LTR recruitment of not only ATF/CREB factors but also CBP/p300. HBZ promotes transcription of Foxp3, CCR4, and T-cell immunoreceptor with Ig and ITIM domains (TIGIT). Thus, HBZ is critical for the immunophenotype of infected cells and ATL cells. HBZ also functions in its RNA form. HBZ RNA suppresses apoptosis and promotes proliferation of T cells. Since HBZ RNA is not recognized by cytotoxic T cells, HTLV-1 has a clever strategy for avoiding immune detection. HBZ plays central roles in maintaining infected T cells in vivo and determining their immunophenotype.
Asunto(s)
Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Leucemia-Linfoma de Células T del Adulto/virología , Proteínas Nucleares/genética , Proteínas de Unión al ARN/genética , Factores de Transcripción/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proliferación Celular , Regulación Viral de la Expresión Génica , Genes pX/genética , Infecciones por HTLV-I/fisiopatología , Infecciones por HTLV-I/virología , Humanos , Proteínas de los Retroviridae/genética , Linfocitos T/inmunologíaRESUMEN
Recent experiments provide sound arguments in favor of the in vivo expression of the AntiSense Protein (ASP) of HIV-1. This putative protein is encoded on the antisense strand of the provirus genome and entirely overlapped by the env gene with reading frame -2. The existence of ASP was suggested in 1988, but is still controversial, and its function has yet to be determined. We used a large dataset of â¼23,000 HIV-1 and SIV sequences to study the origin, evolution, and conservation of the asp gene. We found that the ASP ORF is specific to group M of HIV-1, which is responsible for the human pandemic. Moreover, the correlation between the presence of asp and the prevalence of HIV-1 groups and M subtypes appeared to be statistically significant. We then looked for evidence of selection pressure acting on asp Using computer simulations, we showed that the conservation of the ASP ORF in the group M could not be due to chance. Standard methods were ineffective in disentangling the two selection pressures imposed by both the Env and ASP proteins-an expected outcome with overlaps in frame -2. We thus developed a method based on careful evolutionary analysis of the presence/absence of stop codons, revealing that ASP does impose significant selection pressure. All of these results support the idea that asp is the 10th gene of HIV-1 group M and indicate a correlation with the spread of the pandemic.
Asunto(s)
Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/genética , Pandemias , Proteínas Virales/genética , Secuencia de Bases , Secuencia Conservada/genética , Evolución Molecular , Genoma Viral , Infecciones por VIH/genética , Filogenia , Sistemas de Lectura/genética , Selección GenéticaRESUMEN
There are four human T-lymphotropic viruses (HTLV-1, 2, 3, 4) that have emerged from the transmission of simian viruses. HTLV-1 was the first retrovirus to be shown to be responsible for a human pathology. The expression of retroviral genes depends mostly on their 5'LTR, but it was revealed that HTLV have a promoter in their 3'LTR, capable of transcription from the antisense strand of their genome. These transcripts can be translated into proteins named HBZ, APH-2, APH-3 and APH-4. Antisense transcription in HTLV-1 and its encoded protein HBZ have been thoroughly studied and it has been suggested that HBZ plays an important role in viral replication and the development of ATL. Very few studies have been conducted on antisense transcription from the three other viruses, although it is likely that these genes are also implicated in viral replication.
