RESUMEN
Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV ('Berlin patient'). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by mRNA electroporation, a gentle and truly transient gene-transfer technique. CCR5-Uco-TALEN mediated high-rate CCR5 knockout (>90% in PM1 and >50% in primary T cells) combined with low off-target activity, as assessed by flow cytometry, next-generation sequencing and a newly devised, very convenient gene-editing frequency digital-PCR (GEF-dPCR). GEF-dPCR facilitates simultaneous detection of wild-type and gene-edited alleles with remarkable sensitivity and accuracy as shown for the CCR5 on-target and CCR2 off-target loci. CCR5-edited cells were protected from infection with HIV-derived lentiviral vectors, but also with the wild-type CCR5-tropic HIV-1BaL strain. Long-term exposure to HIV-1BaL resulted in almost complete suppression of viral replication and selection of CCR5-gene edited T cells. In conclusion, we have developed a novel TALEN for the targeted, high-efficiency knockout of CCR5 and a useful dPCR-based gene-editing detection method.
Asunto(s)
Desoxirribonucleasas/genética , Técnicas de Inactivación de Genes/métodos , Receptores CCR5/genética , Línea Celular , Reparación del ADN por Unión de Extremidades , Desoxirribonucleasas/química , Electroporación , VIH/fisiología , Infecciones por VIH/genética , Humanos , Mutación , Reacción en Cadena de la Polimerasa , ARN Mensajero , Linfocitos T/virología , TransfecciónRESUMEN
The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate-limitations can be at the mono-, but also at the di- and triphosphorylation steps. We developed a nucleoside triphosphate (NTP) delivery system (TriPPPro-approach). In this approach, NTPs are masked by two bioreversible units at the γ-phosphate. Using a procedure involving H-phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme-triggered delivery of NTPs was demonstrated by pig liver esterase, in human T-lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The TriPPPro-compounds of some HIV-inactive nucleoside analogues showed marked anti-HIV activity. For cellular uptake studies, a fluorescent TriPPPro-compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH/efectos de los fármacos , Nucleósidos/farmacología , Polifosfatos/farmacología , Profármacos/farmacología , Nucleótidos de Timina/farmacología , Animales , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacocinética , Linfocitos T CD4-Positivos/virología , Línea Celular , Permeabilidad de la Membrana Celular , Esterasas/metabolismo , Infecciones por VIH/tratamiento farmacológico , Humanos , Nucleósidos/química , Nucleósidos/metabolismo , Nucleósidos/farmacocinética , Polifosfatos/química , Polifosfatos/metabolismo , Polifosfatos/farmacocinética , Profármacos/química , Profármacos/metabolismo , Profármacos/farmacocinética , Porcinos , Nucleótidos de Timina/síntesis química , Nucleótidos de Timina/química , Nucleótidos de Timina/metabolismoRESUMEN
Stable integration of HIV proviral DNA into host cell chromosomes, a hallmark and essential feature of the retroviral life cycle, establishes the infection permanently. Current antiretroviral combination drug therapy cannot cure HIV infection. However, expressing an engineered HIV-1 long terminal repeat (LTR) site-specific recombinase (Tre), shown to excise integrated proviral DNA in vitro, may provide a novel and highly promising antiviral strategy. We report here the conditional expression of Tre-recombinase from an advanced lentiviral self-inactivation (SIN) vector in HIV-infected cells. We demonstrate faithful transgene expression, resulting in accurate provirus excision in the absence of cytopathic effects. Moreover, pronounced Tre-mediated antiviral effects are demonstrated in vivo, particularly in humanized Rag2â»/â»Î³câ»/â» mice engrafted with either Tre-transduced primary CD4⺠T cells, or Tre-transduced CD34⺠hematopoietic stem and progenitor cells (HSC). Taken together, our data support the use of Tre-recombinase in novel therapy strategies aiming to provide a cure for HIV.
