Hanne Vanderstraeten

Quantification of reverse transcriptase activity by real-time PCR as a fast and accurate method for titration of HIV, lenti- and retroviral vectors

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.

CLL cells respond to B-Cell receptor stimulation with a microRNA/mRNA signature associated with MYC activation and cell cycle progression.

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.

Expression of ZAP70 in chronic lymphocytic leukaemia activates NF‐κB signalling

Chronic lymphocytic leukaemia (CLL) is a disease with a highly variable prognosis. The clinical course can however be predicted thanks to prognostic markers. Poor prognosis is associated with expression of a B cell receptor (BCR) from unmutated immunoglobulin variable heavy‐chain genes (IGHV) and expression of zeta‐associated protein of 70 kDa (ZAP70). The reason why ZAP70 expression is associated with poor prognosis and whether the protein has a direct pathogenic function is at present unknown. By transfer of ZAP70 to CLL cells, we show here that expression of ZAP70 in CLL cells leads to increased expression of the nuclear factor (NF)‐κB target genes interleukin‐1β (IL1B), IL6 and IL8 upon BCR triggering. This could be blocked by inhibition of NF‐κB signalling through inhibition of IκB kinases (IKK). Transcriptome analysis identified a NF‐κB RELA signature imposed by ZAP70 expression in BCR‐stimulated CLL cells. We conclude that ZAP70 acts directly as an amplifier of NF‐κB signalling in CLL cells which could be an underlying mechanism for its association with poor prognosis and which may represent a therapeutic target.

Primate lentiviral Nef proteins deregulate T-cell development by multiple mechanisms

BackgroundA 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.ResultsWe 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.ConclusionsOur 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.

Lipoprotein lipase SNPs rs13702 and rs301 correlate with clinical outcome in chronic lymphocytic leukemia patients

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and is characterized by a heterogeneous clinical course. This variability in clinical course has spiked the search for prognostic markers able to predict patient evolution at the moment of diagnosis. Markers demonstrated to be of value are the mutation status of the immunoglobulin heavy chain variable region genes (IGHV) and lipoprotein lipase (LPL) expression. High LPL mRNA expression has been associated with short treatment free (TFS) and decreased overall survival (OS) in CLL. The LPL SNPs rs301 (T<C), rs328 (C<G) and rs13702 (T<C) have been associated with various metabolic disorders, but the association with CLL evolution is unknown. Here, in a cohort of 248 patients, we show that patients with the LPL SNP rs13702 wild-type T/T genotype had significantly shorter OS than patients with C/C and T/C genotypes (median time until CLL related death: 90 and 156 months respectively, p=0.008). The same was observed for LPL SNP rs301 (median time until CLL related death T/T: 102 and C/C, T/C: 144 months, p=0.03). Both SNPs rs301 and rs13702 were significantly associated with each other and notably, no association was found between IGHV status and presence of the SNP genotypes, indicating that these LPL SNPs are reliable prognostic markers that could add extra prognostic and predictive information to classical markers and help to improve the management of CLL.

The human immunodeficiency virus (HIV) Rev-binding protein (HRB) is a co-factor for HIV-1 Nef-mediated CD4 downregulation

Human immunodeficiency virus type 1 (HIV-1)-mediated CD4 downregulation is an important determinant of viral replication in vivo. Research on cellular co-factors involved in this process could lead to the identification of potential therapeutic targets. We found that CD4 surface levels were significantly higher in HIV-1-infected cells knocked-down for the HIV Rev-binding protein (HRB) compared with control cells. HRB knock-down affected CD4 downregulation induced by Nef but not by HIV-1 Vpu. Interestingly, the knock-down of the related protein HRBL (HRB-like), but not of the HRB interaction partner EPS15 (epidermal growth factor receptor pathway substrate 15), increased CD4 levels in Vpu-expressing cells significantly. Both of these proteins are known to be involved in HIV-1-mediated CD4 downregulation as co-factors of HIV-1 Nef. These results identify HRB as a previously unknown co-factor for HIV-1 Nef-mediated CD4 downregulation and highlight differences with the related protein HRBL, which affects the CD4 downregulation in a dual role as co-factor of both HIV-1 Nef and Vpu.

Vpx-Independent Lentiviral Transduction and shRNA-Mediated Protein Knock-Down in Monocyte-Derived Dendritic Cells

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.

HIV-1 infection induces a type I IFN response in primary CD4+ T cells

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.

