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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.

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.

Background: The B-cell receptor (BCR) has a key role in the cross-talk between chronic lymphocytic leukaemia (CLL) cells and the tissue microenvironment, which favours disease progression by promoting proliferation and drug resistance. In vitro studies on downstream signalling and functional effects of CLL BCR ligation often report contradictory results, in part owing to the lack of a standardised stimulation protocol. Our aim was to define a biologically relevant and robust in vitro stimulation method with regard to cellular phenotypic and transcriptional responses. Methods: We evaluated mRNA (FOS, MYC, LPL) and protein (CD54, CD19, CD62L, CD184) expression of genes modulated by BCR triggering in immunoglobulin heavy-chain variable region genes (IGHV)-mutated and -unmutated CLL cells, after stimulation using soluble or immobilised anti-IgM antibodies from different suppliers. Results: The effect of BCR stimulation on gene and protein expression was comparable in all CLL patients, irrespective of IGHV mutation status. However, immobilised anti-IgM stimulation elicited clear and robust changes in gene and protein expression, whereas the response to soluble anti-IgM was far less obvious. Conclusions: These data indicate that the method of BCR stimulation is of major importance regarding responsiveness of CLL cells in the context of the tumour microenvironment, whereas genetic differences in the BCR pathway are less critical.

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.

Chronic lymphocytic leukemia (CLL) consists of at least two major prognostic subgroups, characterized by different cellular and molecular markers. This observation sparked studies on the function and clinical importance of these markers. In order to address their function adequately, an efficient and reliable method for gene transfer is needed. In this study, we compared efficiency and utility of different gene transfer techniques in CLL. Lenti-, retro- and adenoviral transduction did not yield appreciable numbers of marker gene enhanced green fluorescent protein (EGFP) positive CLL cells, despite various prestimulation protocols. Efficient transgene expression was observed after nucleofection of CLL cells with plasmid DNA, at the expense of low survival rates. After optimization, electroporation of in vitro transcribed mRNA yielded up to 90% EGFP+CLL cells without affecting survival. Transgene expression remained detectable for at least 2 weeks after electroporation. Furthermore, we could demonstrate overexpression of ZAP70 and of a ZAP70-EGFP fusion protein after electroporation with ZAP70 or ZAP70-EGFP mRNA. We conclude that mRNA electroporation is a novel and straightforward method for highly efficient gene transfer in CLL. The application of this technique should facilitate functional studies on CLL cells, as well as clinical research.

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 HIV-1 gene nef is important for progression toward AIDS and cellular depletion of the infected thymus. Expression of the Nef protein alone impairs human thymopoiesis. Here, we performed a structure-function analysis of the Nef protein by comparing the effect on T-cell development of different nef alleles, either wild type or defective for selected functions, expressed by human thymocytes. We show that Nef-mediated impaired thymopoiesis is not due to altered surface marker trafficking, nor dependent on oligomerization of Nef. By contrast, mutations in the myristoylation site and in signaling sites of Nef, ie, sites important for interaction with phosphofurin acidic cluster sorting protein-1 (PACS-1), Src homology domain 3 (SH3) domains, and p21-activated kinase 2 (PAK2), were found to be critical for its effect on T-cell development. These results point to sites in Nef to target therapeutically for restoration of thymopoiesis in HIV infection.

Background Down-modulation of the CD4 receptor is one of the hallmarks of HIV-1 infection and it is believed to confer a selective replicative advantage to the virus in vivo. This process is mainly mediated by three viral proteins: Env, Vpu and Nef. To date, the mechanisms that lead to CD4 depletion from the surface of infected cells during HIV-1 infection are still only partially characterized. In this study, we sought to identify and characterize cellular host factors in HIV-1-induced CD4 down-modulation. Results To identify host factors involved in CD4 down-regulation, we used a whole genome-targeting shRNA lentiviral library in HeLa CD4+ cells expressing Nef as an inducer of CD4 down-modulation. We identified 55 genes, mainly encoding for proteins involved in various steps of clathrin-mediated endocytosis. For confirmation and further selection of the hits we performed several rounds of validation, using individual shRNA lentiviral vectors with a different target sequence for gene knock-down in HIV-1-infected T cells. By this stringent validation set-up, we could demonstrate that the knock-down of DNM3 (dynamin 3), SNX22 (sorting nexin 22), ATP6AP1 (ATPase, H+ Transporting, Lysosomal Accessory Protein 1), HRBL (HIV-Rev binding protein Like), IDH3G (Isocitrate dehydrogenase), HSP90B1 (Heat shock protein 90 kDa beta member 1) and EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15) significantly increases CD4 levels in HIV-infected SupT1 T cells compared to the non-targeting shRNA control. Moreover, EPS15, DNM3, IDH3G and ATP6AP1 knock-down significantly decreases HIV-1 replication in T cells. Conclusions We identified seven genes as cellular co-factors for HIV-1-mediated CD4 down-regulation in T cells. The knock-down of four out of seven of these genes also significantly reduces HIV-1 replication in T cells. Next to a role in HIV-mediated CD4 down-regulation, these genes might however affect HIV-1 replication in another way. Our findings give insights in the HIV-1-mediated CD4 down-regulation at the level of the plasma membrane and early endosomes and identify four possible new HIV-1 replication co-factors. Electronic supplementary material The online version of this article (doi:10.1186/s12977-014-0118-4) contains supplementary material, which is available to authorized users.

