RESUMEN
Diffuse large B-cell lymphoma (DLBCL) is a commonly diagnosed, aggressive non-Hodgkin's lymphoma. While R-CHOP chemoimmunotherapy is potentially curative, about 40% of DLBCL patients will fail, highlighting the need to identify biomarkers to optimize management. SAMHD1 has a dNTPase-independent role in promoting resection to facilitate DNA double-strand break (DSB) repair by homologous recombination. We evaluated the relationship of SAMHD1 levels with sensitivity to DSB-sensitizing agents in DLBCL cells and the association of SAMHD1 expression with clinical outcomes in 79 DLBCL patients treated with definitive therapy and an independent cohort dataset of 234 DLBCL patients. Low SAMHD1 expression, Vpx-mediated, or siRNA-mediated degradation/depletion in DLBCL cells was associated with greater sensitivity to doxorubicin and PARP inhibitors. On Kaplan-Meier log-rank survival analysis, low SAMHD1 expression was associated with improved overall survival (OS), which on subset analysis remained significant only in patients with advanced stage (III-IV) and moderate to high risk (2-5 International Prognostic Index (IPI)). The association of low SAMHD1 expression with improved OS remained significant on multivariate analysis independent of other adverse factors, including IPI, and was validated in an independent cohort. Our findings suggest that SAMHD1 expression mediates doxorubicin resistance and may be an important prognostic biomarker in advanced, higher-risk DLBCL patients.
RESUMEN
DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. The interaction of the catalytic subunit of DNA-PK (DNA-PKcs) with the Ku70/Ku80 heterodimer on DSBs leads to DNA-PK activation; however, it is not known if upstream signaling events govern this activation. Here, we reveal a regulatory step governing DNA-PK activation by SIRT2 deacetylation, which facilitates DNA-PKcs localization to DSBs and interaction with Ku, thereby promoting DSB repair by NHEJ. SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR. SIRT2 deacetylase activity facilitates DNA-PKcs interaction with Ku and localization to DSBs and promotes DNA-PK activation and phosphorylation of downstream NHEJ substrates. Moreover, targeting SIRT2 with AGK2, a SIRT2-specific inhibitor, augments the efficacy of IR in cancer cells and tumors. Our findings define a regulatory step for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ. Furthermore, our data suggest that SIRT2 inhibition may be a promising rationale-driven therapeutic strategy for increasing the effectiveness of radiation therapy.
Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas Quinasas , ADN/genética , ADN/metabolismo , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Proteína Quinasa Activada por ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Autoantígeno Ku/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinasas/genética , Sirtuina 2/genética , Sirtuina 2/metabolismo , HumanosRESUMEN
Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) has a dNTPase-independent function in promoting DNA end resection to facilitate DNA double-strand break (DSB) repair by homologous recombination (HR); however, it is not known if upstream signaling events govern this activity. Here, we show that SAMHD1 is deacetylated by the SIRT1 sirtuin deacetylase, facilitating its binding with ssDNA at DSBs, to promote DNA end resection and HR. SIRT1 complexes with and deacetylates SAMHD1 at conserved lysine 354 (K354) specifically in response to DSBs. K354 deacetylation by SIRT1 promotes DNA end resection and HR but not SAMHD1 tetramerization or dNTPase activity. Mechanistically, K354 deacetylation by SIRT1 promotes SAMHD1 recruitment to DSBs and binding to ssDNA at DSBs, which in turn facilitates CtIP ssDNA binding, leading to promotion of genome integrity. These findings define a mechanism governing the dNTPase-independent resection function of SAMHD1 by SIRT1 deacetylation in promoting HR and genome stability.