RESUMEN
BACKGROUND: CD8+ T cells recognize HIV-1 epitopes translated from a gene's primary reading frame (F1) and any one of its five alternative reading frames (ARFs) in the forward (F2, F3) or reverse (R1-3) directions. The 3' end of HIV-1's proviral coding strand contains a conserved sequence that is directly overlapping but antiparallel to the env gene (ARF R2) and encodes for a putative antisense HIV-1 protein called ASP. ASP expression has been demonstrated in vitro using HIV-transfected cell lines or infected cells. Although antibodies to ASP were previously detected in patient sera, T cell recognition of ASP-derived epitopes has not been evaluated. We therefore investigated the ex vivo and in vitro induction of ASP-specific T cell responses as a measure of immune recognition and protein expression during HIV-1 infection. RESULTS: A panel of overlapping peptides was initially designed from the full-length ASP sequence to perform a global assessment of T cell responses. Recognition of ASP-derived antigens was evaluated in an IFN-γELISpot assay using PBMCs from HIV-1 seropositive and seronegative individuals. Eight of 25 patients had positive responses to ASP antigens and none of the seronegative donors responded. As a complimentary approach, a second set of antigens was designed using HLA-I binding motifs and affinities. Two ASP-derived peptides with high predicted binding affinities for HLA-A*02 (ASP-YL9) and HLA-B*07 (ASP-TL10) were tested using PBMCs from HIV-1 seropositive and seronegative individuals who expressed the matching HLA-I-restricting allele. We found that HLA-I-restricted ASP peptides were only recognized by CD8+ T cells from patients with the relevant HLA-I and did not induce responses in any of the seronegative donors or patients who do not express the restrictive HLA alleles. Further, ASP-YL9-specific CD8+ T cells had functional profiles that were similar to a previously described HLA-A*02-restricted epitope (Gag-SL9). Specific recognition of ASP-YL9 by CD8+ T cells was also demonstrated by tetramer staining using cells from an HLA-A*02 HIV-infected patient. CONCLUSION: Our results provide the first description of CD8+ T cell-mediated immune responses to ASP in HIV-1-infected patients, demonstrating that ASP is expressed during infection. Our identification of epitopes within ASP has implications for designing HIV vaccines.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Expresión Génica , Antígenos VIH/inmunología , VIH-1/inmunología , VIH-1/fisiología , Proteínas Virales/inmunología , Replicación Viral , Adulto , Anciano , Células Cultivadas , Estudios de Cohortes , Ensayo de Immunospot Ligado a Enzimas , Femenino , Antígenos VIH/biosíntesis , Humanos , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Proteínas Virales/biosíntesisRESUMEN
UNLABELLED: Brain-derived neurotrophic factor (BDNF) is a neurotrophin that promotes neuronal proliferation, survival, and plasticity. These effects occur through autocrine and paracrine signaling events initiated by interactions between secreted BDNF and its high-affinity receptor, TrkB. A BDNF/TrkB autocrine/paracrine signaling loop has additionally been implicated in augmenting the survival of cells representing several human cancers and is associated with poor patient prognosis. Adult T-cell leukemia (ATL) is a fatal malignancy caused by infection with the complex retrovirus human T-cell leukemia virus type 1 (HTLV-1). In this study, we found that the HTLV-1-encoded protein HBZ activates expression of BDNF, and consistent with this effect, BDNF expression is elevated in HTLV-1-infected T-cell lines compared to uninfected T cells. Expression of TrkB is also higher in HTLV-1-infected T-cell lines than in uninfected T cells. Furthermore, levels of both BDNF and TrkB mRNAs are elevated in peripheral blood mononuclear cells (PBMCs) from ATL patients, and ATL patient sera contain higher concentrations of BDNF than sera from noninfected individuals. Finally, chemical inhibition of TrkB signaling increases apoptosis in HTLV-1-infected T cells and reduces phosphorylation of glycogen synthase kinase 3ß (GSK-3ß), a downstream target in the signaling pathway. These results suggest that HBZ contributes to an active BDNF/TrkB autocrine/paracrine signaling loop in HTLV-1-infected T cells that enhances the survival of these cells. IMPORTANCE: Infection with human T-cell leukemia virus type 1 (HTLV-1) can cause a rare form of leukemia designated adult T-cell leukemia (ATL). Because ATL patients are unresponsive to chemotherapy, this malignancy is fatal. As a retrovirus, HTLV-1 integrates its genome into a host cell chromosome in order to utilize host factors for replication and expression of viral proteins. However, in infected cells from ATL patients, the viral genome is frequently modified to block expression of all but a single viral protein. This protein, known as HBZ, is therefore believed to modulate cellular pathways necessary for the leukemic state and the chemotherapeutic resistance of the cell. Here we provide evidence to support this hypothesis. We found that HBZ promotes a BDNF/TrkB autocrine/paracrine signaling pathway that is known to enhance the survival and chemotherapeutic resistance of other types of cancer cells. It is possible that inhibition of this pathway may improve treatments for ATL.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Transformación Celular Viral , Interacciones Huésped-Patógeno , Virus Linfotrópico T Tipo 1 Humano/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Linfocitos T/virología , Proteínas Virales/metabolismo , Supervivencia Celular , Humanos , Receptor trkB , Proteínas de los Retroviridae , Linfocitos T/fisiologíaRESUMEN