Success of gene therapy for diseases affecting the T cell lineage depends on the thymic repopulation by genetically engineered hematopoietic progenitor cells (HPC). Although it has been shown that retrovirally transduced HPC can repopulate the thymus, little information is available on the effect of the culture protocol. Moreover, for expansion of the number of HPC, cytokine supplemented culture is needed. Here, we transduced purified human umbilical cord blood (CB) CD34+ cells in cultures supplemented with various combinations of the cytokines thrombopoietin (TPO), stem cell factor (SCF), flt3/flk-2 ligand (FL), interleukin-3 (IL-3) and IL-6, and investigated thymus-repopulating ability of gene-marked HPC in vitro. Irrespective of the cytokine cocktail used, transduced CD34+CD38- CB cells, expressing the marker green fluorescent protein (GFP) encoded by the MFG-GFP retrovirus, have both superior proliferative and thymus-repopulating potential compared with transduced CD34+CD38+ CB cells. Effectively transduced GFP+CD34+CD38- HPC, cultured for 3 or 17 days, more readily generated T cells than GFP- HPC from the same culture. The reverse was true in the case of CD34+CD38+ HPC cultures. Finally, our results indicate that the number of GFP+ T cell progenitors actually increased during culture of CD34+CD38- HPC, in a magnitude that is determined by the cytokine cocktail used during culture.

Highly purified human CD34+ hemopoietic precursor cells differentiate into mature T cells when seeded in vitro in isolated fetal thymic lobes of SCID mice followed by fetal thymus organ culture (FTOC). Here, this chimeric human-mouse FTOC was used to address the role of IL-9 and of the α-chain of the IL-9 receptor (IL-9Rα) in early human T cell development. We report that addition of the mAb AH9R7, which recognizes and blocks selectively the human high affinity α-chain of the IL-9R, results in a profound reduction of the number of human thymocytes. Analysis of lymphoid subpopulations indicates that a highly reduced number of cells undergo maturation from CD34+ precursor cells toward CD4+CD3−CD8−CD1+ progenitor cells and subsequently toward CD4+CD8+ double positive (DP) thymocytes. Addition of IL-9 to the FTOC resulted in an increase in cell number, without disturbing the frequencies of the different subsets. These data suggest that IL-9Rα signaling is critical in early T lymphoid development.

Thymic repopulation by transplanted hematopoietic progenitor cells (HPC) is likely to be important for long-term immune reconstitution and for successful gene therapy of diseases affecting the T-cell lineage. However, the T-cell progenitor potential of HPC, cultured in vitro for cell number expansion and gene transfer remains largely unknown. Here, we cultured highly purified human umbilical cord blood (CB) CD34+CD38− or CD34+CD38+ cells for up to 5 weeks in stroma-free cultures supplemented with various combinations of the cytokines thrombopoietin (TPO), stem cell factor (SCF), flt3/flk-2 ligand (FL), interleukin-3 (IL-3), and IL-6 and investigated thymus-repopulating ability of expanded cells in vitro and in vivo. After up to 5 weeks of culture in IL-3 + SCF + IL-6 or TPO + FL + SCF supplemented medium, the progeny of CD34+CD38− CB cells generated T cells and natural killer cells in the thymus. Limiting dilution experiments demonstrated increase in the number of T-cell progenitors during culture. After 3 weeks of culture, gene marked CD34+CD38− CB cells injected in the human thymus fragment transplanted in severe combined immunodeficient (SCID) mice (SCID-hu) generated thymocytes expressing the retroviral encoded marker gene GFP in vivo. Thus, our results show that the progeny of CD34+CD38− CB cells cultured for extensive periods, harbor thymus-repopulating cells that retain T-cell progenitor potential after expansion and gene transfer.

Human umbilical cord blood (UCB) hematopoietic stem cells (HSC) receive increased attention as a possible target for gene-transfer in gene therapy trials. Diseases affecting the lymphoid lineage, as adenosine deaminase (ADA) deficiency and acquired immunodeficiency syndrome (AIDS) could be cured by gene therapy. However, the T-cell progenitor potential of these HSC after gene-transfer is largely unknown and was up to now not testable in vitro. We show here that highly purified CD34++ Lineage marker-negative (CD34++Lin−) UCB cells generate T, natural killer (NK), and dendritic cells in a severe combined immunodeficient mouse fetal thymus organ culture (FTOC). CD34++Lin− and CD34++CD38−Lin− UCB cells express the retroviral encoded marker gene Green Fluorescent Protein (GFP) after in vitro transduction with MFG-GFP retroviral supernatant. Transduced cells were still capable of generating T, NK, and dendritic cells in the FTOC, all expressing high levels of GFP under control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat promotor. We thus present an in vitro assay for thymic T-cell development out of transduced UCB HSC, using GFP as a marker gene.

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.

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.

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.

Summary 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.

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.

Background HIV infection of the thymus results in a decreased output of naive T cells. We previously showed that expression of the Nef protein alone is sufficient to disturb human thymopoiesis. Additional structure-function studies with mutant Nef alleles suggested a role of PAK2 in this process. In this study we evaluated the effect of Nef alleles from different clinical HIV-1, HIV-2 and SIV isolates and Nef mutants which were specifically mutated in the PAK2 interaction surface for their effect on T cell development.

Background Rho GTPases are involved in the regulation of many cell functions, including some related to the actin cytoskeleton. Different Rho GTPases have been shown to be important for T-cell development in mice. However, their role in human T-cell development has not yet been explored.Design and Methods We examined the expression and activation of Rho GTPases along different stages of T-cell development in the human thymus. Early stage human thymocytes were transduced with constitutively active and dominant negative mutants of different Rho GTPases to explore their role in human T-cell development, as analyzed in fetal thymus organ cultures. The use of these mutants as well as Rho GTPase-specific inhibitors allowed us to explore the role of GTPases in thymocyte migration.Results We found that the expression of several Rho GTPases is differently regulated during successive stages of T-cell development in man, suggesting a specific role in human thymopoiesis. In chimeric fetal thymus organ culture, T-cell development was not or only mildly affected by expression of dominant negative Rac1 and Rac2, but was severely impaired in the presence of dominant negative Cdc42, associated with enhanced apoptosis and reduced proliferation. Kinetic analysis revealed that Cdc42 is necessary in human T-cell development both before and after expression of the pre-T-cell receptor. Using inhibitors and retrovirally transferred mutants of the aforementioned Rho GTPases, we showed that only Rac1 is necessary for migration of different thymocyte subsets, including the early CD34+ fraction, towards stromal cell-derived factor-1α. Constitutively active mutants of Rac1, Rac2 and Cdc42 all impaired migration towards stromal cell-derived factor-1α and T-cell development to different degrees.Conclusions This is the first report on Rho GTPases in human T-cell development, showing the essential role of Cdc42. Our data suggest that enhanced apoptotic death and reduced proliferation rather than disturbed migration explains the decreased thymopoiesis induced by dominant negative Cdc42.