Asunto(s)
Terapia Genética/métodos , Infecciones por VIH , Duplicado del Terminal Largo de VIH , VIH-1/metabolismo , Integrasas/metabolismo , Provirus/metabolismo , Animales , Vectores Genéticos , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/terapia , VIH-1/genética , Humanos , Integrasas/genética , Ratones , Ratones Noqueados , Provirus/genética , Transducción Genética , Quimera por Trasplante , Integración Viral/genéticaRESUMEN
Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Recombinasas/metabolismo , VIH-1/fisiología , Provirus/genética , Duplicado del Terminal Largo de VIH/genética , Infecciones por VIH/terapia , Vectores Genéticos/genéticaRESUMEN
Despite scientific evidence originating from two patients published to date that CCR5Δ32/Δ32 hematopoietic stem cell transplantation (HSCT) can cure human immunodeficiency virus type 1 (HIV-1), the knowledge of immunological and virological correlates of cure is limited. Here we characterize a case of long-term HIV-1 remission of a 53-year-old male who was carefully monitored for more than 9 years after allogeneic CCR5Δ32/Δ32 HSCT performed for acute myeloid leukemia. Despite sporadic traces of HIV-1 DNA detected by droplet digital PCR and in situ hybridization assays in peripheral T cell subsets and tissue-derived samples, repeated ex vivo quantitative and in vivo outgrowth assays in humanized mice did not reveal replication-competent virus. Low levels of immune activation and waning HIV-1-specific humoral and cellular immune responses indicated a lack of ongoing antigen production. Four years after analytical treatment interruption, the absence of a viral rebound and the lack of immunological correlates of HIV-1 antigen persistence are strong evidence for HIV-1 cure after CCR5Δ32/Δ32 HSCT.
Asunto(s)
Infecciones por VIH , VIH-1 , Trasplante de Células Madre Hematopoyéticas , Masculino , Humanos , Animales , Ratones , Persona de Mediana Edad , VIH-1/genética , Infecciones por VIH/genética , Infecciones por VIH/terapiaRESUMEN
Recently, it has been shown that human ejaculate enhances human immunodeficiency virus 1 (HIV-1) infectivity. Enhancement of infectivity is conceived to be mediated by amyloid filaments from peptides that are proteolytically released from prostatic acid phosphatase (PAP), termed Semen-derived Enhancer of Virus Infection (SEVI). The aim of this study was to test the range of HIV-1 infectivity enhancing properties of a large number of individual semen samples (n = 47) in a TZM-bl reporter cell HIV infection system. We find that semen overall increased infectivity to 156% of the control experiment without semen, albeit with great inter- and intraindividual variability (range -53%-363%). Using transmission electron microscopy, we provide evidence for SEVI fibrils in fresh human semen for the first time. Moreover, we confirm that the infectivity enhancing property can be inhibited by the major green tea ingredient epigallocatechin-3-gallate (EGCG) at non-toxic concentrations. The median inhibition of infection by treatment with 0.4 mM EGCG was 70.6% (p < 0.0001) in our cohort. Yet, there were substantial variations of inhibition and in a minority of samples, infectivity enhancement was not inhibited by EGCG treatment at all. Thus, topical application of EGCG may be a feasible additional measure to prevent the sexual transmission of HIV. However, the reasons for the variability in the efficacy of the abrogation of semen-mediated enhancement of HIV-1 infectivity and EGCG efficacy have to be elucidated before therapeutic trials can be conducted.
RESUMEN
Peptide fragments, derived from prostatic acidic phosphatase, are secreted in large amounts into human semen and form amyloid fibrils. These fibrillar structures, termed semen-derived enhancer of virus infection (SEVI), capture HIV virions and direct them to target cells. Thus, SEVI appears to be an important infectivity factor of HIV during sexual transmission. Here, we are able to demonstrate that epigallocatechin-3-gallate (EGCG), the major active constituent of green tea, targets SEVI for degradation. Furthermore, it is shown that EGCG inhibits SEVI activity and abrogates semen-mediated enhancement of HIV-1 infection in the absence of cellular toxicity. Therefore, EGCG appears to be a promising supplement to antiretroviral microbicides to reduce sexual transmission of HIV-1.
