RESUMEN
Lentiviruses are suitable to transfer potential therapeutic genes into non-replicating cells such as neurons, but systematic in vivo studies on transduction of neural cells within the complete brain are missing. We analysed the distribution of transduced cells with respect to brain structure, virus tropism, numbers of transduced neurons per brain, and influence of the Vpx or Vpr accessory proteins after injection of vectors based on SIVsmmPBj, HIV-2, and HIV-1 lentiviruses into the right striatum of the mouse brain. Transduced cells were found ipsilaterally around the injection canal, in corpus striatum and along corpus callosum, irrespective of the vector type. All vectors except HIV-2SEW transduced also single cells in the olfactory bulb, hippocampus, and cerebellum. Vector HIV-2SEW was the most neuron specific. However, vectors PBjSEW and HIV-1SEW transduced more neurons per brain (means 41,299 and 32,309) than HIV-2SEW (16,102). In the presence of Vpx/Vpr proteins, HIV-2SEW(Vpx) and HIV-1SEW(Vpr) showed higher overall transduction efficiencies (30,696 and 27,947 neurons per brain) than PBjSEW(Vpx) (6636). The distances of transduced cells from the injection canal did not differ among the viruses but correlated positively with the numbers of transduced neurons. The presence of Vpx/Vpr did not increase the numbers of transduced neurons. Parental virus type and the vector equipment seem to influence cellular tropism and transduction efficiency. Thus, precision of injection and choice of virus pseudotype are not sufficient when targeted lentiviral vector transduction of a defined brain cell population is required.
Asunto(s)
Encéfalo/virología , Vectores Genéticos/farmacocinética , VIH-1/metabolismo , VIH-2/metabolismo , Lentivirus/genética , Virus de la Inmunodeficiencia de los Simios/metabolismo , Transducción Genética/métodos , Tropismo Viral , Animales , Encéfalo/metabolismo , Células Cultivadas , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , VIH-1/genética , VIH-2/genética , Lentivirus/metabolismo , Ratones , Ratones Endogámicos C57BL , Embarazo , Investigación Cualitativa , Virus de la Inmunodeficiencia de los Simios/genéticaRESUMEN
Foamy viruses (FV) are retroviruses that are widely distributed in primate and non-primate animal species. We tested here FV with capsids of simian and non-simian origin for sensitivity to interferon-ß (IFN-ß). Our data show significant inhibition of FV by IFN-ß early in infection of human HOS and THP-1 but not of HEK293T cells. The post-entry restriction of FV was not mediated by the interferon-induced MxB protein that was recently identified as a capsid-interacting restriction factor targeting Human immunodeficiency virus (HIV) before integration. Neither the ectopic expression of MxA or MxB in HEK293T cells nor the lack of MxB expression in CRISPR/CAS MxB THP-1 knockout cells impacted the infection of the tested FV. IFN-ß treated THP-1 and THP-1 KO MxB cells showed the same extend of restriction to FV. Together, the data demonstrate that IFN-ß inhibits FV early in infection and that MxB is not a restriction factor of FV.
Asunto(s)
Interferón beta/metabolismo , Proteínas de Resistencia a Mixovirus/metabolismo , Spumavirus/inmunología , Línea Celular , Humanos , Proteínas de Resistencia a Mixovirus/deficienciaRESUMEN
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
Myeloid blood cells are largely resistant to infection with human immunodeficiency virus type 1 (HIV-1). Recently, it was reported that Vpx from HIV-2/SIVsm facilitates infection of these cells by counteracting the host restriction factor SAMHD1. Here, we independently confirmed that Vpx interacts with SAMHD1 and targets it for ubiquitin-mediated degradation. We found that Vpx-mediated SAMHD1 degradation rendered primary monocytes highly susceptible to HIV-1 infection; Vpx with a T17A mutation, defective for SAMHD1 binding and degradation, did not show this activity. Several single nucleotide polymorphisms in the SAMHD1 gene have been associated with Aicardi-Goutières syndrome (AGS), a very rare and severe autoimmune disease. Primary peripheral blood mononuclear cells (PBMC) from AGS patients homozygous for a nonsense mutation in SAMHD1 (R164X) lacked endogenous SAMHD1 expression and support HIV-1 replication in the absence of exogenous activation. Our results indicate that within PBMC from AGS patients, CD14+ cells were the subpopulation susceptible to HIV-1 infection, whereas cells from healthy donors did not support infection. The monocytic lineage of the infected SAMHD1 -/- cells, in conjunction with mostly undetectable levels of cytokines, chemokines and type I interferon measured prior to infection, indicate that aberrant cellular activation is not the cause for the observed phenotype. Taken together, we propose that SAMHD1 protects primary CD14+ monocytes from HIV-1 infection confirming SAMHD1 as a potent lentiviral restriction factor.