Human T-cell leukemia virus types 3 and 4 (HTLV-3 and HTLV-4) are recently isolated retroviruses. We have previously characterized HTLV-3- and HTLV-4-encoded antisense genes, termed APH-3 and APH-4, respectively, which, in contrast to HBZ, the HTLV-1 homologue, do not contain a typical bZIP domain (M. Larocque É Halin, S. Landry, S. J. Marriott, W. M. Switzer, and B. Barbeau, J. Virol. 85:12673-12685, 2011, doi:10.1128/JVI.05296-11). As HBZ differentially modulates the transactivation potential of various Jun family members, the effect of APH-3 and APH-4 on JunD-, c-Jun-, and JunB-mediated transcriptional activation was investigated. We first showed that APH-3 and APH-4 upregulated the transactivation potential of all tested Jun family members. Using an human telomerase catalytic subunit (hTERT) promoter construct, our results also highlighted that, unlike HBZ, which solely modulates hTERT expression via JunD, both APH-3 and APH-4 acted positively on the transactivation of the hTERT promoter mediated by tested Jun factors. Coimmunoprecipitation experiments demonstrated that these Jun proteins interacted with APH-3 and APH-4. Although no activation domain was identified for APH proteins, the activation domain of c-Jun was very important in the observed upregulation of its activation potential. We further showed that APH-3 and APH-4 required their putative bZIP-like domains and corresponding leucine residues for interaction and modulation of the transactivation potential of Jun factors. Our results demonstrate that HTLV-encoded antisense proteins behave differently, and that the bZIP-like domains of both APH-3 and APH-4 have retained their interaction potential for Jun members. These studies are important in assessing the differences between HBZ and other antisense proteins, which might further contribute to determining the role of HBZ in HTLV-1-associated diseases. IMPORTANCE HBZ, the antisense transcript-encoded protein from HTLV-1, is now well recognized as a potential factor for adult T-cell leukemia/lymphoma development. In order to better appreciate the mechanism of action of HBZ, comparison to antisense proteins from other HTLV viruses is important. Little is known in relation to the seemingly nonpathogenic HTLV-3 and HTLV-4 viruses, and studies of their antisense proteins are limited to our previously reported study (M. Larocque É Halin, S. Landry, S. J. Marriott, W. M. Switzer, and B. Barbeau, J. Virol. 85:12673-12685, 2011, doi:10.1128/JVI.05296-11). Here, we demonstrate that Jun transcription factors are differently affected by APH-3 and APH-4 compared to HBZ. These intriguing findings suggest that these proteins act differently on viral replication but also on cellular gene expression, and that highlighting their differences of action might lead to important information allowing us to understand the link between HTLV-1 HBZ and ATL in infected individuals.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , ADN sin Sentido/genética , Virus Linfotrópico T Tipo 3 Humano/genética , Virus Linfotrópico T Tipo 3 Humano/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Activación Transcripcional/genética , Animales , Células COS , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Deltaretrovirus/genética , Deltaretrovirus/metabolismo , Células HEK293 , Células HeLa , Humanos , Regiones Promotoras Genéticas/genética , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-jun/genética , Telomerasa/genética , Telomerasa/metabolismo , Transcripción Genética/genética , Regulación hacia Arriba/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
HIV-1 proteins are synthesized from a single transcript in an unspliced form or following splicing, but the existence of an antisense protein (ASP) expressed from an antisense polyadenylated transcript has been suggested. Difficulties linked to the detection of this protein in mammalian cells led us to codon optimize its cDNA. Codon-optimized ASP was indeed efficiently detected in various transfected cell lines following flow cytometry and confocal microscopy analyses. Western blot analyses also led to the detection of optimized ASP in transfected cells but also provided evidence of its instability and high multimerization potential. ASP was mainly distributed in the cytoplasm in a punctate manner, which was reminiscent of autophagosomes. In agreement with this observation, a significant increase in ASP-positive cells and loss of its punctate distribution was observed in transfected cells when autophagy was inhibited at early steps. Induction of autophagy was confirmed by Western blot analyses that showed an ASP-mediated increase in levels of LC3b-II and Beclin 1, as well as colocalization and interaction between ASP and LC3. Interestingly, Myc-tagged ASP was detected in the context of proviral DNA following autophagy inhibition with a concomitant increase in the level and punctate distribution of LC3b-II. Finally, 3-methyladenine treatment of transfected or infected U937 cells decreased extracellular p24 levels in wild-type proviral DNA and to a much lesser extent in ASP-mutated proviral DNA. This study provides the first detection of ASP in mammalian cells by Western blotting. ASP-induced autophagy might explain the inherent difficulty in detecting this viral protein and might justify its presumed low abundance in infected cells.