Asunto(s)
Fármacos Anti-VIH/farmacología , Catequina/análogos & derivados , Infecciones por VIH/prevención & control , VIH-1 , Semen/efectos de los fármacos , Amiloide/antagonistas & inhibidores , Amiloide/metabolismo , Camellia sinensis/química , Catequina/farmacología , Células Cultivadas , Infecciones por VIH/transmisión , Humanos , Masculino , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/metabolismo , Semen/virologíaRESUMEN
Upon posttranslational activation, the eukaryotic initiation factor-5A (eIF-5A) transports a subset of mRNAs out of the nucleus to the ribosomes for translation. Activation of the protein is an evolutionary highly conserved process that is unique to eIF-5A, the conversion of a lysine to a hypusine. Instrumental for the synthesis of hypusine is the first of two enzymatic reactions mediated by deoxyhypusine synthase (DHS). We show that DHS of wheat and the pathogenic fungus Fusarium graminearum, which causes one of the most destructive crop diseases worldwide, are transcriptionally upregulated during their pathogenic interaction. Although DHS of wheat, fungus, and human can be equally inhibited by the inhibitor CNI-1493 in vitro, application during infection of wheat and maize flowers results in strong inhibition of the pathogen without interference with kernel development. Our studies provide a novel strategy to selectively inhibit fungal growth without affecting plant growth. We identified fungal DHS as a target for the development of new inhibitors, for which CNI-1493 may serve as a lead substance.
Asunto(s)
Fusarium/enzimología , Hidrazonas/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Enfermedades de las Plantas/microbiología , Triticum/microbiología , Zea mays/microbiología , Clonación Molecular , Fusarium/efectos de los fármacos , Fusarium/genética , Fusarium/patogenicidad , Genes Fúngicos/genética , Genes de Plantas/genética , Germinación/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Factores de Iniciación de Péptidos/efectos de los fármacos , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/efectos de los fármacos , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/fisiología , Triticum/enzimología , Triticum/genética , Zea mays/enzimología , Zea mays/genética , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Adeno-associated viruses (AAV) are considered non-pathogenic in humans, and thus have been developed into powerful vector platforms for in vivo gene therapy. Although the various AAV serotypes display broad tropism, frequently infecting multiple tissues and cell types, vectors for specific and efficient targeting of human CD4+ T lymphocytes are largely missing. In fact, a substantial translational bottleneck exists in the field of therapeutic gene transfer that would require in vivo delivery into peripheral disease-related lymphocytes for subsequent genome editing. To solve this issue, capsid modification for retargeting AAV tropism, and in turn improving vector potency, is considered a promising strategy. Here, we genetically modified the minor AAV2 capsid proteins, VP1 and VP2, with a set of novel nanobodies with high-affinity for the human CD4 receptor. These novel vector variants demonstrated improved targeting of human CD4+ cells, including primary human peripheral blood mononuclear cells (PBMC) and purified human CD4+ T lymphocytes. Thus, the technical approach presented here provides a promising strategy for developing specific gene therapy vectors, particularly targeting disease-related peripheral blood CD4+ leukocytes.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Proteínas de la Cápside/genética , Dependovirus/genética , Vectores Genéticos/administración & dosificación , Leucocitos Mononucleares/metabolismo , Anticuerpos de Dominio Único/química , Transducción Genética , Linfocitos T CD4-Positivos/inmunología , Técnicas de Transferencia de Gen , Células HEK293 , Células HeLa , Humanos , Leucocitos Mononucleares/inmunologíaRESUMEN
The guanosine-adenosine-rich exonic splicing enhancer (GAR ESE) identified in exon 5 of the human immunodeficiency virus type-1 (HIV-1) pre-mRNA activates either an enhancer-dependent 5' splice site (ss) or 3' ss in 1-intron reporter constructs in the presence of the SR proteins SF2/ASF2 and SRp40. Characterizing the mode of action of the GAR ESE inside the internal HIV-1 exon 5 we found that this enhancer fulfils a dual splicing regulatory function (i) by synergistically mediating exon recognition through its individual SR protein-binding sites and (ii) by conferring 3' ss selectivity within the 3' ss cluster preceding exon 5. Both functions depend upon the GAR ESE, U1 snRNP binding at the downstream 5' ss D4 and the E42 sequence located between these elements. Therefore, a network of cross-exon interactions appears to regulate splicing of the alternative exons 4a and 5. As the GAR ESE-mediated activation of the upstream 3' ss cluster also is essential for the processing of intron-containing vpu/env-mRNAs during intermediate viral gene expression, the GAR enhancer substantially contributes to the regulation of viral replication.
