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Cells sense a wide variety of signals and respond by adopting complex transcriptional states. Most single-cell profiling is carried out today at cellular baseline, blind to cells’ potential spectrum of functional responses. Exploring the space of cellular responses experimentally requires access to a large combinatorial perturbation space. Single-cell genomics coupled with multiplexing techniques provide a useful tool for characterizing cell states across several experimental conditions. However, current multiplexing strategies require programmatic handling of many samples in macroscale arrayed formats, precluding their application in large-scale combinatorial analysis. Here, we introduce StimDrop, a method that combines antibody-based cell barcoding with parallel droplet processing to automatically formulate cell population × stimulus combinations in a microfluidic device. We applied StimDrop to profile the effects of 512 sequential stimulation conditions on human dendritic cells. Our results demonstrate that priming with viral ligands potentiates hyperinflammatory responses to a second stimulus, and show transcriptional signatures consistent with this phenomenon in myeloid cells of patients with severe COVID-19.

In this study, we report that the TLR4 ligand, LPS, and TLR3 ligand polyinosinic:polycytidylic acid failed to activate IRF3 or STAT1 in bone marrow–derived macrophages (BMMs) isolated from two independently generated lines of Rosa26-integrated Cas9-expressing C57BL/6J (B6) mice. RNA-sequencing analysis reveals that hundreds to thousands of genes including IFN-stimulated genes were differentially expressed in BMMs from these Cas9 strains compared with B6 upon LPS stimulation. Furthermore, the NF-κB signaling axis and TRIF-mediated necroptosis were also strongly reduced in response to LPS and polyinosinic:polycytidylic acid. In contrast, there were no defects in the responses of BMMs to ligands of the RIG-I, STING, TLR2, TLR9, and IFN receptors. Defects in TLR3 and TLR4 signaling were observed in mice with the B6 but not 129 background, and when Cas9 was integrated at the Rosa26 but not H11 locus. However, integration at the Rosa26 site, CAG promoter–driven Cas9 or eGFP were not individually sufficient to cause the defect. Taken together, the results of this study suggest a putative TRIF-mediated defect in TLR-3/4 signaling in BMMs from commercially available and widely used B6–Cas9–expressing mice.

STING is an intracellular sensor of cyclic di-nucleotides involved in response to pathogen- or self-derived DNA that induces protective immunity, or if dysregulated, autoimmunity. STING trafficking is tightly linked to its activity. We aimed to systematically characterize genes regulating STING trafficking and to define their impact on STING responses. Based on proximity-ligation proteomics and genetic screens, an ESCRT complex containing HGS, VPS37A and UBAP1 was found to be required for STING degradation and signaling shutdown. Analogous to phosphorylated STING creating a platform for IRF3 recruitment, oligomerization-driven STING ubiquitination by UBE2N formed a platform for ESCRT recruitment at the endosome, responsible for STING signaling shutdown. A UBAP1 mutant that underlies human spastic paraplegia and disrupts ESCRT function led to STING-dependent type I IFN responses at the steady-state, defining ESCRT as a homeostatic regulator of STING signaling.

Defects in the immune defences induced by the protein interferon are associated with some severe cases of COVID-19. An analysis of patients’ blood samples sheds light on how antibodies might contribute to these defects. A type of antibody Fc domain could limit interferon-mediated responses.

SUMMARYEvidence indicates that transposable elements (TEs) stimulate innate sensing pathways in various pathologies but it is not clear whether they are sensed during normal physiological responses. Here we show that, during activation with an exogenous pathogen associated molecular pattern (PAMP), dendritic cells (DCs) epigenetically remodel heterochromatin at TEs by depleting the methyltransferase Suv39h1 and reducing histone-3 lysine-9 trimethylation (H3K9me3). TLR4 signaling activates TE expression to enhance innate responses through the DNA sensor cGAS. Cytosolic cGAS-bound DNA comprised LINE1 TEs as the predominant endogenous ligands. Concordantly, LINE1 inhibition attenuated the type-I IFN response to LPS and rescued influenza virus infection. We propose that in healthy cells, exogenous PAMPs epigenetically activate self-derived PAMPs (LINE1) that engage cGAS to enhance responses. These data explain why pathogens employ redundant and broad innate immune countermeasures, to suppress activation of host PAMPs and illustrate a hitherto unappreciated role for host genome-derived PAMPs in response to pathogens.