Asunto(s)
Autofagia , Infecciones por VIH/fisiopatología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/metabolismo , ARN Viral/genética , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , VIH-1/aislamiento & purificación , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , ARN Viral/metabolismo , Alineación de Secuencia , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
To study the transcriptional activity of the HIV-1 LTR, we constructed a vector containing Renilla and Firefly luciferase genes under the control of the LTR (wild-type or mutated version) and oriented in a manner that allowed them to be transcribed in opposite directions. We found that the HIV-1 LTR acted as a bidirectional promoter, which activity was controlled by NF-κB- and Sp1-binding sites in both orientations. We next analyzed with this reporter vector the bidirectional promoter activity of the HTLV-1 LTR and showed that this LTR also possessed a bidirectional transcriptional activity. Interestingly, Sp1-binding elements were also involved in the control of HTLV-1 bidirectional transcription. Moreover, both retroviral trans-activators, Tat and Tax, could preferentially activate sense transcription with no or limited effect on the extent of antisense transcription. We also cloned into this plasmid the MLV LTR and found that the LTR of a simple retrovirus also possessed bidirectional transcriptional activity. This reporter vector represents a powerful tool to analyze the bidirectional transcriptional activity of retrovirus LTRs.
Asunto(s)
Genes Reporteros , Vectores Genéticos , VIH/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Secuencias Repetidas Terminales/genética , Activación Transcripcional/genética , Animales , Clonación Molecular , Productos del Gen tat/genética , Productos del Gen tax/genética , Humanos , Ratones , FN-kappa B/metabolismo , Células 3T3 NIH , Regiones Promotoras Genéticas , TransfecciónRESUMEN
The homologous cellular coactivators p300 and CBP contain intrinsic lysine acetyl transferase (termed HAT) activity. This activity is responsible for acetylation of several sites on the histones as well as modification of transcription factors. In a previous study, we found that HBZ, encoded by the Human T-cell Leukemia Virus type 1 (HTLV-1), binds to multiple domains of p300/CBP, including the HAT domain. In this study, we found that HBZ inhibits the HAT activity of p300/CBP through the bZIP domain of the viral protein. This effect correlated with a reduction of H3K18 acetylation, a specific target of p300/CBP, in cells expressing HBZ. Interestingly, lower levels of H3K18 acetylation were detected in HTLV-1 infected cells compared to non-infected cells. The inhibitory effect of HBZ was not limited to histones, as HBZ also inhibited acetylation of the NF-κB subunit, p65, and the tumor suppressor, p53. Recent studies reported that mutations in the HAT domain of p300/CBP that cause a defect in acetylation are found in certain types of leukemia. These observations suggest that inhibition of the HAT activity by HBZ is important for the development of adult T-cell leukemia associated with HTLV-1 infection.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas Virales/metabolismo , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Acetilación , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Línea Celular , Curcumina/farmacología , Inhibidores Enzimáticos/farmacología , Células HeLa , Histonas/metabolismo , Humanos , Dominios y Motivos de Interacción de Proteínas , Proteínas de los Retroviridae , Linfocitos T/metabolismo , Linfocitos T/virología , Factores de Transcripción/metabolismo , Proteínas Virales/química , Factores de Transcripción p300-CBP/química , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Telomerase activity in cancer cells is dependent on the transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene, encoding the catalytic subunit of human telomerase. We have shown previously that HTLV-1 basic leucine zipper (HBZ), a viral regulatory protein encoded by the human retrovirus, human T-cell leukemia virus, type 1 (HTLV-1) cooperates with JunD to enhance hTERT transcription in adult T-cell leukemia (ATL) cells. Menin, the product of the tumor-suppressor MEN-1 gene, also interacts with JunD, represses its transcriptional activity and downregulates telomerase expression. The main objective of this study was to examine how menin and HBZ get involved in the regulation of hTERT transcription. In this study, we report that JunD and menin form a repressor complex of hTERT transcription in HBZ-negative cells. Conversely, in HBZ-positive cells, the formation of a JunD/HBZ/menin ternary complex and the recruitment of p300 histone acetyl transferase activity by HBZ lead to a decreased activity of the JunD-menin suppressor unit that correlates with the activation of hTERT transcription. Silencing HBZ or menin expression in ATL cells confirms that these proteins are differentially involved in telomerase regulation. These results propose that HBZ, by impeding the tumor-suppressor activity of menin, functions as a leukemogenic cofactor to upregulate gene transcription and promote JunD-mediated leukemogenesis.