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The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that facilitates the acidification of intracellular compartments in eukaryotic cells and plays an important role in receptor-mediated endocytosis, intracellular trafficking processes, and protein degradation. In this study we show that the C-terminal fragment of 350 residues of the regulatory subunit H (V1H) of the V-ATPase shares structural and functional homologies with the β-chains of adaptor protein complexes. Moreover, the fragment is similar to a region in the β-subunit of COPI coatomer complexes, which suggests the existence of a shared domain in these three different families of proteins. For β-adaptins, this fragment binds to cytoplasmic di-leucine–based sorting motifs such as in HIV-1 Nef that mediate endocytic trafficking. Expression of this fragment in cells blocks the internalization of transmembrane proteins, which depend on di-leucine–based motifs, whereas mutation of the consensus sequence GEY only partly diminishes the recognition of the sorting motif. Based on recent structural analysis, our results suggest that the di-leucine-binding domain consists of a HEAT or ARM repeat protein fold.

Background The Delta/Notch like EGF-related receptor (DNER) is a single-pass transmembrane protein with characteristic EGF-like repeats in the extracellular domain, similar to those of the Notch receptor and its ligand Delta. DNER is an activator of the Notch signaling pathway, which plays a major role in cell fate determination and differentiation. In our previous study, an overexpression of DNER was observed in lesional areas of human osteoarthritic articular cartilage when compared to unaffected zones of the same tissue. Objectives Based on these findings we analyzed the role of DNER in the chondrogenic differentiation of human mesenchymal stem cells (hMSC). Methods HMSCs were nucleofected with an overexpression vector for DNER and a control vector. Chondrogenic differentiation of the hMSCs was performed in a pellet mass culture system for 28 days. The culture medium was supplemented with transforming growth factor beta 3 (TGF-β3) and dexamethasone. Overexpression of DNER and the effect on specific chondrogenic markers like aggrecan, collagen type 1 and 2, Sox9, collagen type 10 were analyzed with immunohistochemical staining and semi-quantitative real-time PCR. Chondrogenesis of the hMSCs was monitored using alcian blue staining. Results Alcian blue staining increased over time assuming chondrogenic differentiation of the hMSCs. Histologically, an overexpression of DNER was detected 7 days after nucleofection and remained stable during chondrogenesis, whereas no staining of DNER could be detected in the mock-transfected cells (ctr cells). Collagen 1 expression was higher in the ctr cells at the beginning of chondrogenesis compared to the pDNER-nucleofected cells (pDNER cells) and decreased in both over time. Collagen 2 was slightly expressed from day 21 in the ctr and pDNER cells. Sox9 was greater expressed in the ctr cells at the beginning of chondrogenesis and decreased in a time-dependent manner. In pDNER cells the Sox9 expression increased at day 14 and remained stable over time. The collagen 10 level is constantly higher in the pDNER nucleofected cells compared to the ctr cells. In both, the expression increased in a time-dependent manner. Aggrecan expression increased during chondrogenesis in the ctr and pDNER cells but the expression was higher in the pDNER nucleofected cells on day 28. The semi-quantitative real-time PCR data confirmed the overexpression of DNER 2 days after nucleofection and the effect, that collagen 1 expression is downregulated and collagen 2 expression upregulated in the pDNER transfected cells compared to control until day 14. Conclusions Since DNER overexpression led to a downregulation of collagen type 1 in the hMSCs, while the expression of collagen type 2 and aggrecan were upregulated, our results support the idea that DNER might positively influence chondrogenesis and exert a reparative role during OA pathophysiology. This promotion might be regulated via an intermediate overexpression of collagen type 10. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5616

Ankyrin repeats are well-known structural modules that mediate interactions between a wide spectrum of proteins. The regulatory factor X with ankyrin repeats (RFXANK) is a subunit of a tripartite RFX complex that assembles on promoters of major histocompatibility complex class II (MHC II) genes. Although it is known that RFXANK plays a central role in the nucleation of RFX, it was not clear how its ankyrin repeats mediate the interactions within the complex and with other proteins. To answer this question, we modeled the RFXANK protein and determined the variable residues of the ankyrin repeats that should contact other proteins. Site-directed alanine mutagenesis of these residues together with in vitro and in vivo binding studies elucidated how RFXAP and CIITA, which simultaneously interact with RFXANK in vivo, bind to two opposite faces of its ankyrin repeats. Moreover, the binding of RFXAP requires two separate surfaces on RFXANK. One of them, which is located in the ankyrin groove, is severely affected in the FZA patient with the bare lymphocyte syndrome. This genetic disease blocks the expression of MHC II molecules on the surface of B cells. By pinpointing the interacting residues of the ankyrin repeats of RFXANK, the mechanism of this subtype of severe combined immunodeficiency was revealed.