Asunto(s)
Roturas del ADN de Doble Cadena , Sirtuina 1 , Sirtuina 1/genética , Sirtuina 1/metabolismo , Reparación del ADN , Recombinación Homóloga , ADN de Cadena Simple , ADNRESUMEN
The current standard-of-care treatment for glioblastoma includes DNA damaging agents, γ-irradiation (IR) and temozolomide (TMZ). These treatments fail frequently and there is limited alternative strategy. Therefore, identifying a new therapeutic target is urgently needed to develop a strategy that improves the efficacy of the existing treatments. Here, we report that tumor samples from GBM patients express a high level of SAMHD1, emphasizing SAMHD1's importance. The depletion of SAMHD1 using virus-like particles containing Vpx, VLP(+Vpx), sensitized two independent GBM cell lines (LN-229 and U-87) to veliparib, a well-established PARP inhibitor, and slowed cell growth in a dose-dependent manner. In the mouse GBM xenograft model, Vpx-mediated SAMHD1 depletion reduced tumor growth and SAMHD1 knockout (KO) improved survival. In combination with IR or TMZ, SAMHD1 KO and exposure to 50% growth inhibitory dose (gID50) of VLP(+Vpx) displayed a synergistic effect, resulting in impaired HR, and improved LN-229 cells' sensitivity to TMZ and IR. In conclusion, our finding demonstrates that SAMHD1 promotes GBM resistance to treatment, and it is a plausible therapeutic target to improve the efficacy of TMZ and IR in GBM. Furthermore, we show that Vpx could be a potential therapeutic tool that can be utilized to deplete SAMHD1 in GBM.
RESUMEN
Failure to achieve pathologic complete response is associated with poor prognosis in breast cancer patients following neoadjuvant chemotherapy (NACT). However, prognostic biomarkers for clinical outcome are unclear in this patient population. Cyclin-dependent kinase 9 (CDK9) is often dysregulated in breast cancer, and its deficiency results in genomic instability. We reviewed the records of 84 breast cancer patients from Emory University's Winship Cancer Institute who had undergone surgical resection after NACT and had tissue available for tissue microarray analysis (TMA). Data recorded included disease presentation, treatment, pathologic response, overall survival (OS), locoregional recurrence free survival (LRRFS), distant-failure free survival (DFFS), recurrence-free survival (RFS), and event-free survival (EFS). Immunohistochemistry was performed on patient samples to determine CDK9 expression levels after NACT. Protein expression was linked with clinical data to determine significance. In a Cox proportional hazards model, using a time-dependent covariate to evaluate the risk of death between groups beyond 3 years, high CDK9 expression was significantly associated with an increase in OS (HR: 0.26, 95% CI: 0.07-0.98, p=0.046). However, Kaplan-Meier curves for OS, LRRFS, DFFS, RFS, and EFS did not reach statistical significance. The results of this study indicate that CDK9 may have a potential role as a prognostic biomarker in patients with breast cancer following NACT. However, further validation studies with increased sample sizes are needed to help elucidate the prognostic role for CDK9 in the management of these patients.
RESUMEN
DNA double-strand break (DSB) repair by homologous recombination (HR) is initiated by CtIP/MRN-mediated DNA end resection to maintain genome integrity. SAMHD1 is a dNTP triphosphohydrolase, which restricts HIV-1 infection, and mutations are associated with Aicardi-Goutières syndrome and cancer. We show that SAMHD1 has a dNTPase-independent function in promoting DNA end resection to facilitate DSB repair by HR. SAMHD1 deficiency or Vpx-mediated degradation causes hypersensitivity to DSB-inducing agents, and SAMHD1 is recruited to DSBs. SAMHD1 complexes with CtIP via a conserved C-terminal domain and recruits CtIP to DSBs to facilitate end resection and HR. Significantly, a cancer-associated mutant with impaired CtIP interaction, but not dNTPase-inactive SAMHD1, fails to rescue the end resection impairment of SAMHD1 depletion. Our findings define a dNTPase-independent function for SAMHD1 in HR-mediated DSB repair by facilitating CtIP accrual to promote DNA end resection, providing insight into how SAMHD1 promotes genome integrity.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Recombinación Homóloga , Proteína 1 que Contiene Dominios SAM y HD/genética , Roturas del ADN de Doble Cadena , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Proteína 1 que Contiene Dominios SAM y HD/deficiencia , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , TransfecciónRESUMEN
Sirtuin 2 (SIRT2) is a sirtuin family deacetylase, which maintains genome integrity and prevents tumorigenesis. Although Sirt2 deficiency in mice leads to tumorigenesis, the functional significance of somatic SIRT2 mutations in human tumors is unclear. Using structural insight combined with bioinformatics and functional analyses, we show that naturally occurring cancer-associated SIRT2 mutations at evolutionarily conserved sites disrupt its deacetylation of DNA-damage response proteins by impairing SIRT2 catalytic activity or protein levels but not its localization or binding with substrate. We observed that these SIRT2 mutant proteins fail to restore the replication stress sensitivity, impairment in recovery from replication stress, and impairment in ATR-interacting protein (ATRIP) focus accumulation of SIRT2 deficiency. Moreover, the SIRT2 mutant proteins failed to rescue the spontaneous induction of DNA damage and micronuclei of SIRT2 deficiency in cancer cells. Our findings support a model for SIRT2's tumor-suppressive function in which somatic mutations in SIRT2 contribute to genomic instability by impairing its deacetylase activity or diminishing its protein levels in the DNA-damage response. In conclusion, our work provides a mechanistic basis for understanding the biological and clinical significance of SIRT2 mutations in genome maintenance and tumor suppression.
