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SummarySeveral 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.

The function of dendritic cells (DCs) in the immune system is based on their ability to sense and present foreign antigens. Powerful tools to research DC function and to apply in cell-based immunotherapy are either silencing or overexpression of genes achieved by lentiviral transduction. To date, efficient lentiviral transduction of DCs or their monocyte derived counterparts (MDDCs) required high multiplicity of infection (MOI) or the exposure to the HIV-2/SIV protein Vpx to degrade viral restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1). Here we present a Vpx-independent method for efficient (>95%) transduction of MDDCs at lower MOI. The protocol can be used both for ectopic gene expression and knock-down. Introducing shRNA targeting viral entry receptor CD4 and restriction factor SAMHD1 into MDDCs resulted in down-regulation of targeted proteins and, consequently, expected impact on HIV infection. This protocol for MDDCs transduction is robust and free of the potential risk arising from the use of Vpx which creates a virus infection-prone environment, potentially dangerous in clinical setting.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12977-014-0070-3) contains supplementary material, which is available to authorized users.

Chronic lymphocytic leukemia (CLL) is a disease with variable clinical outcome. Several prognostic factors such as the immunoglobulin heavy chain variable genes (IGHV) mutation status are linked to the B-cell receptor (BCR) complex, supporting a role for triggering the BCR in vivo in the pathogenesis. The miRNA profile upon stimulation and correlation with IGHV mutation status is however unknown. To evaluate the transcriptional response of peripheral blood CLL cells upon BCR stimulation in vitro, miRNA and mRNA expression was measured using hybridization arrays and qPCR. We found both IGHV mutated and unmutated CLL cells to respond with increased expression of MYC and other genes associated with BCR activation, and a phenotype of cell cycle progression. Genome-wide expression studies showed hsa-miR-132-3p/hsa-miR-212 miRNA cluster induction associated with a set of downregulated genes, enriched for genes modulated by BCR activation and amplified by Myc. We conclude that BCR triggering of CLL cells induces a transcriptional response of genes associated with BCR activation, enhanced cell cycle entry and progression and suggest that part of the transcriptional profiles linked to IGHV mutation status observed in isolated peripheral blood are not cell intrinsic but rather secondary to in vivo BCR stimulation.

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.

Background The Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF) region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region. Results The dysfunctional HIV group O8 nef allele was restored to the consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2) and non-canonical (B2 and C1422) HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF→AAA) and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef’s association with the cellular p21-activated kinase 2 (PAK2), the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN) were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck. Conclusion Based on these findings, we propose that this highly conserved three amino acid VGF motif together with the acidic cluster and the proline-rich motif form a previously unrecognized amphipathic surface on Nef. This surface appears to be essential for the majority of Nef functions and thus represents a prime target for the pharmacological inhibition of Nef.

Infection by Human Immunodeficiency Virus is difficult to treat thanks to its persistent viral reservoir and to its high rate of mutation that allows appearance of resistance to the available treatment. An approach to discover drugable targets is the identification of cellular partner proteins interacting with the virus during its life cycle. We used RNAi technology to identify new HIV partners in the host cytoskeleton since it has been shown that cytoskeleton components and the irregulators have a role during several steps of HIV life cycle. By transducingseveral Tcell lines such as Jurkat CD4 CCR5, Jurkat E6-1 and SupT1 with lentiviral vectors expressings hRNA sequences, we silenced different target genes, members of pathways involved inactinrearrangement. By infection with HIV-NL4.3-eGFP reporter virus we evaluated HIV replication ratesin transducedcells. Surprisingly, the infection rate affected by the specific knock-down was dependent on the cell line used. lndeed, ashRNAtransduced in one cell line affected infection differently to what it did in another. Moreover, we observed that transduction on itself with a control vector expressinga scrambled shRNA sequence affected HIV infection rate in some but not all cell lines. Therefore, to obtain relevant results in screening co-factors for HIV infection, we turned toprimary cells, the naturaltargets of the virus in vivo. We optimized combinedlentiviral transduction and HIV infection in cultured peripheral blood CD4+ lymphocytes. In this setting, transduction with scrambled shRNA expressing lentivirus did not affect HIV replication, providing us a platform to assay gene-knockdown likely to generate the most relevant information for natural HIV infection invivo.