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Regulación Neoplásica de la Expresión Génica , Leucemia de Células T/patología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Telomerasa/genética , Proteínas Virales/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/antagonistas & inhibidores , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Western Blotting , Proliferación Celular , Inmunoprecipitación de Cromatina , Proteína p300 Asociada a E1A/genética , Células HeLa , Humanos , Inmunoprecipitación , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-jun/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas de los Retroviridae , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa/metabolismo , Transcripción Genética , Células Tumorales Cultivadas , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/genéticaRESUMEN
Human T lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). ATLL is a severe malignancy with no effective treatment. HTLV-1 regulatory proteins Tax and HTLV-1 basic leucine zipper factor (HBZ) play a major role in ATLL development, by interfering with cellular functions such as CD4(+) T-cell survival. In this study, we observed that the expression of Bfl-1, an antiapoptotic protein of the Bcl-2 family, is restricted to HTLV-1-infected T-cell lines and to T-cells expressing both Tax and HBZ proteins. We showed that Tax-induced bfl-1 transcription through the canonical NF-κB pathway. Moreover, we demonstrated that Tax cooperated with c-Jun or JunD, but not JunB, transcription factors of the AP-1 family to stimulate bfl-1 gene activation. By contrast, HBZ inhibited c-Jun-induced bfl-1 gene activation, whereas it increased JunD-induced bfl-1 gene activation. We identified one NF-κB, targeted by RelA, c-Rel, RelB, p105/p50, and p100/p52, and two AP-1, targeted by both c-Jun and JunD, binding sites in the bfl-1 promoter of T-cells expressing both Tax and HBZ. Analyzing the potential role of antiapoptotic Bcl-2 proteins in HTLV-1-infected T-cell survival, we demonstrated that these cells are differentially sensitive to silencing of Bfl-1, Bcl-x(L), and Bcl-2. Indeed, both Bfl-1 and Bcl-x(L) knockdowns decreased the survival of HTLV-1-infected T-cell lines, although no cell death was observed after Bcl-2 knockdown. Furthermore, we demonstrated that Bfl-1 knockdown sensitizes HTLV-1-infected T-cells to ABT-737 or etoposide treatment. Our results directly implicate Bfl-1 and Bcl-x(L) in HTLV-1-infected T-cell survival and suggest that both Bfl-1 and Bcl-x(L) represent potential therapeutic targets for ATLL treatment.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Leucemia-Linfoma de Células T del Adulto/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Adulto , Antineoplásicos Fitogénicos/farmacología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Compuestos de Bifenilo/farmacología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Supervivencia Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Etopósido/farmacología , Femenino , Técnicas de Silenciamiento del Gen , Productos del Gen tax/genética , Genes jun/genética , Células HeLa , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucemia-Linfoma de Células T del Adulto/dietoterapia , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/patología , Ligasas/genética , Ligasas/metabolismo , Masculino , Antígenos de Histocompatibilidad Menor , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Nitrofenoles/farmacología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas c-rel , Proteínas de los Retroviridae , Sulfonamidas/farmacología , Factor de Transcripción ReIB/genética , Factor de Transcripción ReIB/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
In this study, an antisense luciferase-expressing human immunodeficiency virus type 1 (HIV-1) molecular clone was used to infect primary cells. We found that antisense transcription activity from the 3' long terminal repeat (LTR) was significantly more abundant in monocyte-derived cells than in activated T lymphocytes. Moreover, by analyzing antisense transcription in infected monocyte-derived dendritic cells (MDDCs), we observed that the majority of HIV-1-infected MDDCs with significant antisense transcription activity did not produce Gag. We also confirmed that the negative-strand-encoded antisense protein (ASP) was expressed in monocyte-derived cells.