Asunto(s)
Empalme Alternativo , VIH-1/genética , Sitios de Empalme de ARN , ARN Viral/química , Secuencias Reguladoras de Ácido Ribonucleico , Adenosina/análisis , Sitios de Unión , Exones , Guanosina/análisis , Células HeLa , Humanos , Intrones , Proteínas Nucleares/metabolismo , Precursores del ARN/química , ARN Mensajero/química , Proteínas de Unión al ARN , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Factores de Empalme Serina-Arginina , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The introduction of highly active antiretroviral therapy (HAART) has significantly decreased morbidity and mortality among patients infected with HIV-1. However, HIV-1 can acquire resistance against all currently available antiretroviral drugs targeting viral reverse transcriptase, protease, and gp41. Moreover, in a growing number of patients, the development of multidrug-resistant viruses compromises HAART efficacy and limits therapeutic options. Therefore, it is an ongoing task to develop new drugs and to identify new targets for antiretroviral therapy. Here, we identified the guanylhydrazone CNI-1493 as an efficient inhibitor of human deoxyhypusine synthase (DHS). By inhibiting DHS, this compound suppresses hypusine formation and, thereby, activation of eukaryotic initiation factor 5A (eIF-5A), a cellular cofactor of the HIV-1 Rev regulatory protein. We demonstrate that inhibition of DHS by CNI-1493 or RNA interference efficiently suppressed the retroviral replication cycle in cell culture and primary cells. We show that CNI-1493 inhibits replication of macrophage- and T cell-tropic laboratory strains, clinical isolates, and viral strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Moreover, no measurable drug-induced adverse effects on cell cycle transition, apoptosis, and general cytotoxicity were observed. Therefore, human DHS represents a novel and promising drug target for the development of advanced antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.
Asunto(s)
Fármacos Anti-VIH/farmacología , Infecciones por VIH/enzimología , Lisina/análogos & derivados , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Fármacos Anti-VIH/química , Células Cultivadas , Resistencia a Múltiples Medicamentos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Productos del Gen rev/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Hidrazonas/química , Hidrazonas/farmacología , Cinética , Lisina/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/virología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Fenotipo , Interferencia de ARN , Linfocitos T/efectos de los fármacos , Linfocitos T/virología , Activación Transcripcional/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
Malaria, sleeping sickness, Chagas' disease, Aleppo boil, and AIDS are among the tropical diseases causing millions of infections and cases of deaths per year because only inefficient chemotherapy is available. Since the targeting of the enzymes of the polyamine pathway may provide novel therapy options, we aimed to inhibit the deoxyhypusine hydroxylase, which is an important step in the biosynthesis of the eukaryotic initiation factor 5A. In order to identify new lead compounds, piperidines were produced and biologically evaluated. The 3,5-diethyl piperidone-3,5-dicarboxylates 11 and 13 substituted with 4-nitrophenyl rings in the 2 and 6 positions were found to be active against Trypanosoma brucei brucei and Plasmodium falciparum combined with low cytotoxicity against macrophages. The corresponding monocarboxylates are only highly active against the T. brucei brucei. The piperidine oximether 53 demonstrated the highest plasmodicidal activity. Moreover, compounds 11 and 53 were also able to inhibit replication of HIV-1.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Antimaláricos/síntesis química , Piperidinas/síntesis química , Tripanocidas/síntesis química , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Antimaláricos/química , Antimaláricos/farmacología , Línea Celular , Éteres/síntesis química , Éteres/química , Éteres/farmacología , VIH-1/efectos de los fármacos , Humanos , Leishmania major/efectos de los fármacos , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Oximas/síntesis química , Oximas/química , Oximas/farmacología , Piperidinas/química , Piperidinas/farmacología , Plasmodium falciparum/efectos de los fármacos , Compuestos de Espiro/síntesis química , Compuestos de Espiro/química , Compuestos de Espiro/farmacología , Relación Estructura-Actividad , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Replicación Viral/efectos de los fármacosAsunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Fármacos Anti-VIH/uso terapéutico , Drogas en Investigación/uso terapéutico , VIH-1/efectos de los fármacos , Integrasas/uso terapéutico , Terapia Molecular Dirigida , Provirus/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