The accessory Nef protein of HIV and SIV is essential for viral pathogenesis, yet it is perplexing in its multitude of molecular functions. In this review we analyse the structure–function relationships of motifs recently proposed to play roles in aspects of Nef modification, signalling and trafficking, and thereby to impinge on the ability of the virus to survive in, and to manipulate, its cellular host. Based on the full-length structure assembly of HIV Nef, we correlate surface accessibility with secondary structure elements and sequence conservation. Motifs involved in Nef-mediated CD4 and MHC I downregulation are located in flexible regions of Nef, suggesting that the formation of the transient trafficking complexes involved in these processes depends on the recognition of primary sequences. In contrast, the interaction sites for signalling molecules that contain SH3 domains or the p21-activated kinases are associated with the well folded core domain, suggesting the recognition of highly structured protein surfaces.

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIVmac239for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity.

The activation of Nef-associated kinase (NAK) by Nef from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. This induction occurs via the guanine nucleotide exchange factor Vav and the small GTPases Rac1 and Cdc42. In this study, we identified NAK as p21-activated kinase 1 (PAK1). PAK1 bound to Nef in vitro and in vivo. Moreover, the induction of cytoskeletal rearrangements such as the formation of trichopodia, the activation of Jun N-terminal kinase, and the increase of viral production were blocked by an inhibitory peptide that targets the kinase activity of PAK1 (PAK1 83-149). These results identify NAK as PAK1 and emphasize the central role its kinase activity plays in cytoskeletal rearrangements and cellular signaling by Nef.

Transcriptional transactivators (Tat) from human immunodeficiency and equine infectious anemia viruses (HIV and EIAV) interact with their transactivation response elements (TAR) to increase the rates of viral transcription. Whereas the human cyclin T1 is required for the binding of Tat to TAR from HIV, it is unknown how Tat from EIAV interacts with its TAR. Furthermore, Tat from EIAV functions in equine and canine cells but not in human cells. In this study, we present sequences of cyclins T1 from horse and dog and demonstrate that their N-terminal 300 residues rescue the transactivation of Tat from EIAV in human cells. Although human and equine cyclins T1 bind to this Tat, only the equine cyclin T1 supports the binding of Tat to TAR from EIAV. Finally, a reciprocal exchange of the valine for the leucine at position 29 in human and equine cyclins T1, respectively, renders the human cyclin T1 active and the equine cyclin T1 inactive for Tat transactivation from EIAV. Thus, the collaboration between a specific cyclin T1 and Tat for their high-affinity interaction with TAR is a common theme of lentiviral transactivation.

The structure of the complex of Ras with the Ras-binding domain of its effector RalGDS (RGS-RBD), the first genuine Ras-effector complex, has been solved by X-ray crystallography. As with the Rap-RafRBD complex (Nasser et al., 1995), the interaction is via an inter-protein β-sheet between the switch I region of Ras and the second strand of the RGS-RBD sheet, but the details of the interactions in the interface are remarkably different. Mutational studies were performed to investigate the contribution of selected interface residues to the binding affinity. Gel filtration experiments show that the Ras·RGS-RBD complex is a monomer. The results are compared to a recently determined structure of a similar complex using a Ras mutant (Huang et al., 1998) and are discussed in relation to partial loss-of-function mutations and the specificity of Ras versus Rap binding.

Cellular signaling downstream of Ras is highly diversified and may involve many different effector molecules. A potential candidate is AF6 which was originally identified as a fusion to ALL-1 in acute myeloid leukemia. In the present work the interaction between Ras and AF6 is characterized and compared with other effectors. The binding characteristics are quite similar to Raf and RalGEF, i.e. nucleotide dissociation as well as GTPase-activating protein activity are inhibited, whereas the intrinsic GTPase activity of Ras is unperturbed by AF6 binding. Particularly, the dynamics of interaction are similar to Raf and RalGEF with a lifetime of the Ras. AF6 complex in the millisecond range. As probed by 31P NMR spectroscopy one of two major conformational states of Ras is stabilized by the interaction with AF6. Looking at the affinities of AF6 to a number of Ras mutants in the effector region, a specificity profile emerges distinct from that of other effector molecules. This finding may be useful in defining the biological function of AF6 by selectively switching off other pathways downstream of Ras using the appropriate effector mutant. Notably, among the Ras-related proteins AF6 binds most tightly to Rap1A which could imply a role of Rap1A in AF6 regulation.

