RESUMO
The central memory T cell (TCM) model forms a unique HIV-1 latency model based on primary cells that closely resemble in vivo TCM. The virus employed in this model is based on an engineered vector incapable of replication after initial infection. We show that despite this strategy, replication competent viral particles are released into the culture medium due to recombination between overlapping sequences of the env deleted HIV genome that is co-transfected with intact env. This finding emphasizes the need for careful data analysis and interpretation if similar constructs are employed and urges for additional caution during laboratory work.
Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Latência Viral , Replicação Viral/genética , Linfócitos T CD4-Positivos/virologia , DNA Viral/genética , Genes env , Vetores Genéticos/genética , HIV-1/genética , HumanosRESUMO
BACKGROUND: A nef gene is present in all primate lentiviral genomes and is important for high viral loads and progression to AIDS in human or experimental macaque hosts of HIV or SIV, respectively. In these hosts, infection of the thymus results in a decreased output of naive T cells that may contribute to the development of immunodeficiency. We have previously shown that HIV-1 subtype B Nef proteins can block human T-cell development. However, the underlying mechanism(s) and the conservation of this Nef function between different groups of HIV and SIV remained to be determined. RESULTS: We investigated whether reduction of thymic output is a conserved function of highly divergent lentiviral Nef proteins including those from both types of human immunodeficiency viruses (HIV-1 and HIV-2), their direct simian counterparts (SIVcpz, SIVgor and SIVsmm, respectively), and some additional SIV strains. We found that expression of most of these nef alleles in thymocyte progenitors impaired T-cell development and reduced thymic output. For HIV-1 Nef, binding to active p21 protein (Cdc42/Rac)-activated kinase (PAK2) was a major determinant of this function. In contrast, selective disruption of PAK2 binding did not eliminate the effect on T-cell development of SIVmac239 Nef, as was shown by expressing mutants in a newly discovered PAK2 activating structural motif (PASM) constituted by residues I117, H121, T218 and Y221, as well as previously described mutants. Rather, down-modulation of cell surface CD3 was sufficient for reduced thymic output by SIVmac Nef, while other functions of SIV Nefs contributed. CONCLUSIONS: Our results indicate that primate lentiviral Nef proteins impair development of thymocyte precursors into T cells in multiple ways. The interaction of HIV-1 Nef with active PAK2 by HIV-1 seem to be most detrimental, and downregulation of CD3 by HIV-2 and most SIV Nef proteins sufficient for reduced thymic output. Since the reduction of thymic output by Nef is a conserved property of divergent lentiviruses, it is likely to be relevant for peripheral T-cell depletion in poorly adapted primate lentiviral infections.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Linfócitos T/fisiologia , Timócitos/fisiologia , Proteínas Virais Reguladoras e Acessórias/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Animais , Camundongos , Camundongos SCID , Técnicas de Cultura de Órgãos , Linfócitos T/efeitos dos fármacos , Timócitos/efeitos dos fármacos , TimoRESUMO
The majority of T cells encountered by HIV-1 are non-activated and do not readily allow productive infection. HIV-1 Vpr is highly abundant in progeny virions, and induces signalling and HIV-1 LTR transcription. We hence hypothesized that Vpr might be a determinant of non-activated T-cell infection. Virion-delivered Vpr activated nuclear factor of activated T cells (NFAT) through Ca(2+) influx and interference with the NFAT export kinase GSK3ß. This leads to NFAT translocation and accumulation within the nucleus and was required for productive infection of unstimulated primary CD4(+) T cells. A mutagenesis approach revealed correlation of Vpr-mediated NFAT activation with its ability to enhance LTR transcription and mediate cell cycle arrest. Upon NFAT inhibition, Vpr did not augment resting T-cell infection, and showed reduced G2/M arrest and LTR transactivation. Altogether, Vpr renders unstimulated T cells more permissive for productive HIV-1 infection and stimulates activation of productively infected as well as virus-exposed T cells. Therefore, it could be involved in the establishment and reactivation of HIV-1 from viral reservoirs and might have an impact on the levels of immune activation, which are determinants of HIV-1 pathogenesis.