New host factors and pathways involved in CD4 downregulation in HIV-1 infected cells

Background: Downregulation of the CD4 receptor is one of the hallmarks of HIV infection. The virus has evolved redundant mechanisms to remove the receptor from the cell surface and accelerate its degradation, mainly mediated by three viral proteins: Vpu, Env and Nef. We were interested in the discovery of pathways and human proteins involved in the process, which eventually could represent new drug targets. Materials and methods: A genome-wide short-hairpin RNA (shRNA) screening using a SBI shRNA lentiviral interference delivery system library compatible with the GeneChip® Human Genome U133 Plus 2.0 Array (Affymetrix) was performed in HeLa CD4+ cells expressing the Nef protein introduced by retroviral transduction. CD4 surface levels were measured by flow cytometry. The read-out in the screen showed the rescue of the CD4-high phenotype despite Nef expression. shRNA sequences enriched in the CD4-high cells compared to the CD4-low cells were identified and filtered via pathway analysis. For the confirmation and further selection of the hits two cell lines in different conditions were used: 1. HeLa CD4+ cells expressing Nef after retroviral transduction (similar to previous screening effort). 2. SupT1 lymphocytic cells infected with replication competent HIV-1 encoding a GFP reporter. 3. SupT1 cells expressing Nef or Vpu after retroviral transduction. In all three experimental set-ups, the cells were selectively knocked-down for each of the hits individually after transduction with a different set of shRNA encoding lentiviral vectors (Mission Consortium, Sigma Aldrich) prior to HIV-1 infection or retroviral transduction. Results: The genome-wide screen with the SBI library was repeated 4 times to obtain a final list of 75 genes as a first selection of possible new host co-factors in CD4 downregulation by Nef. Of these, 22 proteins were confirmed independently with individual Mission consortium vectors in the same cell line. Eight proteins contributed to CD4 downregulation in HIV-1 infected SupT1 cells. The host factors identified show differential effect on CD4 surface levels in SupT1 cells expressing either HIV-1 Vpu or Nef proteins individually, that together determine CD4 levels on infected cells. These proteins are mainly involved in endosomal and trans Golgi network (TGN) trafficking. Conclusion: Several host proteins involved in endosomal and TGN trafficking differentially affect Nef or Vpu mediated CD4 down-regulation in HIV1-1 infected cells.

Efficient, Vpx independent shRNA transduction of monocyte derived dendritic cells as a tool for studying HIV transmission to primary T-cells

Background: Dendritic cells present at mucosal sites are considered initial targets for HIV transmission. Due to SAMHD1 expression, they are highly resistant to HIV infection themselves, but act as shuttles for viral transmission to T cells. Knock-down of HIV host cellular partners is a powerful method in HIV research. At present, lentiviral shRNA delivery to Monocyte Derived Dendritic Cells (MDDC) requires the use of virus-like particles carrying the Vpx protein in order to alleviate SAMHD1 mediated restriction. Such an approach results in the desired high transduction efficiency, but simultaneously abrogates resistance to HIV thus allowing for viral replication which might obscure the actual outcomes of MDDC-HIV interactions. We present a method for efficient shRNA transduction of MDDC for subsequent studies of HIV transmission to CD4+ T-cells, without the need for Vpx. Methods: MDDC were obtained by culturing primary monocytes for 6 days in presence of lL-4 and GM-CSF. At day 1 after monocyte isolation, cells were transduced with shRNA encoding lentiviral vectors (Sigma MISSION®) at MOI of 1 (titration on HEK293T cell line) by addition of polybrene and using spinoculation. Transduction efficiency was determined by measuring the fraction of eGFP expression (encoded by the lentiviral vector) by flow cytometry. At day 5 post transduction, cells were exposed to NL4.3 HIV virus harboring a marker gene. Twenty four hours later the cells were extensively washed with culture medium to remove unbound virus and autologous, primary CD4+ T cells were added at 1:2 ratio. Infection of T-cells in MDDC-T-cell cocultures was subsequently monitored by flow cytometry to detect marker gene expressing cells in low scatter and/or CD3+ population. Results: Efficient transduction of MDDC (≈95%) as judged by eGFP expression was obtained. This high efficiency avoids puromycin selection and therefore simplifies the protocol while preventing possible side effects. Notably, the transduced MDDC did not show a marked increase in CD80, CD83 and CD86 expression levels. Transduced MDDC are able to capture and transmit the virus to T cells. We have observed a positive correlation between shRNA mediated downregulation of certain MDDC surface markers and transmission of HIV to CD4+ T cells as compared to cells transduced with a control vector encoding a scrambled shRNA sequence not known to target any human gene. Conclusions: We have established a method for lentiviral encoded shRNA transductions allowing for studies of MDDC cellular factors involved in HIV transmission to CD4+ T cells without the need to alleviate SAMHD1 induced restriction. Transduced MDDC remain immature as judged by expression of CD80, CD83 and CD86 cell surface receptors. Preliminary results provide validation of the approach by positively correlating downregulation of MDDC surface markers with trans-infection of CD4+ T cells.