Asunto(s)
Inestabilidad Genómica , Modelos Moleculares , Mutación , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Sirtuina 2/metabolismo , Acetilación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Biocatálisis , Línea Celular , Biología Computacional , Secuencia Conservada , Reparación del ADN , Bases de Datos Genéticas , Regulación Neoplásica de la Expresión Génica , Humanos , Micronúcleos con Defecto Cromosómico , Mutación Missense , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patología , Conformación Proteica , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Sirtuina 2/antagonistas & inhibidores , Sirtuina 2/química , Sirtuina 2/genéticaRESUMEN
The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase checkpoint pathway maintains genome integrity; however, the role of the sirtuin 2 (SIRT2) acetylome in regulating this pathway is not clear. We found that deacetylation of ATR-interacting protein (ATRIP), a regulatory partner of ATR, by SIRT2 potentiates the ATR checkpoint. SIRT2 interacts with and deacetylates ATRIP at lysine 32 (K32) in response to replication stress. SIRT2 deacetylation of ATRIP at K32 drives ATR autophosphorylation and signaling and facilitates DNA replication fork progression and recovery of stalled replication forks. K32 deacetylation by SIRT2 further promotes ATRIP accumulation to DNA damage sites and binding to replication protein A-coated single-stranded DNA (RPA-ssDNA). Collectively, these results support a model in which ATRIP deacetylation by SIRT2 promotes ATR-ATRIP binding to RPA-ssDNA to drive ATR activation and thus facilitate recovery from replication stress, outlining a mechanism by which the ATR checkpoint is regulated by SIRT2 through deacetylation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Puntos de Control del Ciclo Celular/genética , Replicación del ADN , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Proteína de Replicación A/genética , Sirtuina 2/genética , Acetilación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular Tumoral , Daño del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Fosforilación , Unión Proteica , Proteína de Replicación A/metabolismo , Transducción de Señal , Sirtuina 2/metabolismoRESUMEN
HIV-1 proviral DNA integration into host chromosomal DNA is only partially completed by the viral integrase, leaving two single-stranded DNA gaps with 5'-end mismatched viral DNA flaps. It has been inferred that these gaps are repaired by the cellular DNA repair machinery. Here, we investigated the efficiency of gap repair at integration sites in different HIV-1 target cell types. First, we found that the general gap repair machinery in macrophages was attenuated compared with that in dividing CD4(+) T cells. In fact, the repair in macrophages was heavily reliant upon host DNA polymerase ß (Pol ß). Second, we tested whether the poor dNTP availability found in macrophages is responsible for the delayed HIV-1 proviral DNA integration in this cell type because the Km value of Pol ß is much higher than the dNTP concentrations found in macrophages. Indeed, with the use of a modified quantitative AluI PCR assay, we demonstrated that the elevation of cellular dNTP concentrations accelerated DNA gap repair in macrophages at HIV-1 proviral DNA integration sites. Finally, we found that human monocytes, which are resistant to HIV-1 infection, exhibited severely restricted gap repair capacity due not only to the very low levels of dNTPs detected but also to the significantly reduced expression of Pol ß. Taken together, these results suggest that the low dNTP concentrations found in macrophages and monocytes can restrict the repair steps necessary for HIV-1 integration.