Background In mammals, tissue or organ damage often results in fibrotic scar formation, which in turn may impair proper organ function. Many urodele amphibians are capable of regenerating different tissues including lenses, heart, and spinal cord or even entire body parts like limbs or tails without any functional impairment. We previously established the red-spotted newt Notophthalmus viridescens as a model organism to study endogenous knee joint regeneration in adult vertebrates. After surgical removal of articular cartilage from the femur or after intra-articular collagenase injection, newts displayed osteoarthritis (OA)-like symptoms, including joint instability and luxation. However, after approximately 3 months the joint function was completely restored in the treated animals. To evaluate the underlying molecular mechanisms guiding this regenerative process, a cDNA approach was used in this study. Methods A cDNA array derived from regenerating newt myocardium containing 100000 normalised expressed sequence tags (ESTs) was carried out after surgically and collagenase-induced knee damage in newts to identify key players involved in knee joint regeneration. Several ESTs were found to be regulated during the regenerative process. By using public databases, several deregulated candidate genes were identified and selected for further analysis on mRNA (Real‑Time PCR) and protein level (immunohistochemistry). Results In the cDNA array, a number of genes was found to be deregulated during the course of knee joint regeneration, including several matricellular proteins like tenascin-C (TN-C), periostin and osteonectin/SPARC. Among them, TN-C showed the strongest upregulation during the regenerative process and was selected for further studies. On the mRNA level, a strong upregulation of TN-C mRNA was found in both models: While in the collagenase model TN-C was upregulated at 10 days and 40 days after OA induction, TN-C expression ceased in the surgically-induced OA model at day 40. In immunohistochemical analyses, TN-C expression was restricted to the periosteum 10 days after damage. As knee joint regeneration progressed, TN-C was also found in regenerating cartilage (40 days after damage). Conclusions TN-C is involved in the regenerative process after OA induction in the newt. In vitro siRNA knockdown of TN-C might help to identify the molecular pathways involved. Analysis of the selected candidate genes and identification of underlying pathways guiding regeneration might ease the development of new treatment modalities of OA in humans.

The electrical signalling properties of neurons originate largely from the gating properties of their ion channels. N-type inactivation of voltage-gated potassium (Kv) channels is the best-understood gating transition in ion channels, and occurs by a 'ball-and-chain' type mechanism. In this mechanism an N-terminal domain (inactivation gate), which is tethered to the cytoplasmic side of the channel protein by a protease-cleavable chain, binds to its receptor at the inner vestibule of the channel, thereby physically blocking the pore. Even when synthesized as a peptide, ball domains restore inactivation in Kv channels whose inactivation domains have been deleted. Using high-resolution nuclear magnetic resonance (NMR) spectroscopy, we analysed the three-dimensional structure of the ball peptides from two rapidly inactivating mammalian K. channels (Raw3 (Kv3.4) and RCK4 (Kv1.4)). The inactivation peptide of Raw3 (Raw3-IP) has a compact structure that exposes two phosphorylation sites and allows the formation of an intramolecular disulphide bridge between two spatially close cysteine residues. Raw3-IP exhibits a characteristic surface charge pattern with a positively charged, a hydrophobic, and a negatively charged region. The RCK4 inactivation peptide (RCK4-IP) shows a similar spatial distribution of charged and uncharged regions, but is more flexible and less ordered in its amino-terminal part.