Asunto(s)
VIH-1/genética , Monocitos/virología , ARN sin Sentido/genética , Transcripción Genética , Células Cultivadas , Genes Virales , Duplicado del Terminal Largo de VIH , HumanosRESUMEN
Infection with the human T-cell leukemia virus type 1 (HTLV-1) results in a variety of diseases including adult T-cell leukemia (ATL), a fatal malignancy characterized by the uncontrolled proliferation of virally infected CD4(+) T cells. The HTLV-1 basic leucine zipper factor (HBZ) is believed to contribute to development and maintenance of ATL. Unlike the other HTLV-1 genes, the hbz gene is encoded on the complementary strand of the provirus and therefore is not under direct control of the promoter within the 5' long terminal repeat (LTR) of the provirus. This promoter can undergo inactivating genetic or epigenetic changes during the course of ATL that eliminates expression of all viral genes except that of hbz. In contrast, repressive modifications are not known to occur on the hbz promoter located in the 3' LTR, and hbz expression has been consistently detected in all ATL patient samples. Although Sp1 regulates basal transcription from the HBZ promoter, other factors that activate transcription remain undefined. In this study, we used a proviral reporter construct deleted of the 5' LTR to show that HBZ upregulates its own expression through cooperation with JunD. Activation of antisense transcription was apparent in serum-deprived cells in which the level of JunD was elevated, and elimination of JunD expression by gene knockout or shRNA-mediated knockdown abrogated this effect. Activation through HBZ and JunD additionally required Sp1 binding at the hbz promoter. These data favor a model in which JunD is recruited to the promoter through Sp1, where it heterodimerizes with HBZ thereby enhancing its activity. Separately, hbz gene expression led to an increase in JunD abundance, and this effect correlated with emergence of features of transformed cells in immortalized fibroblasts. Overall, our results suggest that JunD represents a novel therapeutic target for the treatment of ATL.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Regulación Viral de la Expresión Génica , Virus Linfotrópico T Tipo 1 Humano/genética , Leucemia-Linfoma de Células T del Adulto/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN sin Sentido/genética , Secuencias Repetidas Terminales , Proteínas Virales/metabolismo , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Línea Celular , Virus Linfotrópico T Tipo 1 Humano/fisiología , Humanos , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/virología , Ratones , Unión Proteica , Proteínas Proto-Oncogénicas c-jun/genética , ARN sin Sentido/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Regulación hacia Arriba , Proteínas Virales/genéticaRESUMEN
A determinant of human T-lymphotropic virus-1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) development is the HTLV-1-infected cell burden. Viral proteins Tax and HBZ, encoded by the sense and antisense strands of the pX region, respectively, play key roles in HTLV-1 persistence. Tax drives CD4(+)-T cell clonal expansion and is the immunodominant viral antigen recognized by the immune response. Valproate (2-n-propylpentanoic acid, VPA), a histone deacetylase inhibitor, was thought to trigger Tax expression, thereby exposing the latent HTLV-1 reservoir to immune destruction. We evaluated the impact of VPA on Tax, Gag, and HBZ expressions in cultured lymphocytes from HTLV-1 asymptomatic carriers and HAM/TSP patients. Approximately one-fifth of provirus-positive CD4(+) T cells spontaneously became Tax-positive, but this fraction rose to two-thirds of Tax-positive-infected cells when cultured with VPA. Valproate enhanced Gag-p19 release. Tax- and Gag-mRNA levels peaked spontaneously, before declining concomitantly to HBZ-mRNA increase. VPA enhanced and prolonged Tax-mRNA expression, whereas it blocked HBZ expression. Our findings suggest that, in addition to modulating Tax expression, another mechanism involving HBZ repression might determine the outcome of VPA treatment on HTLV-1-infected-cell proliferation and survival.