Asunto(s)
ADN Polimerasa beta/metabolismo , Desoxirribonucleótidos/metabolismo , VIH-1/fisiología , Macrófagos/metabolismo , Provirus/fisiología , Integración Viral/fisiología , Células Cultivadas , Reparación del ADN , Femenino , Humanos , Macrófagos/virología , MasculinoRESUMEN
HIV-1 reverse transcriptase (RT) frequently incorporates ribonucleoside triphosphates (rNTPs) during proviral DNA synthesis, particularly under the limited dNTP conditions found in macrophages. We investigated the mechanistic impacts of an rNMP embedded in DNA templates on HIV-1 RT-mediated DNA synthesis. We observed that the template-embedded rNMP induced pausing of RT and delayed DNA synthesis kinetics at low macrophage dNTP concentrations but not at high T cell dNTP concentrations. Although the binding affinity of RT to the rNMP-containing template-primer was not altered, the dNTP incorporation kinetics of RT were significantly reduced at one nucleotide upstream and downstream of the rNMP site, leading to pause sites. Finally, HIV-1 RT becomes more error-prone at rNMP sites with an elevated mismatch extension capability but not enhanced misinsertion capability. Together these data suggest that rNMPs embedded in DNA templates may influence reverse transcription kinetics and impact viral mutagenesis in macrophages.
Asunto(s)
ADN Viral/biosíntesis , Desoxirribonucleótidos/química , Transcriptasa Inversa del VIH/química , VIH-1/enzimología , Provirus/enzimología , Ribonucleótidos/química , Sistema Libre de Células , ADN Viral/química , ADN Viral/genética , Desoxirribonucleótidos/genética , Desoxirribonucleótidos/metabolismo , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/genética , Cinética , Mutación , Provirus/genética , Ribonucleótidos/genética , Ribonucleótidos/metabolismoRESUMEN
BACKGROUND: SAMHD1 is a triphosphohydrolase that restricts the replication of HIV-1 and SIV in myeloid cells. In macrophages and dendritic cells, SAMHD1 restricts virus replication by diminishing the deoxynucleotide triphosphate pool to a level below that which supports lentiviral reverse transcription. HIV-2 and related SIVs encode the accessory protein Vpx to induce the proteasomal degradation of SAMHD1 following virus entry. While SAMHD1 has been shown to restrict HIV-1 and SIV, the breadth of its restriction is not known and whether other viruses have a means to counteract the restriction has not been determined. RESULTS: We show that SAMHD1 restricts a wide array of divergent retroviruses, including the alpha, beta and gamma classes. Murine leukemia virus was restricted by SAMHD1 in macrophages yet removal of SAMHD1 did not alleviate the block to infection because of an additional block to viral nuclear import. Prototype foamy virus (PFV) and Human T cell leukemia virus type I (HTLV-1) were the only retroviruses tested that were not restricted by SAMHD1. PFV reverse transcribes predominantly prior to entry and thus is unaffected by the dNTP level in the target cell. It is possible that HTLV-1 has a mechanism to render the virus resistant to SAMHD1-mediated restriction. CONCLUSION: The results suggest that SAMHD1 has broad anti-retroviral activity against which most viruses have not found an escape.
Asunto(s)
Macrófagos/virología , Proteínas de Unión al GTP Monoméricas/farmacología , Células Mieloides/virología , Retroviridae/efectos de los fármacos , Retroviridae/patogenicidad , Replicación Viral/efectos de los fármacos , Línea Celular , Células Dendríticas/metabolismo , Células Dendríticas/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Células Jurkat , Macrófagos/inmunología , Proteínas de Unión al GTP Monoméricas/metabolismo , Células Mieloides/metabolismo , Retroviridae/clasificación , Retroviridae/fisiología , Proteína 1 que Contiene Dominios SAM y HDRESUMEN
Unlike activated CD4(+) T cells, resting CD4(+) T cells are highly resistant to productive HIV-1 infection. Early after HIV-1 entry, a major block limits reverse transcription of incoming viral genomes. Here we show that the deoxynucleoside triphosphate triphosphohydrolase SAMHD1 prevents reverse transcription of HIV-1 RNA in resting CD4(+) T cells. SAMHD1 is abundantly expressed in resting CD4(+) T cells circulating in peripheral blood and residing in lymphoid organs. The early restriction to infection in unstimulated CD4(+) T cells is overcome by HIV-1 or HIV-2 virions into which viral Vpx is artificially or naturally packaged, respectively, or by addition of exogenous deoxynucleosides. Vpx-mediated proteasomal degradation of SAMHD1 and elevation of intracellular deoxynucleotide pools precede successful infection by Vpx-carrying HIV. Resting CD4(+) T cells from healthy donors following SAMHD1 silencing or from a patient with Aicardi-Goutières syndrome homozygous for a nonsense mutation in SAMHD1 were permissive for HIV-1 infection. Thus, SAMHD1 imposes an effective restriction to HIV-1 infection in the large pool of noncycling CD4(+) T cells in vivo. Bypassing SAMHD1 was insufficient for the release of viral progeny, implicating other barriers at later stages of HIV replication. Together, these findings may unveil new ways to interfere with the immune evasion and T cell immunopathology of pandemic HIV-1.