Background The adult red-spotted newt ( notophthalmus viridescens ) is able to regenerate damaged knee joints after local injury involving surgically induced defects, and collagenase induced joint instability. The mechanism behind this capacity that lacks in mammals is currently not understood. Objectives Investigating the molecular process of joint regeneration in newts and deducing the potential gene candidate Methods Transcriptome analysis with cDNA array derived from regenerating newt myocardium was carried out after surgical and collagenase, induced knee damage. We validated the differentially expressed genes in both instances using qRT-PCR. We conducted immunohistochemistry (IHC)and in situ hybridization (ISH) to investigate spatial distribution of the most promising deregulated genes. Results 88 from 260 significantly regulated gene transcripts had homology to gene transcripts in public DNA database. We selected the deregulated genes retinoic acid receptor response element 3 (RARR), periostin, tenascin-C (TN-C), olfactomedin, osteonektin, decorin, gamma B crystalline (GBC), thymosin β 4 and complement factor B for further analysis after qPCR-confirmation of deregulation. Tenascin C, the most upregulated gene, locates to the periosteum and perichondrium in newts and is additionally strongly expressed in the undifferentiated regeneration tissue after joint injury. We are currently analyzing the other candidate genes. Conclusions We found several gene candidates which may be central to the newts capacity for joint regeneration by employing cDNA array derived from regenerating newt myocardium. TN-C may be an important mediator of joint formation. References Matthias Geyer, Thilo Borchardt, Carina Schreiyack, et al. Endogenous regenration after collagenase -induced knee joint damage in the adult newt notophthalmus viridescens. Ann Rheum Dis (2010). doi:10.1136/ard.2009.126136. James W. Godwin, Jeremy P. Brockes. Regeneration, tissue injury and the immune response. J. Anat. (2006) 209: 423-432 Thilo Borchardt, Mario Looso, Marc Bruckskotten, et al. Analysis of newly established EST databases reveals similarity between heart regeneration in newt and fish. BMC Genomic (2010): 11:4 Disclosure of Interest None Declared

Controlling the hydrolysis rate of GTP bound to guanine nucleotide binding proteins is crucial for the right timing of many biological processes. Theoretical, structural, and functional studies have demonstrated that in p21ras the substrate of the reaction, GTP itself, plays a central role by acting as the base catalyst. This substrate-assisted reaction mechanism was analyzed with the help of linear free energy relationships (LFERs). Here we present experimental data that further support the proposed mechanism. We extend the LFER analysis to a wide range of oncogenic as well as nontransforming Ras mutants. It is illustrated that almost all Ras variants follow the observed LFER and thus also the same reaction path. Further, the reduced GTPase reaction rate that characterizes the oncogenic effect of many of the p21 mutants found in human tumors seems to be a consequence of a slightly reduced pKa of the gamma-phosphate group of bound GTP. Factors causing a pKa deviation of just 0.5 unit are enough to slow the intrinsic GTPase reaction rate significantly, and the system may exhibit as a consequence of this an oncogenic potential. Interestingly, we also found oncogenic mutations that do not follow the regular LFER. This suggests that the oncogenic effect of distinct Ras mutants has a different physical origin. The results presented might aid in the design of drugs aimed at reactivating the GTPase reaction of many oncogenic p21ras mutants. We also analyzed the stimulated GTPase reaction of p21ras by the GTPase activating protein (GAP) and the GTPase reaction of Rap1A, a Ras-related GTP binding protein, with similar approaches. The corresponding results indicate that the GAP-stimulated GTPase as well as the Rap1A-catalyzed reaction seem to follow the same substrate-assisted reaction mechanism. However, the correlation coefficient for the GAP-catalyzed reaction is different from the corresponding coefficient for the intrinsic reaction. While the intrinsic reaction exhibits a Brønsted slope of beta = 2.1, the corresponding value for the GAP-activated reaction is beta = 4.9.

Recombinant N-methyl-D-aspartate receptors composed of NR1/NR2A subunits were expressed in Xenopus oocytes to analyse the voltage-dependent and use-dependent channel blocking activity of argiotoxin636. Functional assays demonstrate that the toxin competes with other open channel blockers such as Mg2+ and MK-801. Direct binding or competition assays using radiolabeled ligands and isolated rat brain membranes, in contrast, reveal no specific binding or yield binding constants which differ by orders of magnitude from the IC50 values of the functional assays. One explanation is that argiotoxin636 does not bind with high affinity to the inhibitory site in the N-methyl-D-aspartate-receptor channel under in vitro conditions when membranes are depolarised. The structure of argiotoxin636 was investigated by NMR spectroscopy. In solution the positively charged argiotoxin636 acquires an extended conformation and its dimensions might allow permeation deep into the channel. In the absence of direct structural information on the channel protein, the detailed analysis of blockade in conjunction with structural information, as provided here, may be of aid in the deduction of structural features of glutamate-receptor channel ion pores.