RNA interference (RNAi), mediated by short double-stranded RNAs, is a powerful mechanism for posttranscriptional gene silencing. Sustained expression of short hairpin RNA (shRNA) can be accomplished in mammalian cells by viral delivery systems. Using lentiviral constructs, stable gene silencing is established both in dividing and non-dividing cells. Targeting one single gene can lead to the development of escape mutants or may be insufficient to silence redundant pathways. Therefore, simultaneous targeting of multiple genes may be necessary. We have generated a lentiviral vector-based system for expression of multiple shRNAs from a single viral vector, which also encodes an EGFP reporter protein. We show that knock-down of each single gene from multiple target vectors is achieved at an efficiency comparable to that obtained after transduction using single target viral vectors. In this way, we were able to knock-down several members of the human Rho-family GTPases in T cells. Double and triple knock-down persisted after multiple passages of the cells. The ability to inhibit two or more genes simultaneously from one single expression vector further widens the application spectrum of RNAi, both in functional studies and therapeutic strategies.

Human immunodeficiency virus (HIV) Nef is a membrane-associated protein decreasing surface expression of CD4, CD28, and major histocompatibility complex class I on infected cells. We report that Nef strongly down-modulates surface expression of the β-chain of the CD8αβ receptor by accelerated endocytosis, while CD8 α-chain expression is less affected. By mutational analysis of the cytoplasmic tail of the CD8 β-chain, an FMK amino acid motif was shown to be critical for Nef-induced endocytosis. Although independent of CD4, endocytosis of the CD8 β-chain was abrogated by the same mutations in Nef that affect CD4 down-regulation, suggesting common molecular interactions. The ability to down-regulate the human CD8 β-chain was conserved in HIV-1, HIV-2, and simian immunodeficiency virus SIVmac239 Nef and required an intact AP-2 complex. The Nef-mediated internalization of receptors, such as CD4, major histocompatibility complex class I, CD28, and CD8αβ, may contribute to the subversion of the host immune system and progression towards AIDS.

Human immunodeficiency virus (HIV)-infected individuals develop an acquired immune deficiency syndrome (AIDS) due to loss in their lymphocyte numbers and cellular defects in T cells and antigen-presenting cells (APC). HIV infection of the thymus results in deficient replenishment of the peripheral naive T-cell pool. The HIVnef gene was shown to be important for progression towards AIDS and cellular depletion of the infected thymus. Here, we demonstrate by retroviral gene transfer that nef expression, in the absence of other HIV genes, impaired human thymic T-cell development. Thymocytes were generated in reduced numbers and downmodulated CD4 and CD8β cell surface expression. T cells grown from nef-expressing thymocytes were hyperproliferative in vitro upon T-cell receptor triggering. Mature dendritic cells (DC) were functional and had normal surface CD4 levels despite nef expression. Thus, nefexpression alone may contribute to AIDS development by reduced T-cell generation and T-cell hyperresponsiveness.

Background The HIV-1 Vpr protein is a 14 kDa accessory protein, required for efficient replication in macrophages. Vpr is incorporated into HIV virions, believed to participate in the docking of the HIV-1 pre-integration complex to the nucleus and to facilitate it’s transport through the nuclear pore. By inducing G2 arrest, Vpr favors transcription from the HIV-1 LTR, which it also transactivates. We noticed two N-terminal amino acids of the HIV-1, SIVmac and SIVcpz Vpr proteins are fully conserved. This N-terminal motif is predicted to be a NatB substrate motif (i.e., Met-Glu-), expected to lead to full Nt-acetylation of the protein, but it also allows the conservation of the Kozak consensus sequence (A/GCCAUGG), critical for efficient protein translation. Nt-acetylation is one of the most common protein modifications in eukaryotes and is believed to affect protein stability, degradation and function.

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.