The delivery of therapeutic genes for treatment of inherited or infectious diseases frequently requires lentiviral transduction of CD34+ hematopoietic stem and progenitor cells (HSC). Optimized transduction protocols with a therapeutic goal aim to maximize the number of transduction-positive cells while limiting the vector copy number that reach each individual cell. Importantly, the transduced HSC should maintain their "stem-like" properties. Here, we analyzed LentiBOOST™ reagent, a membrane-sealing poloxamer, with respect to enhancing lentiviral transduction of CD34+ peripheral blood stem cells. We demonstrate that inclusion of LentiBOOST™ in a standard HSC transduction protocol yields high transduction efficiencies while preserving the ability of the transduced HSC to differentiate into various hematopoietic lineages. Thus, LentiBOOST™ reagent can significantly improve lentiviral CD34+ HSC transduction protocols with the potential to improve production of gene-modified cell products.
Asunto(s)
Vectores Genéticos/inmunología , VIH-1/genética , Células Madre Hematopoyéticas/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Linfocitos T/inmunología , Transducción Genética/métodos , Antígenos CD34/genética , Antígenos CD34/inmunología , Diferenciación Celular , Línea Celular , Linaje de la Célula/inmunología , Variaciones en el Número de Copia de ADN , Genes Reporteros , Terapia Genética/métodos , Vectores Genéticos/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Células HEK293 , VIH-1/inmunología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Plásmidos/química , Plásmidos/metabolismo , Poloxámero/química , Cultivo Primario de Células , Protaminas/química , Reacción en Cadena en Tiempo Real de la Polimerasa/instrumentación , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Linfocitos T/citología , Linfocitos T/virología , Transducción Genética/instrumentación , TransgenesRESUMEN
BACKGROUND: The HIV-1 Rev protein is a key component in the early to late switch in HIV-1 splicing from early intronless (e.g. tat, rev) to late intron-containing Rev-dependent (e.g. gag, vif, env) transcripts. Previous results suggested that cis-acting sequences and inefficient 5' and 3' splice sites are a prerequisite for Rev function. However, we and other groups have shown that two of the HIV-1 5' splice sites, D1 and D4, are efficiently used in vitro and in vivo. Here, we focus on the efficiency of the HIV-1 3' splice sites taking into consideration to what extent their intrinsic efficiencies are modulated by their downstream cis-acting exonic sequences. Furthermore, we delineate their role in RNA stabilization and Rev function. RESULTS: In the presence of an efficient upstream 5' splice site the integrity of the 3' splice site is not essential for Rev function whereas an efficient 3' splice site impairs Rev function. The detrimental effect of a strong 3' splice site on the amount of Rev-dependent intron-containing HIV-1 glycoprotein coding (env) mRNA is not compensatable by weakening the strength of the upstream 5' splice site. Swapping the HIV-1 3' splice sites in an RRE-containing minigene, we found a 3' splice site usage which was variably dependent on the presence of the usual downstream exonic sequence. The most evident activation of 3' splice site usage by its usual downstream exonic sequence was observed for 3' splice site A1 which was turned from an intrinsic very weak 3' splice site into the most active 3' splice site, even abolishing Rev activity. Performing pull-down experiments with nuclear extracts of HeLa cells we identified a novel ASF/SF2-dependent exonic splicing enhancer (ESE) within HIV-1 exon 2 consisting of a heptameric sequence motif occurring twice (M1 and M2) within this short non-coding leader exon. Single point mutation of M1 within an infectious molecular clone is detrimental for HIV-1 exon 2 recognition without affecting Rev-dependent vif expression. CONCLUSION: Under the conditions of our assay, the rate limiting step of retroviral splicing, competing with Rev function, seems to be exclusively determined by the functional strength of the 3' splice site. The bipartite ASF/SF2-dependent ESE within HIV-1 exon 2 supports cross-talk between splice site pairs across exon 2 (exon definition) which is incompatible with processing of the intron-containing vif mRNA. We propose that Rev mediates a switch from exon to intron definition necessary for the expression of all intron-containing mRNAs.