AURELIA is an advanced program for the computer-aided evaluation of two-, three- and four-dimensional NMR spectra of any type of molecule. It can be used for the analysis of spectra of small molecules as well as for evaluation of complicated spectra of biological macromolecules such as proteins. AURELIA is highly interactive and offers a large number of tools, such as artefact reduction, cluster and multiplet analysis, spin system searches, resonance assignments, automated calculation of volumes in multidimensional spectra, calculation of distances with different approaches, including the full relaxation matrix approach, Bayesian analysis of peak features, correlation of molecular structures with NMR data, comparison of spectra via spectral algebra and pattern match techniques, automated sequential assignments on the basis of triple resonance spectra, and automatic strip calculation. In contrast to most other programs, many tasks are performed automatically.

Despite many advances in understanding the structure and function of GTP-binding proteins the mechanism by which these molecules switch from the GTP-bound on-state to the GDP-bound off-state is still poorly understood. Theoretical studies suggest that the activation of the nucleophilic water which hydrolyzes GTP needs a general base. Such a base could not be located in any of the many GTP-binding proteins. Here we present a unique type of linear free energy relationships that not only supports a mechanism for p21ras in which the substrate GTP itself acts as the catalytic base driving the GTPase reaction but can also help to explain why certain mutants of p21ras are oncogenic and others are not.

The three-dimensional structures and biochemical properties of two mutants of the G-domain (residues 1-166) of p21H-ras, p21 (G12D) and p21 (G12P), have been determined in the triphosphate-bound form using guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp). They correspond to the most frequent oncogenic and the only nononcogenic mutation of Gly-12, respectively. The G12D mutation is the only mutant analyzed so far that crystallizes in a space group different from wild type, and the atomic model of the protein shows the most drastic changes of structure around the active site as compared to wild-type p21. This is due to the interactions of the aspartic acid side chain with Tyr-32, Gln-61, and the gamma-phosphate, which result in reduced mobility of these structural elements. The interaction between the carboxylate group of Asp-12 and the gamma-phosphate is mediated by a shared proton, which we show by 31P NMR measurements to exist in solution as well. The structure of p21 (G12P) is remarkably similar to that of wild-type p21 in the active site, including the position of the nucleophilic water. The pyrrolidine ring of Pro-12 points outward and seems to be responsible for the weaker affinity toward GAP (GTPase-activating protein) and the failure of GAP to stimulate GTP hydrolysis.

Due to ongoing developments of novel agents in the field of biological pharmacotherapy, there are now more arrows available in clinicians' quivers for the treatment of rheumatic conditions. As a consequence, however, clear treatment strategies have to be defined in order to guarantee a qualitatively high and individually stage-adapted, state-of-the-art regimen for affected patients. This review summarizes recent evidence regarding the rationale of using different biological therapies to treat rheumatoid arthritis, the most common inflammatory joint disorder after activated osteoarthritis, and draws an actual picture of a possible standardized therapeutic algorithm without claiming exclusive appropriateness.

Osteoarthritis causes pain and loss of function of affected joints. To date, no causative treatment is available and endogenous regeneration of damaged mammalian joint structures appears impossible. Since amphibians are able to regenerate whole limbs, we hypothesised that they might be able to repair joints after local joint damage. Knee joints of adult newts (Notophthalmus viridescens) were injected with collagenase. The clinical and histological courses were analysed. A parallel in …

Today, two-dimensional NMR spectroscopy has become a well-accepted method for the determination of the three-dimensional structures of biological macromolecules such as proteins in solution. The primary evaluation of the spectroscopic data is very time-consuming and complicated, therefore computer assistance grows more and more important. It is not surprising that a number of groups work in the field of pattern recognition in n-dimensional NMR spectra of macromolecules (n ≥ 2). Correspondingly, a number of different methods were described that can solve at least some special aspects of this problem1–29.

Expression of human endogenous retrovirus K (HERV-K) is associated with germ-cell neoplasia. HERV-K encodes a protein of the Rev/Rex family, cORF, that supports cellular transformation and binds the promyelocytic leukemia zinc finger (PLZF) protein implicated in spermatogenesis. Rev/Rex function invariably depends on multimerization. Here we show that cORF likewise self-associates to form higher-order oligomers. Amino acids (aa) 47–87 in cORF are sufficient, aa 75–87 essential for self-association. Consistently, this domain is predicted to form a hydrophobic α-helix that may represent an oligomerization interface. The existence of a dimerization-competent cORF mutant lacking PLZF-binding activity (cORF47–87) suggests a way of dominant negative inhibition of the proposed tumor susceptibility factor cORF.