Table of contents Oral presentations Session 1: Entry & uncoating O1 Host cell polo-like kinases (PLKs) promote early prototype foamy virus (PFV) replication Irena Zurnic, Sylvia Hütter, Ute Lehmann, Nicole Stanke, Juliane Reh, Tobias Kern, Fabian Lindel, Gesche Gerresheim, Martin Hamann, Erik Müllers, Paul Lesbats, Peter Cherepanov, Erik Serrao, Alan Engelman, Dirk Lindemann O2 A novel entry/uncoating assay reveals the presence of at least two species of viral capsids during synchronized HIV-1 infection Claire Da Silva Santos, Kevin Tartour, Andrea Cimarelli O3 Dynamics of nuclear envelope association and nuclear import of HIV-1 complexes Rya Burdick, Jianbo Chen, Jaya Sastri, Wei-Shau Hu, Vinay Pathak O4 Human papillomavirus protein E4 potently enhances the susceptibility to HIV infection Oliver T. Keppler Session 2: Reverse transcription & integration O5 Structure and function of HIV-1 integrase post translational modifications Karine Pradeau, Sylvia Eiler, Nicolas Levy, Sarah Lennon, Sarah Cianferani, Stéphane Emiliani, Marc Ruff O6 Regulation of retroviral integration by RNA polymerase II associated factors and chromatin structure Vincent Parissi Session 3: Transcription and latency O7 A novel single-cell analysis pipeline to identify specific biomarkers of HIV permissiveness Sylvie Rato, Antonio Rausell, Miguel Munoz, Amalio Telenti, Angela Ciuffi O8 A capsid-dependent integration program linking T cell activation to HIV-1 gene expression Alexander Zhyvoloup, Anat Melamed, Ian Anderson, Delphine Planas, Janos Kriston-Vizi, Robin Ketteler, Chen-Hsuin Lee, Andy Merritt, Petronela Ancuta, Charles Bangham, Ariberto Fassati O9 Characterisation of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome Anthony Rodari, Benoit Van Driessche, Mathilde Galais, Nadége Delacourt, Sylvain Fauquenoy, Caroline Vanhulle, Anna Kula, Arsène Burny, Olivier Rohr, Carine Van Lint O10 Tissue-specific dendritic cells differentially modulate latent HIV-1 reservoirs Thijs van Montfort, Renee van der Sluis, Dave Speijer, Ben Berkhout Session 4: RNA trafficking & packaging O11 A novel cis-acting element affecting HIV replication Bo Meng, Andrzej Rutkowski, Neil Berry, Lars Dölken, Andrew Lever O12 Tolerance of HIV’s late gene expression towards stepwise codon adaptation Thomas Schuster, Benedikt Asbach, Ralf Wagner Session 5: Assembly & release O13 Importance of the tax-inducible actin-bundling protein fascin for transmission of human T cell leukemia virus Type 1 (HTLV-1) Christine Gross, Veit Wiesmann, Martina Kalmer, Thomas Wittenberg, Jan Gettemans, Andrea K. Thoma-Kress O14 Lentiviral nef proteins antagonize TIM-mediated inhibition of viral release Minghua Li, Eric O. Freed, Shan-Lu Liu Session 6: Pathogenesis & evolution O15 SEVI and semen prolong the half-life of HIV-1 Janis Müller, Jan Münch O16 CD169+ macrophages mediate retrovirus trans-infection of permissive lymphocytes to establish infection in vivo Xaver Sewald, Pradeep Uchil, Mark Ladinsky, Jagadish Beloor, Ruoxi Pi, Christin Herrmann, Nasim Motamedi, Thomas Murooka, Michael Brehm, Dale Greiner, Thorsten Mempel, Pamela Bjorkman, Priti Kumar, Walther Mothes O17 Efficient replication of a vpu containing SIVagm construct in African Green Monkeys requires an HIV-1 nef gene Simone Joas, Erica Parrish, Clement Wesley Gnanadurai, Edina Lump, Christina M. Stürzel, Nicholas F. Parrish, Ulrike Sauermann, Katharina Töpfer, Tina Schultheiss, Steven Bosinger, Guido Silvestri, Cristian Apetrei, Nicholas Huot, Michaela Müller-Trutwin, Daniel Sauter, Beatrice H. Hahn, Christiane Stahl-Hennig, Frank Kirchhoff O18 Reprogramming initiates mobilization of endogenous mutagenic LINE-1, Alu and SVA retrotransposons in human induced pluripotent stem cells with consequences for host gene expression Gerald Schumann, Sabine Jung-Klawitter, Nina V. Fuchs, Kyle R. Upton, Martin Muñoz-Lopez, Ruchi Shukla, Jichang Wang, Marta Garcia-Canadas, Cesar Lopez-Ruiz, Daniel J. Gerhardt, Attila Sebe, Ivana Grabundzija, Patricia Gerdes, Sylvia Merkert, Andres Pulgarin, Anja Bock, Ulrike Held, Anett Witthuhn, Alexandra Haase, Ernst J. Wolvetang, Ulrich Martin, Zoltán Ivics, Zsuzsanna Izsvák, J. Garcia-Perez, Geoffrey J. Faulkner O19 NF-κB activation induces expression of human endogenous retrovirus and particle production Tara Hurst, Aris Katzourakis, Gkikas Magiorkinis Session 7a and b: Innate sensing & intrinsic immunity O20 Identification of the phosphatase acting on T592 in SAMHD1 during M/G1 transition Kerstin Schott, Rita Derua, Janna Seifried, Andreas Reuter, Heike Schmitz, Christiane Tondera, Alberto Brandariz-Nuñez, Felipe Diaz-Griffero, Veerle Janssens, Renate König O21 Vpx overcomes a SAMHD1-independent block to HIV reverse transcription that is specific to resting CD4 T cells Hanna-Mari Baldauf, Lena Stegmann, Sarah-Marie Schwarz, Maud Trotard, Margarethe Martin, Gina Lenzi, Manja Burggraf, Xiaoyu Pan, Oliver I. Fregoso, Efrem S. Lim, Libin Abraham, Elina Erikson, Laura Nguyen, Ina Ambiel, Frank Rutsch, Renate König, Baek Kim, Michael Emerman, Oliver T. Fackler, Oliver T. Keppler O22 The role of SAMHD1 in antiviral restriction and immune sensing in the mouse Sabine Wittmann, Rayk Behrendt, Bianca Volkmann, Kristin Eissmann, Thomas Gramberg O23 T cells expressing reduced restriction factors are preferentially infected in therapy naïve HIV-1 patients Sebastian Bolduan, Herwig Koppensteiner, Stefanie Regensburg, Ruth Brack-Werner, Rika Draenert, Michael Schindler O24 cGAS-mediated innate immunity spreads through HIV-1 env-induced membrane fusion sites from infected to uninfected primary HIV-1 target cells Aurélie Ducroux, Shuting Xu, Aparna Ponnurangam, Sergej Franz, Angelina Malassa, Ellen Ewald, Christine Goffinet O25 Perturbation of innate RNA and DNA sensing by human T cell leukemia virus type 1 oncoproteins Sin-Yee Fung, Ching-Ping Chan, Chun-Kit Yuen, Kin-Hang Kok, Chin-Ping Chan, Dong-Yan Jin O26 Induction and anti-viral activity of Interferon α subtypes in HIV-1 infection Ulf Dittmer O27 Vpu-mediated counteraction of tetherin is a major determinant of HIV-1 interferon resistance Dorota Kmiec, Shilpa Iyer, Christina Stürzel, Daniel Sauter, Beatrice Hahn, Frank Kirchhoff O28 DNA repair protein