The step-by-step protocol for performing the experimental steps of CeVICA, a cell-free nanobody selection platform. This protocol describes one round of selection. Multiple rounds of selection can be performed by iterating this protocol and using the output library DNA from the previous round as the input library DNA for the next round.

BackgroundTherapeutically immunosuppressed transplant recipients exhibit attenuated responses to COVID-19 vaccines. To better understand the immune alterations that determined poor vaccine response, we correlated quantities of circulating T and B cell subsets at baseline with longitudinal serologic responses to SARS-CoV-2 mRNA vaccination in heart and lung transplant recipients.MethodsSamples at baseline and at approximately 8 and 30 days after each vaccine dose for 22 heart and lung transplant recipients with no history of COVID-19, four heart and lung transplant recipients with prior COVID-19 infection, and 12 healthy controls undergoing vaccination were analyzed. Anti-spike protein receptor binding domain (RBD) IgG and pseudovirus neutralization activity were measured. Proportions of B and T cell subsets at baseline were comprehensively quantitated.ResultsAt 8-30 days post vaccination, healthy controls displayed robust anti-RBD IgG responses, whereas heart and lung transplant recipients showed minimally increased responses. A parallel absence of activity was observed in pseudovirus neutralization. In contrast, three of four (75%) transplant recipients with prior COVID-19 infection displayed robust responses at levels comparable to controls. Baseline levels of activated PD-1+ HLA-DR+ CXCR5- CD4+ T cells (also known as T peripheral helper [TPH] cells) and CD4+ T cells strongly predicted the ability to mount a response.ConclusionsImmunosuppressed patients have defective vaccine responses but can be induced to generate neutralizing antibodies after SARS-CoV-2 infection. Strong correlations of vaccine responsiveness with baseline TPH and CD4+ T cell numbers highlights a role for T helper activity in B cell differentiation into antibody secreting cells during vaccine response.

Genome-wide association studies have uncovered hundreds of autoimmune disease-associated loci; however, the causal genetic variant(s) within each locus are mostly unknown. Here, we perform high-throughput allele-specific reporter assays to prioritize disease-associated variants for five autoimmune diseases. By examining variants that both promote allele-specific reporter expression and are located in accessible chromatin, we identify 60 putatively causal variants that enrich for statistically fine-mapped variants by up to 57.8-fold. We introduced the risk allele of a prioritized variant (rs72928038) into a human T cell line and deleted the orthologous sequence in mice, both resulting in reduced BACH2 expression. Naïve CD8 T cells from mice containing the deletion had reduced expression of genes that suppress activation and maintain stemness. Our results represent an example of an effective approach for prioritizing variants and studying their physiologically relevant effects.

Genome-wide association studies (GWASs) have uncovered hundreds of autoimmune disease-associated loci; however, the causal genetic variants within each locus are mostly unknown. Here, we perform high-throughput allele-specific reporter assays to prioritize disease-associated variants for five autoimmune diseases. By examining variants that both promote allele-specific reporter expression and are located in accessible chromatin, we identify 60 putatively causal variants that enrich for statistically fine-mapped variants by up to 57.8-fold. We introduced the risk allele of a prioritized variant (rs72928038) into a human T cell line and deleted the orthologous sequence in mice, both resulting in reduced BACH2 expression. Naive CD8 T cells from mice containing the deletion had reduced expression of genes that suppress activation and maintain stemness and, upon acute viral infection, displayed greater propensity to become effector T cells. Our results represent an example of an effective approach for prioritizing variants and studying their physiologically relevant effects.