Asunto(s)
Linfocitos T CD4-Positivos , VIH-1 , Proteínas de Unión al GTP Monoméricas , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/virología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Infecciones por VIH , VIH-1/genética , VIH-1/metabolismo , VIH-2/genética , VIH-2/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/virología , Transcripción Reversa/genética , Proteína 1 que Contiene Dominios SAM y HD , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Virión/genética , Virión/crecimiento & desarrollo , Replicación ViralRESUMEN
Sterile alpha motif domain- and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase that restricts the replication of lentiviruses in myeloid cells by hydrolyzing the cellular deoxynucleotide triphosphates to a level below that which is required for reverse transcription. Human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency viruses (SIVs) encode the accessory protein viral protein X (Vpx) that counteracts SAMHD1. Vpx recruits SAMHD1 to a cullin4A-RING E3 ubiquitin ligase (CRL4), which targets the enzyme for proteasomal degradation. Vpx and SAMHD1 both localize to the nucleus of the cell. We identified the nuclear localization sequence (NLS) of SAMHD1 as a conserved KRPR sequence at amino acid residues 11 to 14. SAMHD1 lacking a functional NLS localized to the cytoplasm but retained its triphosphohydrolase and antiviral activities. However, cytoplasmic SAMHD1 was resistant to Vpx-induced degradation, and its antiviral activity was not counteracted by Vpx. Cytoplasmic SAMHD1 interacted with Vpx and retained it in the cytoplasm. The inhibition of nuclear export with leptomycin B did not impair the ability of Vpx to degrade SAMHD1. These findings suggest that SAMHD1 is targeted by Vpx for ubiquitination and degradation in the nucleus.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Secuencia de Aminoácidos/genética , Proteínas Cullin/metabolismo , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Microscopía Fluorescente , Señales de Localización Nuclear/genética , Plásmidos/genética , Proteína 1 que Contiene Dominios SAM y HD , UbiquitinaciónRESUMEN
Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates.
Asunto(s)
Adenoviridae/genética , ADN Polimerasa Dirigida por ADN/genética , Mutagénesis , Mutación , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Línea Celular , Línea Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Insectos , Cinética , Pulmón/virología , Datos de Secuencia Molecular , Fenotipo , Plásmidos/metabolismo , Replicación ViralRESUMEN
Recently, SAMHD1 has come under intense focus as a host anti-HIV factor. SAMHD1 is a dNTP triphosphohydrolase, which leads to the regulation of DNA metabolism in host cells. HIV-2/SIV (simian immunodeficiency virus) viral protein x (Vpx) has been shown to promote the degradation of SAMHD1. In this study, we examine the kinetics of SAMHD1 degradation, the increase in the dNTP pool level, and the efficiency of proviral DNA synthesis in Vpx+ virus-like particle (VLP)-treated monocyte-derived macrophages (MDMs). Our results indicate a very close temporal link with a reduction in SAMHD1 detected within the first few hours of Vpx+ VLP treatment. This loss of SAMHD1 is followed by a significant increase in cellular dNTP levels by 8 h after Vpx+ VLP addition, ultimately leading to the enhancement of the HIV proviral DNA synthesis rate and HIV infection in MDMs. Finally, the pretreatment of MDMs with the Vpx+ VLPs, which is a widely used protocol, displayed identical proviral DNA synthesis as compared with MDMs co-treated with Vpx+ VLP and HIV vector. These findings further indicate that Vpx degradation of SAMHD1 is sufficiently rapid to enable appropriate progression of reverse transcription in MDMs, even when present at the time of infection. Overall, this study demonstrates a tight interplay between SAMHD1 level, dNTP levels, and HIV proviral DNA synthesis kinetics in MDMs.