Rad18 restricts HIV-1 and LINE-1 life cycle Yasuo Ariumi, Mariko Yasuda-Inoue, Koudai Kawano, Satoshi Tateishi, Priscilla Turelli O29 Natural mutations in IFITM3 allow escape from post-translational regulation and toggle antiviral specificity Alex Compton, Nicolas Roy, Françoise Porrot, Anne Billet, Nicoletta Casartelli, Jacob Yount, Chen Liang, Oliver Schwartz Session 8: Adaptive immunity & immune evasion O30 Observing evolution in HIV-1 infection: phylogenetics and mutant selection windows to infer the influence of the autologous antibody response on the viral quasispecies Carsten Magnus, Lucia Reh, Penny Moore, Therese Uhr, Jacqueline Weber, Lynn Morris, Alexandra Trkola O31 Dose and subtype specific analyses of the anti-HIV effects of IFN-alpha family members Rashel V. Grindberg, Erika Schlaepfer, Gideon Schreiber, Viviana Simon, Roberto F. Speck Session 9: Novel antiviral strategies O32 LEDGIN-mediated inhibition of the integrase-LEDGF/p75 interaction reduces reactivation of residual latent HIV Zeger Debyser, Lenard Vranckx, Jonas Demeulemeester, Suha Saleh, Eric Verdin, Anna Cereseto, Frauke Christ, Rik Gijsbers O33 NKG2D-mediated clearance of reactivated viral reservoirs by natural killer cells O34 Inhibition of HIV reactivation in brain cells by AAV-mediated delivery of CRISPR/Cas9 O35 CRISPR-Cas9 as antiviral: potent HIV-1 inhibition, but rapid virus escape and the subsequent design of escape-proof antiviral strategies Ben Berkhout, Gang Wang, Na Zhao, Atze T. Das Session 10: Recent advances in HIV vaccine development O36 Priming with a potent HIV-1 DNA vaccine frames the quality of T cell and antibody responses prior to a poxvirus and protein boost Benedikt Asbach, Josef Köstler, Beatriz Perdiguero, Mariano Esteban, Bertram L. Jacobs, David C. Montefiori, Celia C. LaBranche, Nicole L. Yates, Georgia D. Tomaras, Guido Ferrari, Kathryn E. Foulds, Mario Roederer, Gary Landucci, Donald N. Forthal, Michael S. Seaman, Natalie Hawkins, Steven G. Self, Sanjay Phogat, James Tartaglia, Susan W. Barnett, Brian Burke, Anthony D. Cristillo, Song Ding, Jonathan L. Heeney, Giuseppe Pantaleo, Ralf Wagner O37 Passive immunisation with a neutralising antibody against HIV-1 Env prevents infection of the first cells in a mucosal challenge rhesus monkey model Christiane Stahl-Hennig, Viktoria Stab, Armin Ensser, Ulrike Sauermann, Bettina Tippler, Dennis Burton, Matthias Tenbusch, Klaus Überla O38 HIV antibody Fc-glycoforms drive B cell affinity maturation Galit Alter, Giuseppe Lofano, Anne-Sophie Dugast, Viraj Kulkarni, Todd Suscovich Poster presentations Topic 1: Entry & uncoating P1 Dynein light chain is required for murine leukemia virus infection Tatiana Opazo, Felipe Barraza, Diego Herrera, Andrea Garces, Tomas Schwenke, Diego Tapia, Jorge Cancino, Gloria Arriagada P2 Peptide paratope mimics of the broadly neutralising HIV-1 antibody b12 Christina Haußner, Dominik Damm, Anette Rohrhofer, Barbara Schmidt, Jutta Eichler P3 Investigating cellular pathways involved in the transmission of HIV-1 between dendritic cells and T cells using RNAi screening techniques Rebecca Midgley, James Wheeldon, Vincent Piguet P4 Co-receptor tropism in HIV-1, HIV-2 monotypic and dual infections Priyanka Khopkar, Megha Rohamare, Smita Kulkarni P5 Characterisation of the role of CIB1 and CIB2 as HIV-1 helper factors Ana Godinho-Santos, Allan Hance, Joao Goncalves, Fabrizio Mammano P6 Buffering deleterious polymorphisms in the highly constrained C2 region of HIV-1 envelope by the flexible V3 domain Romain Gasser, Meriem Hamoudi, Martina Pellicciotta, Zhicheng Zhou, Clara Visdeloup, Philippe Colin, Martine Braibant, Bernard Lagane, Matteo Negroni P7 Entry inhibition of HERV-K(HML-2) by an Env-IgG fusion protein Jula Wamara, Norbert Bannert Topic 2: Reverse transcription & integration P8 The R263K/H51Y resistance substitutions in HIV integrase decreases levels of integrated HIV DNA over time Thibault Mesplede, Nathan Osman, Kaitlin Anstett, Jiaming Calvin Liang, Hanh Thi Pham, Mark Wainberg P9 The Retrovirus Integration Database (RID) Wei Shao, Jigui Shan, Mary Kearney, Xiaolin Wu, Frank Maldarelli, John Mellors, Brian Luke, John Coffin, Stephen Hughes P10 The small molecule 3G11 inhibits HIV-1 reverse transcription Thomas Fricke, Silvana Opp, Caitlin Shepard, Dmitri Ivanov, Baek Kim, Jose Valle-Casuso, Felipe Diaz-Griffero P11 Dual and opposite regulation of HIV-1 integration by hRAD51: impact on therapeutical approaches using homologous DNA repair modulators Vincent Parissi P12 A flexible motif essential for integration by HIV-1 integrase Marine Kanja, Pierre Cappy, Matteo Negroni, Daniela Lener P13 Interaction between HIV-1 integrase and the host protein Ku70: identification of the binding site and study of the influence on integrase-proteasome interplay Ekaterina Knyazhanskaya, Andrey Anisenko, Timofey Zatsepin, Marina Gottikh P14 Normalisation based method for deep sequencing of somatic retroelement integrations in human genome Alexander Komkov, Anastasia Minervina, Gaiaz Nugmanov, Vadim Nazarov, Konstantin Khodosevich, Ilgar Mamedov, Yuri Lebedev Topic 3: Transcription and latency P15 BCA2/RABRING7 restricts HIV-1 transcription by preventing the nuclear translocation of NF-κB Marta Colomer-Lluch, Ruth Serra-Moreno P16 MATR3 post-transcriptional regulation of HIV-1 transcription during latency Ambra Sarracino, Anna Kula, Lavina Gharu, Alexander Pasternak, Carine Van Lint, Alessandro Marcello P17 HIV-1 tat intersects the SUMO pathway to regulate HIV-1 promoter activity Ann Marie McCartin, Anurag Kulkarni, Valentin Le Douce, Virginie Gautier P18 Conservation in HIV-1 Vpr guides tertiary gRNA folding and alternative splicing Ann Baeyens, Evelien Naessens, Anouk Van Nuffel, Karin Weening, Anne-Marie Reilly, Eva Claeys, Wim Trypsteen, Linos Vandekerckhove, Sven Eyckerman, Kris Gevaert, Bruno Verhasselt P19 The majority of reactivatable latent HIV are genetically distinct Hoi Ping Mok, Nicholas Norton, Axel Fun, Jack Hirst, Mark Wills, Andrew Lever P20 Do mutations in the tat exonic splice enhancer contribute to HIV-1 latency? Nicholas Norton, Hoi Ping Mok, Jack Hirst, Andrew Lever P21 Culture-to-Ct: A fast and direct RT-qPCR HIV gene reactivation