Mechanisms underlying severe coronavirus disease 2019 (COVID-19) disease remain poorly understood. We analyze several thousand plasma proteins longitudinally in 306 COVID-19 patients and 78 symptomatic controls, uncovering immune and non-immune proteins linked to COVID-19. Deconvolution of our plasma proteome data using published scRNA-seq datasets reveals contributions from circulating immune and tissue cells. Sixteen percent of patients display reduced inflammation yet comparably poor outcomes. Comparison of patients who died to severely ill survivors identifies dynamic immune-cell-derived and tissue-associated proteins associated with survival, including exocrine pancreatic proteases. Using derived tissue-specific and cell-type-specific intracellular death signatures, cellular angiotensin-converting enzyme 2 (ACE2) expression, and our data, we infer whether organ damage resulted from direct or indirect effects of infection. We propose a model in which interactions among myeloid, epithelial, and T cells drive tissue damage. These datasets provide important insights and a rich resource for analysis of mechanisms of severe COVID-19 disease., Graphical abstract, Filbin et al. use plasma proteomics in 306 coronavirus disease 2019 (COVID-19) patients and 78 symptomatic controls over time to better understand the role of circulating immune cells and tissue cells in inflammation, disease severity, and survival. They propose a model in which interactions among myeloid, epithelial, and T cells drive tissue damage.

BACKGROUND: SARS-CoV-2 plasma viremia has been associated with severe disease and death in COVID-19 in small-scale cohort studies. The mechanisms behind this association remain elusive. METHODS: We evaluated the relationship between SARS-CoV-2 viremia, disease outcome, and inflammatory and proteomic profiles in a cohort of COVID-19 emergency department participants. SARS-CoV-2 viral load was measured using a quantitative reverse transcription PCR–based platform. Proteomic data were generated with Proximity Extension Assay using the Olink platform. RESULTS: This study included 300 participants with nucleic acid test–confirmed COVID-19. Plasma SARS-CoV-2 viremia levels at the time of presentation predicted adverse disease outcomes, with an adjusted OR of 10.6 (95% CI 4.4–25.5, P < 0.001) for severe disease (mechanical ventilation and/or 28-day mortality) and 3.9 (95% CI 1.5–10.1, P = 0.006) for 28-day mortality. Proteomic analyses revealed prominent proteomic pathways associated with SARS-CoV-2 viremia, including upregulation of SARS-CoV-2 entry factors (ACE2, CTSL, FURIN), heightened markers of tissue damage to the lungs, gastrointestinal tract, and endothelium/vasculature, and alterations in coagulation pathways. CONCLUSION: These results highlight the cascade of vascular and tissue damage associated with SARS-CoV-2 plasma viremia that underlies its ability to predict COVID-19 disease outcomes. FUNDING: Mark and Lisa Schwartz; the National Institutes of Health (U19AI082630); the American Lung Association; the Executive Committee on Research at Massachusetts General Hospital; the Chan Zuckerberg Initiative; Arthur, Sandra, and Sarah Irving for the David P. Ryan, MD, Endowed Chair in Cancer Research; an EMBO Long-Term Fellowship (ALTF 486-2018); a Cancer Research Institute/Bristol Myers Squibb Fellowship (CRI2993); the Harvard Catalyst/Harvard Clinical and Translational Science Center (National Center for Advancing Translational Sciences, NIH awards UL1TR001102 and UL1TR002541-01); and by the Harvard University Center for AIDS Research (National Institute of Allergy and Infectious Diseases, 5P30AI060354).

Although mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are less known. Here, we show that nuclear envelope ruptures induce DNA damage that promotes senescence in non-transformed cells and induces an invasive phenotype in human breast cancer cells. We find that the endoplasmic reticulum (ER)-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures that generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.

Malate dehydrogenases (MDHs) sustain tumor growth and carbon metabolism by pathogens including Plasmodium falciparum. However, clinical success of MDH inhibitors is absent, as current small molecule approaches targeting the active site are unselective. The presence of an allosteric binding site at oligomeric interface allows the development of more specific inhibitors. To this end we performed a differential NMR-based screening of 1500 fragments to identify fragments that bind at the oligomeric interface. Subsequent biophysical and biochemical experiments of an identified fragment indicate an allosteric mechanism of 4-(3,4-difluorophenyl) thiazol-2-amine (4DT) inhibition by impacting the formation of the active site loop, located >30 Å from the 4DT binding site. Further characterization of the more tractable homolog 4-phenylthiazol-2-amine (4PA) and 16 other derivatives are also reported. These data pave the way for downstream development of more selective molecules by utilizing the oligomeric interfaces showing higher species sequence divergence than the MDH active site., Romero et al. perform NMR-based screening of 1500 fragments to identify fragments that bind at the oligomeric interface of malate dehydrogenase (MDH). Their study indicates an allosteric mechanism impacting enzymatic activity, paving the way for development of more selective molecules and a starting point for the future development of specific MDH inhibitors.