Asunto(s)
Regiones no Traducidas 3'/genética , Productos del Gen rev/metabolismo , VIH-1/genética , Empalme del ARN , Elementos de Facilitación Genéticos , Exones/genética , Genes rev , VIH-1/metabolismo , Células HeLa , Humanos , Intrones/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
Genome editing using designer nucleases such as transcription activator-like effector nucleases (TALENs) or clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 nucleases is an emerging technology in basic and applied research. Whereas the application of editing tools, namely CRISPR-Cas9, has recently become very straightforward, quantification of resulting gene knockout rates still remains a bottleneck. This is particularly true if the product of a targeted gene is not easily detectable. To address this problem, we devised a novel gene-editing frequency digital PCR (GEF-dPCR) technique. GEF-dPCR exploits two differently labeled probes that are placed within one amplicon at the gene-editing target site to simultaneously detect wild-type and nonhomologous end-joining (NHEJ)-affected alleles. Taking advantage of the principle of dPCR, this enables concurrent quantification of edited and wild-type alleles in a given sample. We propose that our method is optimal for the monitoring of gene-edited cells in vivo, e.g., in clinical settings. Here we describe preparation, design of primers and probes, and setup and analysis of GEF-dPCR. The setup of GEF-dPCR requires up to 2 weeks (depending on the starting point); once the dPCR has been established, the protocol for sample analysis takes <1 d.
Asunto(s)
Sistemas CRISPR-Cas , Endonucleasas/genética , Ingeniería Genética , Reacción en Cadena de la Polimerasa/métodos , Alelos , Secuencia de Bases , Línea Celular , ADN/genética , Reparación del ADN por Unión de Extremidades , Sondas de ADN/genética , Endonucleasas/metabolismo , Ingeniería Genética/métodos , Humanos , Datos de Secuencia Molecular , MutaciónRESUMEN
Current combination antiretroviral therapies (cART) efficiently suppress HIV-1 reproduction in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. To generate an antiviral agent capable of eradicating the provirus from infected cells, we employed 145 cycles of substrate-linked directed evolution to evolve a recombinase (Brec1) that site-specifically recognizes a 34-bp sequence present in the long terminal repeats (LTRs) of the majority of the clinically relevant HIV-1 strains and subtypes. Brec1 efficiently, precisely and safely removes the integrated provirus from infected cells and is efficacious on clinical HIV-1 isolates in vitro and in vivo, including in mice humanized with patient-derived cells. Our data suggest that Brec1 has potential for clinical application as a curative HIV-1 therapy.