screening method using primary T cell culture Valentin Le Douce, Ann Marie McCartin, Virginie Gautier P22 A novel approach to define populations of early silenced proviruses Dalibor Miklik, Filip Senigl, Jiri Hejnar Topic 4: RNA trafficking & packaging P23 Functional analysis of the structure and conformation of HIV-1 genome RNA DIS Jun-ichi Sakuragi, Sayuri Sakuragi, Masaru Yokoyama, Tatsuo Shioda, Hironori Sato P24 Regulation of foamy viral env splicing controls gag and pol expression Jochen Bodem, Rebecca Moschall, Sarah Denk, Steffen Erkelenz, Christian Schenk, Heiner Schaal Topic 5: Assembly & release P25 Transfer of HTLV-1 p8 to target T cells depends on VASP: a novel interaction partner of p8 Norbert Donhauser, Ellen Socher, Sebastian Millen, Heinrich Sticht, Andrea K. Thoma-Kress P26 COL4A1 and COL4A2 are novel HTLV-1 tax targets with a putative role in virus transmission Christine Gross, Sebastian Millen, Melanie Mann, Klaus Überla, Andrea K. Thoma-Kress P27 The C terminus of foamy virus gag protein is required for particle formation, and virus budding: starting assembly at the C terminus? Guochao Wei, Matthew J. Betts, Yang Liu, Timo Kehl, Robert B. Russell, Martin Löchelt P28 Generation of an antigen-capture ELISA and analysis of Rec and Staufen-1 effects on HERV-K(HML-2) virus particle production Oliver Hohn, Saeed Mostafa, Kirsten Hanke, Stephen Norley, Norbert Bannert P29 Antagonism of BST-2/tetherin is a conserved function of primary HIV-2 Env glycoproteins Chia-Yen Chen, Masashi Shingai, Pedro Borrego, Nuno Taveira, Klaus Strebel P30 Mutations in the packaging signal region of the HIV-1 genome cause a late domain mutant phenotype Chris Hellmund, Bo Meng, Andrew Lever P31 p6 regulates membrane association of HIV-1 gag Melanie Friedrich, Friedrich Hahn, Christian Setz, Pia Rauch, Kirsten Fraedrich, Alina Matthaei, Petra Henklein, Maximilian Traxdorf, Torgils Fossen, Ulrich Schubert Topic 6: Pathogenesis & evolution P32 Molecular and structural basis of protein evolution during viral adaptation Aya Khwaja, Meytal Galilee, Akram Alian P33 HIV-1 enhancement and neutralisation by soluble gp120 and its role for the selection of the R5-tropic “best fit” Birco Schwalbe, Heiko Hauser, Michael Schreiber P34 An insertion of seven amino acids in the Env cytoplasmic tail of Human Immunodeficiency Virus type 2 (HIV-2) selected during disease progression enhances viral replication François Dufrasne, Mara Lucchetti, Patrick Goubau, Jean Ruelle P35 Cell-associated HIV-1 unspliced to multiply spliced RNA ratio at 12 weeks ART correlates with markers of immune activation and apoptosis and predicts the CD4 T-cell count at 96 weeks ART Mirte Scherpenisse, Ben Berkhout, Alexander Pasternak P36 Faster progression in non-B subtype HIV-1-infected patients than Korean subclade of subtype B is accompanied by higher variation and no induction of gross deletion in non-B nef gene by Korean red ginseng treatment Young-Keol Cho, Jungeun Kim, Daeun Jeong P37 Aberrant expression of ERVWE1 endogenous retrovirus and overexpression of TET dioxygenases are characteristic features of seminoma Katerina Trejbalova, Martina Benesova, Dana Kucerova, Zdenka Vernerova, Rachel Amouroux, Petra Hajkova, Jiri Hejnar P38 Life history of the oldest lentivirus: characterisation of ELVgv integrations and the TRIM5 selection pattern in dermoptera Daniel Elleder, Tomas Hron, Helena Farkasova, Abinash Padhi, Jan Paces P39 Characterisation of a highly divergent endogenous retrovirus in the equine germ line Henan Zhu, Robert Gifford, Pablo Murcia P40 The emergence of pandemic retroviral infection in small ruminants Maria Luisa Carrozza, Anna-Maria Niewiadomska, Maurizio Mazzei, Mounir Abi-Said, Joseph Hughes, Stéphane Hué, Robert Gifford P41 Near full-length genome (NFLG) Characterisation of HIV-1 subtype B identified in South Africa Adetayo Obasa, Graeme Jacobs, Susan Engelbrecht P42 Acquisition of Vpu-mediated tetherin antagonism by an HIV-1 group O strain Katharina Mack, Kathrin Starz, Daniel Sauter, Matthias Geyer, Frederic Bibollet-Ruche, Christina Stürzel, Marie Leoz, Jean Christophe Plantier, Beatrice H. Hahn, Frank Kirchhoff P43 The human endogenous retrovirus type K is involved in cancer stem cell markers expression and in human melanoma malignancy Ayele Argaw-Denboba, Emanuela Balestrieri, Annalucia Serafino, Ilaria Bucci, Chiara Cipriani, Corrado Spadafora, Paolo Sinibaldi-Vallebona, Claudia Matteucci P44 Natural infection of Indian non-human primates by unique lentiviruses S. Nandi Jayashree, Ujjwal Neogi, Anil K. Chhangani, Shravan Sing Rathore, Bajrang R. J. Mathur P45 Free cervical cancer screening among HIV-positive women receiving antiretroviral treatment in Nigeria Adeyemi Abati P46 Molecular evolutionary status of feline immunodeficiency virus in Turkey B. Taylan Koç, Tuba Çiğdem Oğuzoğlu Topic 7: Innate sensing & intrinsic immunity P47 Cell-to-cell contact with HTLV-1-infected T cells reduces dendritic cell immune functions and contributes to infection in trans. Takatoshi Shimauchi, Stephan Caucheteux, Jocelyn Turpin, Katja Finsterbusch, Charles Bangham, Yoshiki Tokura, Vincent Piguet P48 Deciphering the mechanisms of HIV-1 exacerbation induced by Mycobacterium tuberculosis in monocytes/macrophages Shanti Souriant, Luciana Balboa, Karine Pingris, Denise Kviatcowsky, Brigitte Raynaud-Messina, Céline Cougoule, Ingrid Mercier, Marcelo Kuroda, Pablo González-Montaner, Sandra Inwentarz, Eduardo Jose Moraña, Maria del Carmen Sasiain, Olivier Neyrolles, Isabelle Maridonneau-Parini, Geanncarlo Lugo-Villarino, Christel Vérollet P49 The SAMHD1-mediated inhibition of LINE-1 retroelements is regulated by phosphorylation Alexandra Herrmann, Sabine Wittmann, Caitlin Shepard, Dominique Thomas, Nerea Ferreirós Bouzas, Baek Kim, Thomas Gramberg P50 Activities of nuclear envelope protein SUN2 in HIV infection Xavier Lahaye, Anvita Bhargava, Takeshi Satoh, Matteo Gentili, Silvia Cerboni, Aymeric Silvin, Cécile Conrad, Hakim Ahmed-Belkacem, Elisa C. Rodriguez, Jean-François Guichou, Nathalie Bosquet, Matthieu Piel, Roger Le Grand, Megan King, Jean-Michel Pawlotsky, Nicolas Manel P51 Activation of TLR7/8 with a small molecule agonist induces a novel