Down-modulation of the CD4 receptor expressed on T cells is one of the hallmarks of Human Immunodeficiency Virus (HIV) infection and it is believed to confer to the virus a selective replicative advantage in vivo. HIV has evolved redundant mechanisms to remove the receptor from the cell surface and accelerate its degradation. This process is mainly mediated by three viral proteins: Vpu, Env and Nef. Up to date, the mechanisms that lead to CD4 depletion from the surface of CD4+ T lymphocytes, the natural targets of HIV, are still poorly understood and only partially characterized. We are interested in the discovery of pathways and human proteins involved in the process, in order to eventually find potential new drug targets. To pursue our aim, we first performed a functional screening on HeLa CD4+ cells expressing Nef, using a shRNA lentiviral interference delivery system targeting the whole human genome compatible with an Affymetrix GeneChip Microarray. The read out was the rescue of the CD4 high phenotype, despite Nef expression. The results were analyzed with Affymetrix Expression Console 1.1 software and the web-based gene ontology resource DAVID. After four different screens we obtained a final list of 75 genes enriched in the cells sorted for high surface CD4. These genes appear to code for proteins involved in endocytic trafficking, trans-Golgi trafficking and lysosomal degradation pathways. We decided to validate our hits in primary blood CD4+ T cells, the most relevant for this kind of studies, and we set up a protocol for the transduction of these cells with lentiviral and retroviral vectors, necessary respectively for the expression of the shRN As and the viral proteins Vpu, Nef and Env. The actual down-regulation of the gene expression will be validated via quantitative Real Time PCR and Western Blot and the experiments will be eventually repeated in the context of HIV-1 infection.

Background: Production of type 1 interferon (IFN) in response to viral infection requires the detection of viral nucleic acids or proteins by at least one cellular pattern recognition receptor. HIV-1 is capable of inducing an elaborate IFN response in plasmacytoid dendritic cells (pDCs) through Toll-like receptor mediated recognition of the entering viral RNA genome. However in T cells and macrophages, the main HIV target cells, IFN induction by HIV-1 is considered to be weak or undetectable. Here, we re-evaluate the occurrence of an innate immune response upon HIV-1 infection of primary CD4+ T cells (PBLs). Methods and results: Type 1 IFN induction was evaluated in activated purified PBLs during productive infection with HIV-1. In cells from several donors we observed a clear increase in IFNβ and IFNα mRNA levels, as well as induction of several interferon stimulated genes (ISGs) upon HIV-1 infection. The levels of induction progressed concurrently with the levels of HIV infection in the culture. Secreted type 1 IFN protein biological activity was furthermore detected in supernatants of HIV-1 infected cultures. To rule out residual contaminating pDCs as the source of IFN production in these cultures, we performed additional depletion of these cells from CD4+ T cell populations prior to infection and found that type 1 IFN induction was maintained in the absence of pDCs. Furthermore, we evaluated the biological relevance of the detected levels of type 1 IFN by addition of neutralizing antibodies to IFNβ and IFNα during infection. In presence of the both antibodies, we observed an increase in HIV-1 infection, indicating that the levels of HIV-induced IFN in the T cell cultures are sufficient to have an antiviral effect. To gain more insight in the mechanism of IFN induction by HIV-1, we used inhibitors of reverse transcription, integration and of the HIV-1 protease during single-cycle infection of VSV-pseudotyped HIV-1. We found that integration of the HIV provirus is required for full IFN induction in PBLs, indicating that newly expressed HIV RNA or newly produced HIV proteins are important for evoking an innate immune response in T cells. Conclusion: These data show that activated PBLs are capable of producing relevant levels of type 1 IFN in response to HIV-1 infection and suggest recognition of newly expressed HIV RNA or proteins as a main trigger of the innate response in these cells. Characterization of the responsible innate immune pathway and pattern recognition receptors will be subject of further investigation.