Asunto(s)
Antivirales/farmacología , Evolución Molecular Dirigida/métodos , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Provirus/efectos de los fármacos , Recombinasas/farmacología , Integración Viral/efectos de los fármacos , Animales , Antivirales/metabolismo , Secuencia de Bases , Células Cultivadas , VIH-1/genética , Humanos , Ratones , Datos de Secuencia Molecular , Provirus/genética , Recombinasas/metabolismo , Integración Viral/genéticaRESUMEN
Antiviral therapy of primary and recurrent infections with human cytomegalovirus is reserved for severe manifestations and faces several limitations. Presently candidates for novel drugs with lower adverse side effects and a minimized frequency of resistance formation are under investigation. Here we demonstrate that artesunate, an antimalaria drug with highly valuable pharmacological properties, possesses antiviral activity. A concentration-dependent inhibition of the replication of human cytomegaloviruses with wild-type phenotype was demonstrated in several cell lines. Inhibition was quantified using recombinant green fluorescent protein expressing virus variants. The IC50 values were in the same range for ganciclovir-sensitive and ganciclovir-resistant human cytomegalovirus, as calculated with 5.8+/-0.4 microM and 6.9+/-0.2 microM, respectively. This indicated a strong antiviral potential and a lack of cross-resistance. The optimal antiviral concentrations of artesunate were separable from those inducing cytotoxicity. In addition, the replication of viruses from three genera was seen to be artesunate-sensitive to varying degrees. This suggests a mechanism linked to cellular activation pathways. Both the protein levels and the DNA binding activity of the two virus-induced cellular transcription factors Sp1 and NF-kappaB were found to be markedly reduced in the presence of artesunate. We also analyzed the cellular signaling kinase phosphoinositide 3-kinase, required for the activation of factors such as Sp1 and NF-kappaB in infected fibroblasts. The phosphorylation of two downstream effectors of phosphoinositide 3-kinase, Akt and p70S6K, was markedly inhibited in the presence of artesunate. Thus, artesunate possesses attractive antiviral characteristics which are suggestively based on the interference with essential steps in the host cell kinase cascades.
Asunto(s)
Antivirales/farmacología , Artemisininas , Citomegalovirus/efectos de los fármacos , Ganciclovir/farmacología , Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Sesquiterpenos/farmacología , Artesunato , Células Cultivadas , Citomegalovirus/genética , ADN Viral/efectos de los fármacos , ADN Viral/metabolismo , Farmacorresistencia Viral , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes , Pruebas de Sensibilidad Microbiana , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Quinasas S6 Ribosómicas/metabolismo , Simplexvirus/efectos de los fármacos , Factor de Transcripción Sp1/metabolismo , Ensayo de Placa Viral , Replicación Viral/efectos de los fármacosRESUMEN
The major obstacle to cure infections with human immunodeficiency virus (HIV-1) is integrated proviral genomes, which are not eliminated by antiretroviral therapies (ART). Treatment approaches with latency-reversing agents (LRAs) aim at inducing provirus expression to tag latently-infected cells for clearance through viral cytopathic effects or cytotoxic T cell (CTL) responses. However, the currently tested LRAs reveal evident drawbacks as gene expression is globally induced and viral outgrowth is insecure. Here, we present transcription activator-like effector (TALE) proteins as potent tools to activate HIV-1 specifically. The large variety of circulating HIV-1 strains and, accordingly, integrated proviruses was addressed by the programmable DNA-specificity of TALEs. Using customized engineered TALEs, a substantial transcription activation and viral outgrowth was achieved with cells obtained from different HIV-1 patients. Our data suggest that TALEs may be useful tools in future strategies aimed at removing HIV-1 reservoirs.
Asunto(s)
Infecciones por VIH/metabolismo , VIH-1/fisiología , Factores de Transcripción/metabolismo , Activación Viral , Regulación Viral de la Expresión Génica , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , Humanos , Familia de Multigenes , Especificidad de la Especie , Factores de Transcripción/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Latencia del VirusRESUMEN
The cellular DNA repair hRAD51 protein has been shown to restrict HIV-1 integration both in vitro and in vivo. To investigate its regulatory functions, we performed a pharmacological analysis of the retroviral integration modulation by hRAD51. We found that, in vitro, chemical activation of hRAD51 stimulates its integration inhibitory properties, whereas inhibition of hRAD51 decreases the integration restriction, indicating that the modulation of HIV-1 integration depends on the hRAD51 recombinase activity. Cellular analyses demonstrated that cells exhibiting high hRAD51 levels prior to de novo infection are more resistant to integration. On the other hand, when hRAD51 was activated during integration, cells were more permissive. Altogether, these data establish the functional link between hRAD51 activity and HIV-1 integration. Our results highlight the multiple and opposite effects of the recombinase during integration and provide new insights into the cellular regulation of HIV-1 replication.