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/biosíntesis , Regulación Viral de la Expresión Génica/efectos de los fármacos , Productos del Gen tax/biosíntesis , Virus Linfotrópico T Tipo 1 Humano/fisiología , Ácido Valproico/farmacología , Proteínas Virales/biosíntesis , Elementos sin Sentido (Genética)/efectos de los fármacos , Apoptosis/efectos de los fármacos , Enfermedades Asintomáticas , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Células Cultivadas/efectos de los fármacos , Células Cultivadas/virología , Genes gag , Genes pX , Histona Acetiltransferasas/antagonistas & inhibidores , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/virología , Paraparesia Espástica Tropical , Provirus/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Viral/biosíntesis , ARN Viral/genética , Proteínas de los Retroviridae , Proteínas Virales/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/biosíntesisRESUMEN
Eukaryotic cells employ a broad range of mechanisms to regulate gene expression. Among others, mRNA alternative splicing is a key process. It consists of introns removal from an immature mRNA (pre-mRNA) via a transesterification reaction to create a mature mRNA molecule. Large-scale genomic studies have shown that in the human genome, almost 95% of protein-encoding genes go through alternative splicing and produce transcripts with different exons combinations (and sometimes retained introns), thus increasing the proteome diversity. Considering the importance of RNA regulation in cellular proliferation, survival, and differentiation, alterations in the alternative splicing pathway have been linked to several human cancers, including adult T-cell leukemia/lymphoma (ATL). ATL is an aggressive and fatal malignancy caused by the Human T-cell leukemia virus type 1 (HTLV-1). HTLV-1 genome encodes for two oncoproteins: Tax and HBZ, both playing significant roles in the transformation of infected cells and ATL onset. Here, we review current knowledge on alternative splicing and its link to cancers and reflect on how dysregulation of this pathway could participate in HTLV-1-induced cellular transformation and adult T-cell leukemia/lymphoma development.
Asunto(s)
Virus Linfotrópico T Tipo 1 Humano , Leucemia-Linfoma de Células T del Adulto , Linfoma , Adulto , Empalme Alternativo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Transformación Celular Neoplásica , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucemia-Linfoma de Células T del Adulto/genética , ARN Mensajero/metabolismoRESUMEN
Human T-cell leukemia virus type 1 is the causative agent of HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T-cell leukemia-lymphoma (ATL). The HTLV-1 basic leucine zipper factor (HBZ) has been associated to the cancer-inducing properties of this virus, although the exact mechanism is unknown. In this study, we identified nucleophosmin (NPM1/B23) as a new interaction partner of HBZ. We show that sHBZ and the less abundant uHBZ isoform interact with nucleolar NPM1/B23 in infected cells and HTLV-1 positive patient cells, unlike equivalent antisense proteins of related non-leukemogenic HTLV-2, -3 and-4 viruses. We further demonstrate that sHBZ association to NPM1/B23 is sensitive to RNase. Interestingly, sHBZ was shown to interact with its own RNA. Through siRNA and overexpression experiments, we further provide evidence that NPM1/B23 acts negatively on viral gene expression with potential impact on cell transformation. Our results hence provide a new insight over HBZ-binding partners in relation to cellular localization and potential function on cell proliferation and should lead to a better understanding of the link between HBZ and ATL development.
RESUMEN
BACKGROUND: Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. RESULTS: We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. CONCLUSION: These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP.