Human primary monocytes are critical in controlling immune responses and tissue homeostasis. However, to identify and study molecular components that underlie the function of these cells, there remains a need to develop methods to perturb genes in these cells. Here we report lentiviral-based delivery of dCas9-KRAB and guide RNAs to efficiently silent target genes, such as CD45 and CD209. We further show that sgRNAs against TICAM1 dampen proinflammatory cytokine and interferon expression in response to lipopolysaccharide. When delivered by lentivirus, sgRNAs are incorporated into the genome, thus enabling pooled screening to perturb and identify coding and non-coding elements that contribute to the functions of primary human monocytes.

SummaryCOVID-19 has caused over 1 million deaths globally, yet the cellular mechanisms underlying severe disease remain poorly understood. By analyzing several thousand plasma proteins in 306 COVID-19 patients and 78 symptomatic controls over serial timepoints using two complementary approaches, we uncover COVID-19 host immune and non-immune proteins not previously linked to this disease. Integration of plasma proteomics with nine published scRNAseq datasets shows that SARS-CoV-2 infection upregulates monocyte/macrophage, plasmablast, and T cell effector proteins. By comparing patients who died to severely ill patients who survived, we identify dynamic immunomodulatory and tissue-associated proteins associated with survival, providing insights into which host responses are beneficial and which are detrimental to survival. We identify intracellular death signatures from specific tissues and cell types, and by associating these with angiotensin converting enzyme 2 (ACE2) expression, we map tissue damage associated with severe disease and propose which damage results from direct viral infection rather than from indirect effects of illness. We find that disease severity in lung tissue is driven by myeloid cell phenotypes and cell-cell interactions with lung epithelial cells and T cells. Based on these results, we propose a model of immune and epithelial cell interactions that drive cell-type specific and tissue-specific damage in severe COVID-19.

Antibody engineering technologies face increasing demands for speed, reliability and scale. We develop CeVICA, a cell-free nanobody engineering platform that uses ribosome display for in vitro selection of nanobodies from a library of 1011 randomized sequences. We apply CeVICA to engineer nanobodies against the Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein and identify >800 binder families using a computational pipeline based on CDR-directed clustering. Among 38 experimentally-tested families, 30 are true RBD binders and 11 inhibit SARS-CoV-2 pseudotyped virus infection. Affinity maturation and multivalency engineering increase nanobody binding affinity and yield a virus neutralizer with picomolar IC50. Furthermore, the capability of CeVICA for comprehensive binder prediction allows us to validate the fitness of our nanobody library. CeVICA offers an integrated solution for rapid generation of divergent synthetic nanobodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel nanobody engineering., Faster, higher throughput antibody engineering methods are needed. Here the authors present CeVICA, a cell-free nanobody engineering platform using ribosome display and computational clustering analysis for in vitro selection; they use this to develop nanobodies against the RBD of SARS-CoV-2 spike protein.

T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection; yet the repertoire of naturally processed and presented viral epitopes on HLA class I remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post-infection using mass spectrometry. We found HLA-I peptides derived not only from canonical ORFs, but also from internal out-of-frame ORFs in Spike and Nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and COVID-19 patients that exceeded responses to canonical peptides including some of the strongest epitopes reported to date. Whole proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights as well as the discovery of out-of-frame ORF epitopes will facilitate selection of peptides for immune monitoring and vaccine development., Analysis of the HLA-1 peptidome of SARS-CoV-2 infection identifies peptides derived from canonical and out- of-frame ORFs in viral S and N protein that are not captured by current vaccines and yield potent T cell responses in a mouse model as well as patients with COVID-19.