Asunto(s)
Enfermedades Autoinmunes del Sistema Nervioso/genética , Predisposición Genética a la Enfermedad/genética , Infecciones por VIH/genética , Proteínas de Unión al GTP Monoméricas/deficiencia , Proteínas de Unión al GTP Monoméricas/genética , Células Mieloides/virología , Malformaciones del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Enfermedades Autoinmunes del Sistema Nervioso/virología , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , VIH-1/metabolismo , Humanos , Immunoblotting , Inmunoprecipitación , Receptores de Lipopolisacáridos/metabolismo , Microscopía Confocal , Mutación Missense , Células Mieloides/metabolismo , Malformaciones del Sistema Nervioso/metabolismo , Malformaciones del Sistema Nervioso/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 1 que Contiene Dominios SAM y HD , Espectrometría de Masas en Tándem , TransfecciónRESUMEN
BACKGROUND: The non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM). RESULTS: Mutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG) viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP) kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6. CONCLUSIONS: In sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential.
Asunto(s)
Colon/patología , Productos del Gen nef/química , Activación de Linfocitos , Mutación , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Linfocitos T/inmunología , Secuencias de Aminoácidos , Animales , Células Cultivadas , Colon/virología , Productos del Gen nef/genética , Productos del Gen nef/metabolismo , Humanos , Linfopenia/virología , Macaca nemestrina , Enfermedades de los Monos/inmunología , Enfermedades de los Monos/patología , Enfermedades de los Monos/virología , Fenotipo , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/fisiopatología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/metabolismo , Viremia/virología , Replicación ViralRESUMEN
Lentiviral gene transfer vectors are suitable for genetically modifying non-cycling primary human cells. In this study, we analyzed transduced human dendritic cells (DC) generated by the use of three different GFP-encoding lentiviral vectors, HIV-2 ROD A Δenv-GFP (ROD A), SIVsmm PBj ΔE EGFP (PBj), and SIVmac ΔE EGFP (SIVmac). CD14+ monocytes were isolated from buffy coat, transduced, and differentiated to immature and mature DC. Cytofluometric analysis of DC revealed high transduction efficiencies at MOI 1 for simian immunodeficiency virus (SIV)-derived vectors PBj and SIVmac ranging between 80-90 and 70-90%, respectively. In contrast, transduction with ROD A resulted only in approximately 30%-positive DC at the same MOI. Of note, none of the analyzed vectors affected expression of maturation and/or activation markers. Moreover, transduction with PBj or SIVmac did not induce significant cytokine responses whereas ROD A transduction stimulated weak interferon-alpha responses. SIVmac transduced DC showed normal phagocytosis of antigen and normal allo T cell stimulatory capacity when compared with untreated DC. Thus, the SIVmac lentiviral transduction vector is suitable for efficient genetic modification of human DC without affecting phenotype or function and thus qualifies this vector as a versatile tool for use in basic research.