restriction to HIV-1 infection of monocytes Henning Hofmann, Benedicte Vanwalscappel, Nicolin Bloch, Nathaniel Landau P52 Steady state between the DNA polymerase and Rnase H domain activities of reverse transcriptases determines the sensitivity of retroviruses to inhibition by APOBEC3 proteins Stanislav Indik, Benedikt Hagen P53 HIV restriction in mature dendritic cells is related to p21 induction and p21-mediated control of the dNTP pool and SAMHD1 activity. José Carlos Valle-Casuso, Awatef Allouch, Annie David, Françoise Barré-Sinoussi, Michaela Müller-Trutwin, Monsef Benkirane, Gianfranco Pancino, Asier Saez-Cirion P54 IFITM protens restrict HIV-1 protein synthesis Wing-Yiu Lee, Chen Liang, Richard Sloan P55 Characterisation and functional analysis of the novel restriction factor Serinc5 Bianca Schulte, Silvana Opp, Felipe Diaz-Griffero P56 piRNA sequences are common in Human Endogenous Retroviral Sequences (HERVs): An antiretroviral restriction mechanism? Jonas Blomberg, Luana Vargiu, Patricia Rodriguez-Tomé, Enzo Tramontano, Göran Sperber P57 Ferroportin restricts HIV-1 infection in sickle cell disease Namita Kumari, Tatiana Ammosova, Sharmeen Diaz, Patricia Oneal, Sergei Nekhai P58 APOBEC3G modulates the response to antiretroviral drugs in humanized mice Audrey Fahrny, Gustavo Gers-Huber, Annette Audigé, Roberto F. Speck, Anitha Jayaprakash, Ravi Sachidanandam, Matt Hernandez, Marsha Dillon-White, Viviana Simon P59 High-throughput epigenetic analysis of evolutionarily young endogenous retrovirus presents in the mule deer (Odocoileus hemionus) genome Tomas Hron, Helena Farkasova, Daniel Elleder P60 Characterisation of the expression of novel endogenous retroviruses and immune interactions in a macaque model Neil Berry, Emmanuel Maze, Claire Ham, Neil Almond, Greg Towers, Robert Belshaw P61 HIV-1 restriction by orthologs of SERINC3 and SERINC5 Patrícia de Sousa-Pereira, Joana Abrantes, Massimo Pizzato, Pedro J. Esteves, Oliver T. Fackler, Oliver T. Keppler, Hanna-Mari Baldauf P62 TRIM19/PML restricts HIV infection in a cell type-dependent manner Bianca Volkmann, Tanja Kahle, Kristin Eissmann, Alexandra Herrmann, Sven Schmitt, Sabine Wittmann, Laura Merkel, Nina Reuter, Thomas Stamminger, Thomas Gramberg P63 Recent invasion of the mule deer genome by a retrovirus Helena Farkasova, Tomas Hron, Daniel Elleder P64 Does the antiviral protein SAMHD1 influence mitochondrial function? Ilaria Dalla Rosa, Kate Bishop, Antonella Spinazzola, Harriet Groom P65 cGAMP transfers intercellularly via HIV-1 Env-mediated cell–cell fusion sites and triggers an innate immune response in primary target cells Shuting Xu, Aurélie Ducroux, Aparna Ponnurangam, Sergej Franz, Gabrielle Vieyres, Mathias Müsken, Thomas Zillinger, Angelina Malassa, Ellen Ewald, Veit Hornung, Winfried Barchet, Susanne Häussler, Thomas Pietschmann, Christine Goffinet P66 Pre-infection transcript levels of FAM26F in PBMCS inform about overall plasma viral load in acute and postacute phase after SIV-infection Ulrike Sauermann, Aneela Javed, Nicole Leuchte, Gabriela Salinas, Lennart Opitz, Christiane Stahl-Hennig, Sieghart Sopper P67 Sequence-function analysis of three T cell receptors targeting the HIV-1 p17 epitope SLYNTVATL Christiane Mummert, Christian Hofmann, Angela G. Hückelhoven, Silke Bergmann, Sandra M. Müller-Schmucker, Ellen G. Harrer, Jan Dörrie, Niels Schaft, Thomas Harrer P68 An immunodominant region of the envelope glycoprotein of small ruminant lentiviruses may function as decoy antigen Laure Cardinaux, M.-L. Zahno, H.-R. Vogt, R. Zanoni, G. Bertoni P69 Impact of immune activation, immune exhaustion, broadly neutralising antibodies and viral reservoirs on disease progression in HIV-infected children Maximilian Muenchhoff, Philip Goulder, Oliver Keppler Topic 9: Novel antiviral strategies P70 Identification of natural compounds as new antiviral products by bioassay-guided fractionation Alexandra Herrmann, Stephanie Rebensburg, Markus Helfer, Michael Schindler, Ruth Brack-Werner P71 The PPARG antagonism disconnects the HIV replication and effector functions in Th17 cells Yuwei Zhang, Huicheng Chen, Delphine Planas, Annie Bernier, Annie Gosselin, Jean-Pierre Routy, Petronela Ancuta P72 Characterisation of a multiresistant subtype AG reverse transcriptase: AZT resistance, sensitivity to RNase H inhibitors and inhibitor binding Birgitta Wöhrl, Anna Schneider, Angela Corona, Imke Spöring, Mareike Jordan, Bernd Buchholz, Elias Maccioni, Roberto Di Santo, Jochen Bodem, Enzo Tramontano, Kristian Schweimer P73 Insigths into the acetylation pattern of HDAC inhibitors and their potential role in HIV therapy Christian Schölz, Brian Weinert, Sebastian Wagner, Petra Beli, Yasuyuki Miyake, Jun Qi, Lars Jensen, Werner Streicher, Anna McCarthy, Nicholas Westwood, Sonia Lain, Jürgen Cox, Patrick Matthias, Matthias Mann, James Bradner, Chunaram Choudhary P74 HPV-derived and seminal amyloid peptides enhance HIV-1 infection and impair the efficacy of broadly neutralising antibodies and antiretroviral drugs Marcel Stern, Oliver T. Keppler P75 D(−)lentiginosine inhibits both proliferation and virus expression in cells infected by HTLV-1 in vitro Elena Valletta, Caterina Frezza, Claudia Matteucci, Francesca Marino-Merlo, Sandro Grelli, Anna Lucia Serafino, Antonio Mastino, Beatrice Macchi P76 HIV-1 resistance analyses of the Cape Winelands districts, South Africa Sello Mikasi, Graeme Jacobs, Susan Engelbrecht Topic 10: Recent advances in HIV vaccine development P77 Induction of complex retrovirus antigen-specific immune responses by adenovirus-based vectors depends on the order of vector administration Meike Kaulfuß, Sonja Windmann, Wibke Bayer P78 Direct impact of structural properties of HIV-1 Env on the regulation of the humoral immune response Rebecca Heß, Michael Storcksdieck gen. Bonsmann, Viktoria Stab, Carsten Kirschning, Bernd Lepenies, Matthias Tenbusch, Klaus Überla P79 Lentiviral virus-like particles mediate gerenration of T-follicular helper cells in vitro Anne Kolenbrander, Klaus Überla, Vladimir Temchura P80 Recruitment of HIV-1 Vpr to DNA damage sites and protection of proviral DNA from nuclease activity Kenta Iijima, Junya Kobayashi, Yukihito Ishizaka