T cell-mediated immunity may play a critical role in controlling and establishing protective immunity against SARS-CoV-2 infection; yet the repertoire of viral epitopes responsible for T cell response activation remains mostly unknown. Identification of viral peptides presented on class I human leukocyte antigen (HLA-I) can reveal epitopes for recognition by cytotoxic T cells and potential incorporation into vaccines. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two human cell lines at different times post-infection using mass spectrometry. We found HLA-I peptides derived not only from canonical ORFs, but also from internal out-of-frame ORFs in Spike and Nucleoprotein not captured by current vaccines. Proteomics analyses of infected cells revealed that SARS-CoV-2 may interfere with antigen processing and immune signaling pathways. Based on the endogenously processed and presented viral peptides that we identified, we estimate that a pool of 24 peptides would provide one or more peptides for presentation by at least one HLA allele in 99% of the human population. These biological insights and the list of naturally presented SARS-CoV-2 peptides will facilitate data-driven selection of peptides for immune monitoring and vaccine development.

The recently identified Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. How this novel beta-coronavirus virus, and coronaviruses more generally, alter cellular metabolism to support massive production of ~30 kB viral genomes and subgenomic viral RNAs remains largely unknown. To gain insights, transcriptional and metabolomic analyses are performed 8 hours after SARS-CoV-2 infection, an early timepoint where the viral lifecycle is completed but prior to overt effects on host cell growth or survival. Here, we show that SARS-CoV-2 remodels host folate and one-carbon metabolism at the post-transcriptional level to support de novo purine synthesis, bypassing viral shutoff of host translation. Intracellular glucose and folate are depleted in SARS-CoV-2-infected cells, and viral replication is exquisitely sensitive to inhibitors of folate and one-carbon metabolism, notably methotrexate. Host metabolism targeted therapy could add to the armamentarium against future coronavirus outbreaks., Viruses rely on host metabolism for replication. Here, the authors perform transcriptional and metabolomic analyses at 8 hours after SARS-CoV-2 infection and find that the virus alters host folate and one-carbon metabolism at a post-transcriptional level.

While mutations leading to a fragile envelope of the cell nucleus are well known to cause diseases such as muscular dystrophies or accelerated aging, the pathophysiological consequences of the recently discovered mechanically induced nuclear envelope ruptures in cells harboring no mutation are less known. Here we show that repeated loss of nuclear envelope integrity in nuclei experiencing mechanical constraints promotes senescence in nontransformed cells, and induces an invasive phenotype including increased collagen degradation in human breast cancer cells, both in vitro and in a mouse xenograft model of breast cancer progression. We show that these phenotypic changes are due to the presence of chronic DNA damage and activation of the ATM kinase. In addition, we found that depletion of the cytoplasmic exonuclease TREX1 is sufficient to abolish the DNA damage in mechanically challenged nuclei and to suppress the phenotypes associated with the loss of nuclear envelope integrity. Our results also show that TREX1-dependent DNA damage induced by physical confinement of tumor cells inside the mammary duct drives the progression of in situ breast carcinoma to the invasive stage. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.

Detection of viruses by innate immune sensors induces protective antiviral immunity. The viral DNA sensor cyclic GMP-AMP synthase (cGAS) is necessary for detection of HIV by human dendritic cells and macrophages. However, synthesis of HIV DNA during infection is not sufficient for immune activation. The capsid protein, which associates with viral DNA, has a pivotal role in enabling cGAS-mediated immune activation. We now find that NONO is an essential sensor of the HIV capsid in the nucleus. NONO protein directly binds capsid with higher affinity for weakly pathogenic HIV-2 than highly pathogenic HIV-1. Upon infection, NONO is essential for cGAS activation by HIV and cGAS association with HIV DNA in the nucleus. NONO recognizes a conserved region in HIV capsid with limited tolerance for escape mutations. Detection of nuclear viral capsid by NONO to promote DNA sensing by cGAS reveals an innate strategy to achieve distinction of viruses from self in the nucleus.