Asunto(s)
ADN Viral/metabolismo , Fosfatos de Dinucleósidos/metabolismo , VIH-1/metabolismo , Macrófagos/virología , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación del ADN , Fosfatos de Dinucleósidos/química , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Cinética , Receptores de Lipopolisacáridos/biosíntesis , Modelos Biológicos , Nucleótidos/química , Reacción en Cadena de la Polimerasa/métodos , Proteína 1 que Contiene Dominios SAM y HD , Factores de TiempoRESUMEN
Leishmaniasis is a parasitic disease that is widely prevalent in many tropical and sub-tropical regions of the world. Infection with Leishmania has been recognized to induce a striking acceleration of Human Immunodeficiency Virus Type 1 (HIV-1) infection in coinfected individuals through as yet incompletely understood mechanisms. Cells of the monocyte/macrophage lineage are the predominant cell types coinfected by both pathogens. Monocytes and macrophages contain extremely low levels of deoxynucleoside triphosphates (dNTPs) due to their lack of cell cycling and S phase, where dNTP biosynthesis is specifically activated. Lentiviruses, such as HIV-1, are unique among retroviruses in their ability to replicate in these non-dividing cells due, at least in part, to their highly efficient reverse transcriptase (RT). Nonetheless, viral replication progresses more efficiently in the setting of higher intracellular dNTP concentrations related to enhanced enzyme kinetics of the viral RT. In the present study, in vitro infection of CD14+ peripheral blood-derived human monocytes with Leishmania major was found to induce differentiation, marked elevation of cellular p53R2 ribonucleotide reductase subunit and R2 subunit expression. The R2 subunit is restricted to the S phase of the cell cycle. Our dNTP assay demonstrated significant elevation of intracellular monocyte-derived macrophages (MDMs) dNTP concentrations in Leishmania-infected cell populations as compared to control cells. Infection of Leishmania-maturated MDMs with a pseudotyped GFP expressing HIV-1 resulted in increased numbers of GFP+ cells in the Leishmania-maturated MDMs as compared to control cells. Interestingly, a sub-population of Leishmania-maturated MDMs was found to have re-entered the cell cycle, as demonstrated by BrdU labeling. In conclusion, Leishmania infection of primary human monocytes promotes the induction of an S phase environment and elevated dNTP levels with notable elevation of HIV-1 expression in the setting of coinfection.
Asunto(s)
Desoxirribonucleótidos/metabolismo , Infecciones por VIH , VIH-1/metabolismo , Leishmania major/metabolismo , Leishmaniasis Cutánea , Macrófagos , Proteínas de Ciclo Celular/biosíntesis , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Viral de la Expresión Génica , Infecciones por VIH/complicaciones , Infecciones por VIH/metabolismo , Transcriptasa Inversa del VIH/metabolismo , Humanos , Leishmaniasis Cutánea/complicaciones , Leishmaniasis Cutánea/metabolismo , Macrófagos/metabolismo , Macrófagos/parasitología , Macrófagos/virología , Masculino , Monocitos , Ribonucleótido Reductasas/biosíntesis , Fase SRESUMEN
SAMHD1 restricts the infection of dendritic and other myeloid cells by human immunodeficiency virus type 1 (HIV-1), but in lentiviruses of the simian immunodeficiency virus of sooty mangabey (SIVsm)-HIV-2 lineage, SAMHD1 is counteracted by the virion-packaged accessory protein Vpx. Here we found that SAMHD1 restricted infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs), lowering their concentrations to below those required for the synthesis of the viral DNA by reverse transcriptase (RT). SAMHD1-mediated restriction was alleviated by the addition of exogenous deoxynucleosides. An HIV-1 with a mutant RT with low affinity for dNTPs was particularly sensitive to SAMHD1-mediated restriction. Vpx prevented the SAMHD1-mediated decrease in dNTP concentration and induced the degradation of human and rhesus macaque SAMHD1 but had no effect on mouse SAMHD1. Nucleotide-pool depletion could be a general mechanism for protecting cells from infectious agents that replicate through a DNA intermediate.