The diaphanous-related formin homology 2 domain containing protein 1 (FHOD1) interacts with the Rac GTPase and activates the Rho-ROCK cascade leading to the formation of actin stress fibers. Here, we report the detection of homotypic interactions of FHOD1 in the yeast two-hybrid system, by co-immunoprecipitation and co-localization in mammalian cells. A predicted coiled-coil motif C-terminal to the core FH2 domain, but not the core FH2 domain itself, was critical for self-association of FHOD1. Deletion of both the coiled-coil motif and the core FH2 domain abrogated formation of actin stress fibers and activation of transcription of the serum response element by FHOD1. In contrast, these motifs were dispensable for the physical and functional interaction of FHOD1 with Rac1. Together, these results indicate that oligomerization of FHOD1 via the coiled-coil motif is a critical parameter for its biological activities.

The accessory Nef protein from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. Here we present an assembly of the full length structure of HIV-1 Nef, allele NL4-3, based on the previously solved anchor and core domain structures. The center part of the 33 residue encompassing flexible loop at the C-terminus of Nef, involved in Nef internalization and CD4 endocytosis, has been modelled. The degree of sequence conservation in HIV-1 Nef proteins was determined using a total of 186 different strains from five different subtypes. The sequence conservation has been correlated with the accessible surface area and with secondary structure features for individual residues. The high amount of flexible regions in Nef accounts for the large surface and the multiple interaction sites the protein exhibits.

Summary Simian immunodeficiency virus (SIVsmm) infection of sooty mangabeys ( Cercocebus atys ) is characterized by stable CD4 + T cell counts despite high plasma levels of CCR5-tropic viruses. However, in rare instances, SIVsmm acquires CXCR4 coreceptor tropism and causes severe CD4 + T cell depletion, albeit without clinical signs of immunodeficiency. Here, we show that CXCR4-tropic SIVsmm strains lost their ability to downmodulate TCR-CD3 by evolving unusual Nef mutations that initially reduced (I132V) and subsequently disrupted (I123L and L146F) interaction with the CD3 ζ chain. This coevolution of Env and Nef function suggests that CD3 downmodulation is advantageous for viral replication in activated CCR5 + memory T cells, but not in resting naive CXCR4 + T cells that have not yet undergone TCR-CD3-mediated stimulation. This may explain why HIV-1, which generally lacks the CD3 downmodulation function, commonly switches to CXCR4 usage, whereas this is extremely rare for SIV strains that have retained this Nef activity.

Membrane association is believed to be a prerequisite for the biological activity of the HIV-1 pathogenicity factor Nef. Attachment to cellular membranes as well as incorporation into detergent-insoluble microdomains (lipid rafts) require the N-terminal myristoylation of Nef. However, this modification is not sufficient for sustained membrane association and a specific raft-targeting signal for Nef has not yet been identified. Using live cell confocal microscopy and membrane fractionation analyses, we found that the N-terminal anchor domain (aa 1–61) is necessary and sufficient for efficient membrane binding of Nef from HIV-1 SF2 . Within this domain, highly conserved lysine and arginine residues significantly contributed to Nef's membrane association and localization. Plasma membrane localization of Nef was also governed by an additional membrane-targeting motif between residues 40 and 61. Importantly, two lysines at positions 4 and 7 were not essential for the overall membrane association but critically contributed to Nef's incorporation into lipid raft domains. Cell surface receptor downmodulation was largely unaffected by mutations of all N-terminal basic residues, while the association of Nef with Pak2 kinase activity and its ability to augment virion infectivity correlated with its lysine-mediated raft incorporation. In contrast, all basic residues were required for efficient HIV-1 replication in primary human T lymphocytes but did not contribute to the incorporation of Nef into HIV-1 virions. Together, these results unravel that Nef's membrane association is governed by a complex pattern of signature motifs that differentially contribute to individual Nef activities. The identification of a critical raft targeting determinant and the functional characterization of a membrane-bound, non-raft-associated Nef variant indicate raft incorporation as a regulatory mechanism that determines the biological activity of distinct subpopulations of Nef in HIV-infected cells.