Repairing tears in the nuclear envelope The nuclear envelope segregates genomic DNA from the cytoplasm and regulates protein trafficking between the cytosol and the nucleus. Maintaining nuclear envelope integrity during interphase is considered crucial. However, Raab et al. and Denais et al. show that migrating immune and cancer cells experience frequent and transitory nuclear envelope ruptures when they move through tight spaces (see the Perspective by Burke). The nuclear envelope reseals rapidly during interphase, assisted by components of the ESCRT III membrane-remodeling machinery. Science , this issue pp. 359 and 353 ; see also p. 295

High-throughput screening of samples is the strategy of choice to detect occupational exposure biomarkers, yet it requires a user-friendly apparatus that gives relatively prompt results while ensuring high degrees of selectivity, precision, accuracy and automation, particularly in the preparation process. Miniaturization has attracted much attention in analytical chemistry and has driven solvent and sample savings as easier automation, the latter thanks to the introduction on the market of the three axis autosampler. In light of the above, this contribution describes a novel user-friendly solid-phase microextraction (SPME) off- and on-line platform coupled with gas chromatography and triple quadrupole-mass spectrometry to determine urinary metabolites of polycyclic aromatic hydrocarbons 1- and 2-hydroxy-naphthalene, 9-hydroxy-phenanthrene, 1-hydroxy-pyrene, 3- and 9-hydroxy-benzoantracene, and 3-hydroxy-benzo[a]pyrene. In this new procedure, chromatography&rsquo, s sensitivity is combined with the user-friendliness of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide on-fiber SPME derivatization using direct immersion sampling, moreover, specific isotope-labelled internal standards provide quantitative accuracy. The detection limits for the seven OH-PAHs ranged from 0.25 to 4.52 ng/L. Intra-(from 2.5 to 3.0%) and inter-session (from 2.4 to 3.9%) repeatability was also evaluated. This method serves to identify suitable risk-control strategies for occupational hygiene conservation programs.

Along with CD4 + T lymphocytes, macrophages are a major cellular source of HIV-1 replication and a potential viral reservoir. Following entry and reverse transcription in macrophages, cloaking of the viral cDNA by the HIV-1 capsid limits its cytosolic detection, enabling efficient replication. However, whether incoming HIV-1 particles are sensed by macrophages prior to reverse transcription remains unclear. Here, we show that HIV-1 triggers a broad expression of interferon (IFN)-stimulated genes (ISG) in monocyte-derived macrophages within a few hours after infection. This response does not require viral reverse transcription or the presence of HIV-1 RNA within particles, but viral fusion is essential. This response is elicited by viruses carrying different envelope proteins and thus different receptors to proceed for viral entry. Expression of ISG in response to viral entry requires TBK1 activity and type I IFNs signaling. Remarkably, the ISG response is transient but affects subsequent viral spread. Together, our results shed light on an early step of HIV-1 sensing by macrophages at the level of entry, which confers an early protection through type I IFN signaling and has potential implications in controlling the infection. IMPORTANCE HIV infection is restricted to T lymphocytes and macrophages. HIV-1-infected macrophages are found in many tissues of infected patients, even under antiretroviral therapy, and are considered a viral reservoir. How HIV-1 is detected and what type of responses are elicited upon sensing remain in great part elusive. The kinetics and localization of the production of cytokines such as interferons in response to HIV is of critical importance to understanding how the infection and the immune response are established. Our study provides evidence that macrophages can detect HIV-1 as soon as it enters the cell. Interestingly, this sensing is independent of the presence of viral nucleic acids within the particles but requires their fusion with the macrophages. This triggers a low interferon response, which activates an antiviral program protecting cells against further viral challenge and thus potentially limiting the spread of the infection.