Assuntos
Linfócitos T CD4-Positivos/virologia , Repetição Terminal Longa de HIV , Fatores de Transcrição NFATC/metabolismo , Vírion/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Cálcio/metabolismo , Núcleo Celular/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HEK293 , Células HeLa , Humanos , Células Jurkat , Transcrição Gênica , Vírion/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genéticaRESUMO
To facilitate studies on Vpr function in replicating HIV-1, we aimed to tag the protein in an infectious virus. First we showed that N-, but not C-terminal HA/FLAG tagging of Vpr protein preserves Vpr cytopathicity. Cloning the tags into proviral DNA however ablated viral production and replication. By construction of additional viral variants we could show this defect was not protein- but RNA-dependent and sequence specific, and characterized by oversplicing of the genomic RNA. Simulation of genomic RNA folding suggested that introduction of the tag sequence induced an alternative folding structure in a region enriched in splice sites and splicing regulatory sequences. In silico predictions identified the HA/His6-Vpr tagging in HIV-1 to affect mRNA folding less than HA/FLAG-Vpr tagging. In vitro infectivity and mRNA splice pattern improved but did not reach wild-type values. Thus, sequence-specific insertions may interfere with mRNA splicing, possibly due to altered RNA folding. Our results point to the complexity of viral RNA genome sequence interactions. This should be taken into consideration when designing viral manipulation strategies, for both research as for biological interventions.
Assuntos
Processamento Alternativo/fisiologia , Genoma Viral/fisiologia , HIV-1/metabolismo , Dobramento de RNA/fisiologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Linhagem Celular , HIV-1/genética , Humanos , RNA Mensageiro/genética , RNA Viral/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genéticaRESUMO
Several pattern-recognition receptors sense HIV-1 replication products and induce type I interferon (IFN-I) production under specific experimental conditions. However, it is thought that viral sensing and IFN induction are virtually absent in the main target cells of HIV-1 in vivo. Here, we show that activated CD4+ T cells sense HIV-1 infection through the cytosolic DNA sensor cGAS and mount a bioactive IFN-I response. Efficient induction of IFN-I by HIV-1 infection requires proviral integration and is regulated by newly expressed viral accessory proteins: Vpr potentiates, while Vpu suppresses cGAS-dependent IFN-I induction. Furthermore, Vpr also amplifies innate sensing of HIV-1 infection in Vpx-treated dendritic cells. Our results identify cGAS as mediator of an IFN-I response to HIV-1 infection in CD4+ T cells and demonstrate that this response is modulated by the viral accessory proteins Vpr and Vpu. Thus, viral innate immune evasion is incomplete in the main target cells of HIV-1.
Assuntos
Infecções por HIV/imunologia , Proteínas do Vírus da Imunodeficiência Humana/genética , Interferon Tipo I/imunologia , Nucleotidiltransferases/genética , Proteínas Virais Reguladoras e Acessórias/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Células HEK293 , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/patogenicidade , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Humanos , Imunidade Inata/genética , Interferon Tipo I/genética , Lentivirus/genética , Nucleotidiltransferases/imunologia , Proteínas Virais Reguladoras e Acessórias/imunologia , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Quantification of retroviruses in cell culture supernatants and other biological preparations is required in a diverse spectrum of laboratories and applications. Methods based on antigen detection, such as p24 for HIV, or on genome detection are virus specific and sometimes suffer from a limited dynamic range of detection. In contrast, measurement of reverse transcriptase (RT) activity is a generic method which can be adapted for higher sensitivity using real-time PCR quantification (qPCR-based product-enhanced RT (PERT) assay). We present an evaluation of a modified SYBR Green I-based PERT assay (SG-PERT), using commercially available reagents such as MS2 RNA and ready-to-use qPCR mixes. This assay has a dynamic range of 7 logs, a sensitivity of 10 nU HIV-1 RT and outperforms p24 ELISA for HIV titer determination by lower inter-run variation, lower cost and higher linear range. The SG-PERT values correlate with transducing and infectious units in HIV-based viral vector and replication-competent HIV-1 preparations respectively. This assay can furthermore quantify Moloney Murine Leukemia Virus-derived vectors and can be performed on different instruments, such as Roche Lightcycler® 480 and Applied Biosystems ABI 7300. We consider this test to be an accurate, fast and relatively cheap method for retroviral quantification that is easily implemented for use in routine and research laboratories.