The Cdk12/CycK complex promotes expression of a subset of RNA polymerase II genes, including those of the DNA damage response. CDK12 is among only nine genes with recurrent somatic mutations in high-grade serous ovarian carcinoma. However, the influence of thesemutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations prevent formation of the Cdk12/CycK complex, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations reveals coordinated downregulation of genes critical to the homologous recombination DNA repair pathway. Moreover, we establish that the Cdk12/CycK complex occupies these genes and promotes phosphorylation of RNA polymerase II at Ser2. Accordingly, we demonstrate that the mutant Cdk12 proteins fail to stimulate the faithful DNA double strand break repair via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability underlying the genesis of the cancer.

SummaryAlthough poly(ADP-ribose) polymerase (PARP) inhibitors are active in homologous recombination (HR)-deficient cancers, their utility is limited by acquired resistance after restoration of HR. Here, we report that dinaciclib, an inhibitor of cyclin-dependent kinases (CDKs) 1, 2, 5, and 9, additionally has potent activity against CDK12, a transcriptional regulator of HR. In BRCA-mutated triple-negative breast cancer (TNBC) cells and patient-derived xenografts (PDXs), dinaciclib ablates restored HR and reverses PARP inhibitor resistance. Additionally, we show that de novo resistance to PARP inhibition in BRCA1-mutated cell lines and a PDX derived from a PARP-inhibitor-naive BRCA1 carrier is mediated by residual HR and is reversed by CDK12 inhibition. Finally, dinaciclib augments the degree of response in a PARP-inhibitor-sensitive model, converting tumor growth inhibition to durable regression. These results highlight the significance of HR disruption as a therapeutic strategy and support the broad use of combined CDK12 and PARP inhibition in TNBC.

Summary Objective To elucidate disease-specific molecular changes in osteoarthritis (OA) by analyzing the differential gene expression profile of damaged vs intact cartilage areas within the same joint of patients with OA of the knee using a combination of a novel RNA extraction technique and whole-genome oligonucleotide arrays. Methods The transcriptome of macroscopically affected vs intact articular cartilage as determined by visual assessment was analyzed using an optimized mill-based total RNA isolation directly from the tissue and high density synthetic oligonucleotide arrays. Articular cartilage samples were obtained from patients with OA of the knee. Expression of differentially regulated genes was validated by real-time quantitative polymerase chain reaction and immunohistochemistry. Results The amount of RNA obtained by the optimized extraction procedure was at least 1μg per 500mg of cartilage and fulfilled the common quality requirements. After hybridization onto HG-U133 Plus 2.0 GeneChips (Affymetrix), 28.6–51.7% of the probe sets on the microarray showed a detectable signal above the signal threshold in the individual samples. A subset of 411 transcripts, which appeared to be differentially expressed, was obtained when applying predefined filtering criteria. Of these, six genes were found to be up-regulated in the affected cartilage of all patients, including insulin-like growth factor binding protein 3 (IGFBP-3), wnt-1-inducible signaling protein 1 (WISP-1), aquaporin 1 (AQP-1), delta/notch-like EGF-repeat containing transmembrane (DNER), decay accelerating factor (DAF), complement factor I (IF). Conclusion The optimized methodical approach reported here not only allows to determine area-specific gene expression profiles of intraindividually different low-RNA containing OA cartilage specimens. In addition, this study also revealed novel genes not yet reported to play a role in the pathophysiology of joint destruction in OA.

Infiltration of HIV-1 infected and uninfected monocytes/macrophages in organs and tissues is a general phenomenon observed in progression of AIDS. HIV-1 Nef protein is considered as a progression factor in AIDS, and is released from HIV-1 infected cells. Here we show that extracellular Nef increases migration of monocytes. This effect is (i) concentration dependent, (ii) reaches the order of magnitude of that induced by fMLP or CC chemokine ligand 2 MCP-1, (iii) inhibited by anti-Nef monoclonal antibodies as well as by heating and (iiii) depends on a concentration gradient of Nef. Further, Nef does not elicit monocytic THP-1 cells to express chemokines such as CCL2, CCL3 and CCL4. These data suggest that extracellular Nef may contribute to disease progression as well as HIV-1 spreading through affecting migration of monocytes.

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