Asunto(s)
Células Dendríticas/metabolismo , Vectores Genéticos/genética , Lentivirus/genética , Células Cultivadas , Citometría de Flujo , Humanos , Fagocitosis/genética , Fagocitosis/fisiología , Linfocitos T/citología , Linfocitos T/metabolismo , Transducción GenéticaRESUMEN
SIVagm does not induce disease in its African green monkey (AGM) host. In comparison, the hybrid simian-human immunodeficiency virus SHIV89.6P that carries the HIV env gene induces disease in rhesus macaques more rapidly than the SIVmac parent virus. To address the possibility that this enhancement of disease by HIV env would also occur when present in SIVagm, a full-length SIVagm/89.6Penv chimeric lentivirus genome (termed SHIV-MP) was constructed. SHIV-MP replicated similarly to SIVagm in simian peripheral blood mononuclear cells (PBMCs). In inoculated AGMs, rhesus macaques and pig-tailed (PT) macaques the absolute number of CD4(+) T lymphocytes remained at normal levels. The peak levels of productively infected cells in SHIV-MP-infected monkeys ranged from 10(1) to 10(2) per 10(6) PBMCs, while in SIVagm infected macaques the levels were 10-100-fold higher. The env gene of SHIV89.6P therefore appears insufficient to confer acute pathogenicity to a non-pathogenic primate lentivirus due to poor in vivo replication.
Asunto(s)
Chlorocebus aethiops/virología , Genes env/fisiología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Animales , Línea Celular , Chlorocebus aethiops/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Genes env/genética , VIH-1/fisiología , Humanos , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/fisiología , Carga Viral , Replicación Viral/genética , Replicación Viral/fisiologíaRESUMEN
BACKGROUND: Human primary monocytes are refractory to infection with the human immunodeficiency virus 1 (HIV-1) or transduction with HIV-1-derived vectors. In contrast, efficient single round transduction of monocytes is mediated by vectors derived from simian immunodeficiency virus of sooty mangabeys (SIVsmmPBj), depending on the presence of the viral accessory protein Vpx. METHODS AND FINDINGS: Here we analyzed whether Vpx of SIVsmmPBj is sufficient for transduction of primary monocytes by HIV-1-derived vectors. To enable incorporation of PBj Vpx into HIV-1 vector particles, a HA-Vpr/Vpx fusion protein was generated. Supplementation of HIV-1 vector particles with this fusion protein was not sufficient to facilitate transduction of human monocytes. However, monocyte transduction with HIV-1-derived vectors was significantly enhanced after delivery of Vpx proteins by virus-like particles (VLPs) derived from SIVsmmPBj. Moreover, pre-incubation with Vpx-containing VLPs restored replication capacity of infectious HIV-1 in human monocytes. In monocytes of non-human primates, single-round transduction with HIV-1 vectors was enabled. CONCLUSION: Vpx enhances transduction of primary human and even non-human monocytes with HIV-1-derived vectors, only if delivered in the background of SIVsmmPBj-derived virus-like particles. Thus, for accurate Vpx function the presence of SIVsmmPBj capsid proteins might be required. Vpx is essential to overcome a block of early infection steps in primary monocytes.
Asunto(s)
VIH-1/genética , VIH-1/metabolismo , Monocitos/virología , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación Viral , Animales , Western Blotting , Cercocebus , Vectores Genéticos , Células HeLa , Humanos , Modelos Genéticos , Monocitos/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The coreceptor usage of HIV-1 envelope proteins (Env) is mainly dependent on a defined variable region within the V3-loop of Env. Thus, retroviral vectors derived from murine leukemia virus (MLV), which have been pseudotyped with HIV-1 envelope proteins holding different V3-loops, enable selective gene delivery into either CXCR4 or CCR5 positive cultured cells. Here, we tested the distribution of CD4/CCR5-tropic [MLV(HIV)]-pseudotype vectors in transgenic mice expressing CD4 and either CXCR4 or CCR5 of human origin. The specificity of gene transfer was analyzed by ex vivo transduction of spleen cells as well as after i.v. or i.p. injection of transgenic mice. Expression of the transferred marker gene EGFP and vector sequences could be detected exclusively in lymphocytes expressing (hu)CD4 and (hu)CCR5, whereas MLV vectors pseudotyped with the VSV-G envelope glycoprotein mediated gene transfer in mice of all genotypes investigated. These data demonstrated that cell-specific gene delivery via [MLV(HIV)]-pseudotyped vectors, as previously shown for cultured cells, is also achievable in vivo.