A set of structures designed for the recognition of glucosides has been obtained by systematically destructuring a tripodal aminopyrrolic cage receptor that selectively recognizes octyl-?-D-glucopyranoside (Oct?Glc). NMR spectroscopy and isothermal titration calorimetry binding measurements showed that cleavage of one pillar of the cage was beneficial to the binding properties of the receptor, as long as two residual amino groups of the cleaved pillar were present. Removal of these two residual amino groups produced a dramatic loss of affinity for Oct?Glc of the resulting monocyclic analogue of the parent cage receptor. A significant improvement in the binding ability was achieved by replacing one pillar with two aminopyrrolic hydrogen-bonding arms, despite the loss of a preorganized structure. In contrast to the cage receptor, recognition of Oct?Glc was observed, even in a competitive medium (30% DMF in chloroform). Structural studies in solution, carried out through NMR spectroscopy and molecular modeling calculations, led to the elucidation of the 3D binding modes of the side-armed monocyclic receptors; this highlighted the key role of the amino groups and demonstrated the occurrence of a rotaxane-like complex, which featured the octyl chain of the glucoside threaded through the macrocyclic ring. Comfort is paramount! A set of structures designed for the recognition of glucosides has been obtained by systematically destructuring a tripodal aminopyrrolic cage receptor. A ?-glucoside is more effectively recognized by an adaptive cleft than by a preorganized cage, if a more comfortable fit can be achieved (see picture). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthetic ditopic receptors, designed for the molecular recognition of dimannosides, have been prepared by bridging two monotopic units effectively recognizing mannosides with linkers of the appropriate size and flexibility, endowed with hydrogen-bonding groups. Affinities toward the α and β glycosides of the biologically relevant Manα(1-2)Man disaccharide were measured by NMR spectroscopy and isothermal titration calorimetry (ITC) in polar organic media (30-40 % DMF in chloroform). Significant selectivities and affinities in the micromolar range were observed in most cases, with two newly designed receptors being the most effective receptors of the set, together with a distinct preference of the dimannosides for the (S) enantiomer of the receptor in all cases. A 3D view of the recognition mode was elucidated by a combined NMR spectroscopic/molecular modeling approach, showing the dimannoside included in the cleft of the receptor. Compared to the monotopic precursors, the ditopic receptors showed markedly improved recognition properties, proving the efficacy of the modular receptor design for the recognition of disaccharides.

Aicardi–Goutières syndrome (AGS) provides a monogenic model of nucleic acid‐mediated inflammation relevant to the pathogenesis of systemic autoimmunity. Mutations that impair ribonuclease (RNase) H2 enzyme function are the most frequent cause of this autoinflammatory disorder of childhood and are also associated with systemic lupus erythematosus. Reduced processing of either RNA:DNA hybrid or genome‐embedded ribonucleotide substrates is thought to lead to activation of a yet undefined nucleic acid‐sensing pathway. Here, we establish Rnaseh2b A174T/A174T knock‐in mice as a subclinical model of disease, identifying significant interferon‐stimulated gene (ISG) transcript upregulation that recapitulates the ISG signature seen in AGS patients. The inflammatory response is dependent on the nucleic acid sensor cyclic GMP‐AMP synthase (cGAS) and its adaptor STING and is associated with reduced cellular ribonucleotide excision repair activity and increased DNA damage. This suggests that cGAS/STING is a key nucleic acid‐sensing pathway relevant to AGS, providing additional insight into disease pathogenesis relevant to the development of therapeutics for this childhood‐onset interferonopathy and adult systemic autoimmune disorders.

What are they like? This chapter would like to be a pragmatic user's guide in the non-trivial and yet challenging task of designing effective synthetic receptors for molecular recognition of carbohydrates. In the next pages the reader will go through a critical overview of the recent literature focused on systematic studies concerned with the understanding of the structural and functional features that can lead to effective recognition of carbohydrates.

Viruses pack antiviral mediators Viruses often hijack host proteins for their own use, turning host cells into virion-spewing machines. However, Bridgeman et al. and Gentili et al. now report a sneaky way that the host can fight back (see the Perspective by Schoggins). Host cells that expressed the enzyme cGAS, an innate immune receptor that senses cytoplasmic DNA, packaged the cGAS-generated second messenger cGAMP into virions. Virions could then transfer cGAMP to neighboring cells, triggering an antiviral gene program in these newly infected cells. Such transfer of an antiviral mediator may help to speed up the immune response to put the brakes on viral spread. Science , this issue pp. 1228 and 1232 ; see also p. 1166

In this work we have investigated the binding properties of a new synthetic receptor for phosphate anions that combines metal ion coordination with electrostatic and H-bonding interactions. The described receptor is obtained by assembling an iminodiacetic (IDA) fragment, as a Zn(II) binding site, with a polyamine macrocyclic portion containing two trans-1,2-diaminocyclohexane (DAC) units and a pyrrole ring, as a cationic binding site, into an adaptive structure appropriately spanning the length of di- and tridentate phosphates. Potentiometric measurements together with (1)H and (31)P NMR investigation showed that, in a wide pH range including values of physiological interest, the Zn(II) complex of the receptor binds di- and triphosphates, such as ADP, ATP, pyrophosphate (PP) and triphosphate (TP), far better than monophosphate (MP), and that TP is poorly bound by methyliminodiacetate (MIDA) as a model for the Zn(II) binding site. Besides the excellent selectivity over other phosphates, the affinity for TP is the largest reported to date for Zn(II) complexes in water.