Objective Human CD34 + cord blood (CB) cells are hematopoietic progenitors useful for stem cell transplantation, even after ex vivo expansion. We investigated the effect of tumor necrosis factor (TNF) on lymphoid development from cultured CD34 + CB cells. Materials and Methods Human CD34 + CB cells were cultured in cytokine mixes with or without TNF. Preculture during 60 hours was followed by in vitro differentiation assays, including fetal thymus organ culture and coculture on murine stromal MS-5 cells. In a next step, experiments were extended to CD34 + CD38 − and CD34 + CD38 + CB cells and prolonged preculture. Results Preculture in the presence of TNF improved differentiation into T cells and diminished the ability to generate B cells, while NK potential and myeloid development were unaffected. Sorted CD34 + CD38 − CB cells were more potent T-cell precursors after preculture in TNF, compared to CD34 + CD38 + CB cells. In precultured CD34 + CD38 − CB cells, TNF increased GATA3 but decreased EBF1 expression, in line with the skewed lymphoid differentiation induced by TNF. However, when preculture in the presence of TNF was extended to 1 week, T-cell precursors were lost. Conclusion After short-term culture of CD34 + CB cells in the presence of TNF, T-cell generation is stimulated at the expense of B-cell generation. T-cell progenitors are enriched in the CD34 + CD38 − fraction. These results have implications on the culture conditions to be used for CB CD34 + cells prior to transplantation.

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.