Asunto(s)
Membrana Celular/virología , Regulación Viral de la Expresión Génica , Infecciones por VIH/virología , VIH-1/genética , ARN sin Sentido/genética , ARN Viral/genética , Linfocitos T/virología , Proteínas Virales/genética , Línea Celular , Membrana Celular/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Humanos , Mutación , Transporte de Proteínas , ARN sin Sentido/metabolismo , ARN Viral/metabolismo , Linfocitos T/metabolismo , Proteínas Virales/metabolismoRESUMEN
Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) retroviruses infect T lymphocytes. The minus strand of the HTLV-1 genome encodes HBZ, a protein that could play a role in the development of leukemia in infected patients. Herein, we demonstrate that the complementary strand of the HTLV-2 genome also encodes a protein that we named APH-2 for "antisense protein of HTLV-2." APH-2 mRNA is spliced, polyadenylated, and initiates in the 3'-long terminal repeat at different positions. This transcript was detected in all HTLV-2-infected cell lines and short-term culture of lymphocytes obtained from HTLV-2 African patients tested and in 4 of 15 HTLV-2-infected blood donors. The APH-2 protein is 183 amino acids long, is localized in the cell nucleus, and is detected in vivo. Despite the lack of a consensus basic leucine zipper domain, APH-2 interacts with cyclic adenosine monophosphate-response element binding protein (CREB) and represses Tax2-mediated transcription in Tax2-expressing cells and in cells transfected with an HTLV-2 molecular clone. Altogether, our results demonstrate the existence of an antisense strand-encoded protein in HTLV-2, which could represent an important player in the development of disorders, such as lymphocytosis, which is frequently observed in HTLV-2 patients.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Productos del Gen tax/genética , Virus Linfotrópico T Tipo 2 Humano/fisiología , Empalme del ARN/genética , ARN sin Sentido/genética , Transcripción Genética , Proteínas Virales/genética , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Northern Blotting , Western Blotting , Femenino , Técnica del Anticuerpo Fluorescente , Regulación Viral de la Expresión Génica , Productos del Gen tax/metabolismo , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Células Jurkat , Luciferasas/genética , Luciferasas/metabolismo , Persona de Mediana Edad , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN sin Sentido/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Transfección , Proteínas Virales/metabolismoRESUMEN
Paradigm shifts throughout the history of microbiology have typically been ignored, or met with skepticism and resistance, by the scientific community. This has been especially true in the field of virology, where the discovery of a "contagium vivum fluidum", or infectious fluid remaining after excluding bacteria by filtration, was initially ignored because it did not coincide with the established view of microorganisms. Subsequent studies on such infectious agents, eventually termed "viruses", were met with skepticism. However, after an abundance of proof accumulated, viruses were eventually acknowledged as defined microbiological entities. Next, the proposed role of viruses in oncogenesis in animals was disputed, as was the unique mechanism of genome replication by reverse transcription of RNA by the retroviruses. This same pattern of skepticism holds true for the prediction of the existence of retroviral "antisense" transcripts and genes. From the time of their discovery, it was thought that retroviruses encoded proteins on only one strand of proviral DNA. However, in 1988, it was predicted that human immunodeficiency virus type 1 (HIV-1), and other retroviruses, express an antisense protein encoded on the DNA strand opposite that encoding the known viral proteins. Confirmation came quickly with the characterization of the antisense protein, HBZ, of the human T-cell leukemia virus type 1 (HTLV-1), and the finding that both the protein and its antisense mRNA transcript play key roles in viral replication and pathogenesis. However, acceptance of the existence, and potential importance, of a corresponding antisense transcript and protein (ASP) in HIV-1 infection and pathogenesis has lagged, despite gradually accumulating theoretical and experimental evidence. The most striking theoretical evidence is the finding that asp is highly conserved in group M viruses and correlates exclusively with subtypes, or clades, responsible for the AIDS pandemic. This review outlines the history of the major shifts in thought pertaining to the nature and characteristics of viruses, and in particular retroviruses, and details the development of the hypothesis that retroviral antisense transcripts and genes exist. We conclude that there is a need to accelerate studies on ASP, and its transcript(s), with the view that both may be important, and overlooked, targets in anti-HIV therapeutic and vaccine strategies.