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The membrane-anchored c-Src tyrosine kinase mediates signaling from a wide range of cell surface receptors controlling cell growth, adhesion, and survival. c-Src deregulation is associated with cancer. Dimerization appears to be a novel layer of regulation through a yet unclear mechanism. Binding of c-Src tyrosine kinase to the plasma membrane is mediated by the myristoylated and strongly positively charged N-terminal SH4 domain. Although activation of c-Src is known to require phosphorylation by a second c-Src molecule, electrostatic repulsion between the charged residues was considered to prevent dimerization. Here we show that a cluster of positively charged lysine residues in c-Src SH4 domain not only does not prevent dimerization but, in fact, enhances it through a lipid-mediated process. Dimerization not only depends on the number of positive charges but also on their position and the nature of the charged residues. Replacement of lysine by arginine increases dimerization in vitro and in vivo and, in HEK293T cells, causes a two-fold increase in tyrosine phosphorylation. Lipid mediated protein-protein interactions induced by clusters of basic residues may represent a general mechanism for modulating cell signaling, consistent with the abundance of positively charged residues in the juxta membrane region of many signaling proteins.

Simple Summary The human kinome is composed of about 50 pseudo-kinases with unclear function, because they are predicted to be catalytically inactive; however, they are shown to play an important role in cancer, similar to active kinases. Understanding how these pseudo-kinases promote tumor formation despite their catalytic inactivity is a great challenge, which may lead to innovative anti-cancer therapies. The PEAK1 and 2 pseudo-kinases have emerged as important components of the protein tyrosine kinase pathway implicated in cancer progression. They can signal using a scaffolding mechanism via a conserved split helical dimerization (SHED) module. In this study, we uncovered a similar SHED-dependent oncogenic activity for PEAK3, a recently discovered new member of this family. We also show that this new signaling mechanism may be implicated in acute myeloid leukemia. Abstract The PEAK1 and Pragmin/PEAK2 pseudo-kinases have emerged as important components of the protein tyrosine kinase pathway implicated in cancer progression. They can signal using a scaffolding mechanism that involves a conserved split helical dimerization (SHED) module. We recently identified PEAK3 as a novel member of this family based on structural homology; however, its signaling mechanism remains unclear. In this study, we found that, although it can self-associate, PEAK3 shows higher evolutionary divergence than PEAK1/2. Moreover, the PEAK3 protein is strongly expressed in human hematopoietic cells and is upregulated in acute myeloid leukemia. Functionally, PEAK3 overexpression in U2OS sarcoma cells enhanced their growth and migratory properties, while its silencing in THP1 leukemic cells reduced these effects. Importantly, an intact SHED module was required for these PEAK3 oncogenic activities. Mechanistically, through a phosphokinase survey, we identified PEAK3 as a novel inducer of AKT signaling, independent of growth-factor stimulation. Then, proteomic analyses revealed that PEAK3 interacts with the signaling proteins GRB2 and ASAP1/2 and the protein kinase PYK2, and that these interactions require the SHED domain. Moreover, PEAK3 activated PYK2, which promoted PEAK3 tyrosine phosphorylation, its association with GRB2 and ASAP1, and AKT signaling. Thus, the PEAK1-3 pseudo-kinases may use a conserved SHED-dependent mechanism to activate specific signaling proteins to promote oncogenesis.

The PEAK1 and pragmin/PEAK2 pseudo-kinases have emerged as important components of the protein tyrosine kinase pathway implicated in cancer progression. They can signal by a scaffolding mechanism that involves a conserved split helical dimerization (SHED) module. We recently identified PEAK3 as a novel member of this family based on structural homology; however, its signalling mechanism remains unclear. Here, we found that although it can self-associate, PEAK3 shows higher evolutionary divergence than PEAK1/2. Moreover, PEAK3 protein is strongly expressed in human haematopoietic cells, and is upregulated in acute myeloid leukaemia. Functionally, PEAK3 overexpression in U2OS sarcoma cells enhanced their growth and migratory properties, while its silencing in THP1 leukemic cells reduced these effects. Importantly, an intact SHED module was required for these PEAK3 oncogenic activities. Mechanistically, through a phosphokinase survey, we identified PEAK3 as a novel inducer of AKT signalling, independent of growth factor stimulation. Then, proteomic analyses revealed that PEAK3 interacts with the signalling proteins GRB2 and ASAP1/2 and the protein kinase PYK2, and that these interactions require the SHED domain. Moreover, PEAK3 activated PYK2 to promote AKT signalling. Thus, the PEAK1-3 pseudo-kinases may use a conserved SHED-dependent mechanism to activate specific signalling proteins to promote oncogenesis.

The PEAK family pseudokinases are essential components of tyrosine kinase (TK) pathways that regulate cell growth and adhesion; however, their role in human cancer remains unclear. Here, we report an oncogenic activity of the pseudokinase PEAK2 in colorectal cancer (CRC). Notably, high PRAG1 expression, which encodes PEAK2, was associated with a bad prognosis in CRC patients. Functionally, PEAK2 depletion reduced CRC cell growth and invasion in vitro, while its overexpression increased these transforming effects. PEAK2 depletion also reduced CRC development in nude mice. Mechanistically, PEAK2 expression induced cellular protein tyrosine phosphorylation, despite its catalytic inactivity. Phosphoproteomic analysis identified regulators of cell adhesion and F-actin dynamics as PEAK2 targets. Additionally, PEAK2 was identified as a novel ABL TK activator. In line with this, PEAK2 expression localized at focal adhesions of CRC cells and induced ABL-dependent formation of actin-rich plasma membrane protrusions filopodia that function to drive cell invasion. Interestingly, all these PEAK2 transforming activities were regulated by its main phosphorylation site, Tyr413, which implicates the SRC oncogene. Thus, our results uncover a protumoural function of PEAK2 in CRC and suggest that its deregulation affects adhesive properties of CRC cells to enable cancer progression.