International audience; HIV-2 is less pathogenic for humans than HIV-1 and might provide partial cross-protection from HIV-1-induced pathology. Although both viruses replicate in the T cells of infected patients, only HIV-2 replicates efficiently in dendritic cells (DCs) and activates innate immune pathways. How HIV is sensed in DC is unknown. Capsid-mutated HIV-2 revealed that sensing by the host requires viral cDNA synthesis, but not nuclear entry or genome integration. The HIV-1 capsid prevented viral cDNA sensing up to integration, allowing the virus to escape innate recognition. In contrast, DCs sensed capsid-mutated HIV-1 and enhanced stimulation of T cells in the absence of productive infection. Finally, we found that DC sensing of HIV-1 and HIV-2 required the DNA sensor cGAS. Thus, the HIV capsid is a determinant of innate sensing of the viral cDNA by cGAS in dendritic cells. This pathway might potentially be harnessed to develop effective vaccines against HIV-1.

Dendritic cells (DCs) associate the capacity to sense pathogens with the initiation of adaptive immunity. Pathogens can be sensed through pathogen-associated molecular patterns by pathogen-recognition receptors expressed on host cells. Pathogen-encoded activities can also be sensed when they modify normal host cellular processes. The diversity of pathogen sensors has been highlighted by the identification of several cytosolic sensors involved in the recognition of nucleic acids from pathogens. The number of these pathogen cytosolic sensors has dramatically increased recently. Different DC populations appear to be equipped with distinct sensors but the precise expression pattern and the regulation of these sensors remain to be established, especially in humans. The engagement of sensors in DCs by pathogens leads to antipathogen effects through multiple mechanisms including interferon responses and promotes effector pathways that can shape the adaptive immune response. How the diversity of cytosolic pathogen sensors impacts these processes is incompletely understood. Investigating the expression, regulation, and crosstalk of the sensors should shed light on how pathogen sensing impacts pathogen replication and host immune responses.

Dendritic cells (DCs) associate the capacity to sense pathogens with the initiation of adaptive immunity. Pathogens can be sensed through pathogen-associated molecular patterns by pathogen-recognition receptors expressed on host cells. Pathogen-encoded activities can also be sensed when they modify normal host cellular processes. The diversity of pathogen sensors has been highlighted by the identification of several cytosolic sensors involved in the recognition of nucleic acids from pathogens. The number of these pathogen cytosolic sensors has dramatically increased recently. Different DC populations appear to be equipped with distinct sensors but the precise expression pattern and the regulation of these sensors remain to be established, especially in humans. The engagement of sensors in DCs by pathogens leads to antipathogen effects through multiple mechanisms including interferon responses and promotes effector pathways that can shape the adaptive immune response. How the diversity of cytosolic pathogen sensors impacts these processes is incompletely understood. Investigating the expression, regulation, and crosstalk of the sensors should shed light on how pathogen sensing impacts pathogen replication and host immune responses.

The carbohydrate recognition properties of synthetic tripodal receptors relying on H-bonding interactions have highlighted the crucial role played by the functional groups matching saccharidic hydroxyls. Herein, pyrrole and pyridine, which emerged as two of the most effective H-bonding groups, were quantitatively compared through their isostructural substitution within the architecture of a shape-persistent bicyclic cage receptor. NMR and ITC binding studies gave for the pyrrolic receptor a 20-fold larger affinity toward octyl-β-d-glucopyranoside in CDCl(3), demonstrating the superior recognition properties of pyrrole under conditions in which differences would depend on the intrinsic binding ability of the two groups. The three-dimensional structures of the two glucoside complexes in solution were elucidated by combined NMR and molecular mechanics computational techniques, showing that the origin of the stability difference between the two closely similar complex structures resides in the ability of pyrrole to establish shorter/stronger H-bonds with the glucosidic ligand compared to pyridine.

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