Asunto(s)
VIH-1/fisiología , Proteínas de Unión al GTP Monoméricas/metabolismo , Nucleótidos/metabolismo , Replicación Viral , Animales , Línea Celular , Humanos , Espacio Intracelular/metabolismo , Macaca mulatta , Macrófagos/inmunología , Ratones , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/inmunología , Proteína 1 que Contiene Dominios SAM y HDRESUMEN
Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) has a unique tight binding to dNTP substrates. Structural modeling of Ala-114 of HIV-1 RT suggests that longer side chains at this residue can reduce the space normally occupied by the sugar moiety of an incoming dNTP. Indeed, mutations at Ala-114 decrease the ability of RT to synthesize DNA at low dNTP concentrations and reduce the dNTP-binding affinity (K(d)) of RT. However, the K(d) values of WT and A114C RT remained equivalent with an acyclic dNTP substrate. Finally, mutant A114 RT HIV-1 vectors displayed a greatly reduced transduction in nondividing human lung fibroblasts (HLFs), while WT HIV-1 vector efficiently transduced both dividing and nondividing HLFs. Together these data support that the A114 residue of HIV-1 RT plays a key mechanistic role in the dNTP binding of HIV-1 RT and the unique viral infectivity of target cell types with low dNTP pools.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , VIH-1/fisiología , Nucleósidos/metabolismo , Replicación Viral/fisiología , Sitios de Unión , Regulación Viral de la Expresión Génica/fisiología , Transcriptasa Inversa del VIH/genética , Humanos , Cinética , Modelos Moleculares , Unión Proteica , Especificidad por SustratoRESUMEN
Prolonged highly active anti-retroviral therapy with multiple nucleoside reverse transcriptase inhibitors for the treatment of patients infected with human immunodeficiency virus type 1 (HIV-1) can induce the development of an HIV-1 reverse transcriptase (RT) harboring a dipeptide insertion at the RT fingers domain with a background thymidine analog mutation. This mutation renders viral resistance to multiple nucleoside reverse transcriptase inhibitors. We investigated the effect of the dipeptide fingers domain insertion mutation on strand transfer activity using two clinical RT variants isolated during the pre-treatment and post-treatment of an infected patient, termed pre-drug RT without dipeptide insertion and post-drug RT with Ser-Gly insertion, respectively. First, the post-drug RT displayed elevated strand transfer activity compared to the pre-drug RT, with two different RNA templates. Second, the post-drug RT exhibited less RNA template degradation than the pre-drug RT but higher polymerization-dependent RNase H activity. Third, the post-drug RT had a faster association rate (k(on)) for template binding and a lower equilibrium binding constant K(d) for the template, leading to a template binding affinity tighter than that of the pre-drug RT. The k(off) values for the pre-drug RT and the post-drug RT were similar. Finally, the removal of the dipeptide insertion from the post-drug RT abolished the elevated strand transfer activity and RNase H activity, in addition to the loss of azidothymidine resistance. These biochemical data suggest that the dipeptide insertion elevates strand transfer activity by increasing the interaction of the RT with the RNA donor template, promoting cleavage that generates more invasion sites for the acceptor template during DNA synthesis.
Asunto(s)
Farmacorresistencia Viral Múltiple , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/enzimología , Mutagénesis Insercional , Fármacos Anti-VIH/administración & dosificación , ADN Viral/metabolismo , Dipéptidos/genética , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Humanos , Cinética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , ARN Viral/metabolismoRESUMEN
Terminally differentiated/non-dividing macrophages contain extremely low cellular dNTP concentrations (20-40 nm), compared with activated CD4(+) T cells (2-5 µm). However, our LC-MS/MS study revealed that the non-canonical dUTP concentration (2.9 µm) is â¼60 times higher than TTP in macrophages, whereas the concentrations of dUTP and TTP in dividing human primary lymphocytes are very similar. Specifically, we evaluated the contribution of HIV-1 reverse transcriptase to proviral DNA uracilation under the physiological conditions found in HIV-1 target cells. Indeed, biochemical simulation of HIV-1 reverse transcription demonstrates that HIV-1 RT efficiently incorporates dUTP in the macrophage nucleotide pools but not in the T cell nucleotide pools. Measurement of both pre-steady state and steady state kinetic parameters of dUTP incorporation reveals minimal selectivity of HIV-1 RT for TTP over dUTP, implying that the cellular dUTP/TTP ratio determines the frequency of HIV-1 RT-mediated dUTP incorporation. The RT of another lentivirus, simian immunodeficiency virus, also displays efficient dUTP incorporation in the dNTP/dUTP pools found in macrophages but not in T cells. Finally, 2',3'-dideoxyuridine was inhibitory to HIV-1 proviral DNA synthesis in macrophages but not in T cells. The data presented demonstrates that the non-canonical dUTP was abundant relative to TTP, and efficiently incorporated during HIV-1 reverse transcription, particularly in non-dividing macrophages.