The membrane-anchored Src tyrosine kinase is involved in numerous pathways and its deregulation is involved in human cancer. Our knowledge on Src regulation relies on crystallography, which revealed intramolecular interactions to control active Src conformations. However, Src contains a N-terminal intrinsically disordered unique domain (UD) whose function remains unclear. Using NMR, we reported that UD forms an intramolecular fuzzy complex involving a conserved region with lipid-binding capacity named Unique Lipid-Binding Region (ULBR), which could modulate Src membrane anchoring. Here we show that the ULBR is essential for Src’s oncogenic capacity. ULBR inactive mutations inhibited Src transforming activity in NIH3T3 cells and in human colon cancer cells. It also reduced Src-induced tumor development in nude mice. An intact ULBR was required for MAPK signaling without affecting Src kinase activity nor sub-cellular localization. Phospho-proteomic analyses revealed that, while not impacting on the global tyrosine phospho-proteome in colon cancer cells, this region modulates phosphorylation of specific membrane-localized tyrosine kinases needed for Src oncogenic signaling, including EPHA2 and Fyn. Collectively, this study reveals an important role of this intrinsically disordered region in malignant cell transformation and suggests a novel layer of Src regulation by this unique region via membrane substrate phosphorylation.

The paradigm of developmental regulation by Polycomb group (PcG) proteins posits that they maintain silencing outside the spatial expression domains of their target genes, particularly of Hox genes, starting from mid embryogenesis. The Enhancer of zeste [E(z)] PcG protein is the catalytic subunit of the PRC2 complex, which silences its targets via deposition of the H3K27me3 mark. Here, we studied the ascidian Ciona intestinalis counterpart of E(z). Ci-E(z) is detected by immunohistochemistry as soon as the 2- and 4-cell stages as a cytoplasmic form and becomes exclusively nuclear thereafter, whereas the H3K27me3 mark is detected starting from the gastrula stage and later. Morpholino invalidation of Ci-E(z) leads to the total disappearance of both Ci-E(z) protein and its H3K27me3 mark. Ci-E(z) morphants display a severe phenotype. Strikingly, the earliest defects occur at the 4-cell stage with the dysregulation of cell positioning and mitotic impairment. At later stages, Ci-E(z)-deficient embryos are affected by terminal differentiation defects of neural, epidermal and muscle tissues, by the failure to form a notochord and by the absence of caudal nerve. These major phenotypic defects are specifically rescued by injection of a morpholino-resistant Ci-E(z) mRNA, which restores expression of Ci-E(z) protein and re-deposition of the H3K27me3 mark. As observed by qPCR analyses, Ci-E(z) invalidation leads to the early derepression of tissue-specific developmental genes, whereas late-acting developmental genes are generally down-regulated. Altogether, our results suggest that Ci-E(z) plays a major role during embryonic development in Ciona intestinalis by silencing early-acting developmental genes in a Hox-independent manner.

International audience; We report the characterization of a new Leishmania major gene, lmaj3'nt/nu, encoding a 382 amino acids protein, Lmaj3'NT/NU, that belongs to the 3'nucleotidase/nuclease family. Interestingly, sequence and phylogenetic analysis show that this protein is Leishmania major specific and thus constitutes a new 3'nucleotidase/nuclease subgroup. Lmaj3'NT/NU displays nuclease enzymatic activity and Western blot analysis shows that it is exclusively expressed in promastigotes. Immunofluorescence microscopy using a specific anti-Lmaj3'NT/NU shows that the protein has a plasma membrane localization. Surprisingly, contrary to the previously described Leishmania mexicana 3'NT/NU, lmaj3'nt/nu is not up-regulated when parasites are cultured under purine starvation conditions. Together, these findings suggest Lmaj3'NT/NU may constitute a new important compound of the L. major purine scavenging pathway and could be involved in sandfly parasite survival and colonization.

We report here the isolation in Spodoptera frugiperda (Lepidoptera) of an immune-related protein (hereafter named Spod-11-tox), characterized by imperfectly conserved tandem repeats of 11 cysteine-stabitized alpha beta motifs (CS-alpha beta), the structural scaffold characteristic of invertebrate defensins and scorpion toxins. Spod-11-tox orthologs were only found in Lepidopteran species, suggesting that this new protein family (named X-tox) is specific to this insect order. Moreover, phylogenetic analysis suggests that X-tox proteins represent a new class of proteins restricted to Lepidoptera and likely derived from Lepidopteran defensins. In S. frugiperda, analysis of gene expression revealed that spod-11-tox is rapidly induced by infection. However, and conversely to what is known for most insect antimicrobial peptides (AMP), spod-11-tox is mainly expressed in blood cells. Moreover, recombinant Spod-11-tox produced in the Sf9 cell line does not show any antimicrobial activity. Altogether, these results suggest that although X-tox proteins are derived from defensins, they may play a different and stilt unknown rote in Lepidoptera immune response

Interleukin-15 (IL-15) is crucial for the generation of multiple lymphocyte subsets (natural killer (NK), NK-T cells, and memory CD8 T cells), and transpresentation of IL-15 by monocytes and dendritic cells has been suggested to be the dominant activating process of these lymphocytes. We have previously shown that a natural soluble form of IL-15R alpha chain corresponding to the entire extracellular domain of IL-15R alpha behaves as a high affinity IL-15 antagonist. In sharp contrast with this finding, we demonstrate in this report that a recombinant, soluble sushi domain of IL-15R alpha, which bears most of the binding affinity for IL-15, behaves as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and protection from apoptosis) through the IL-15R beta/gamma heterodimer, whereas it does not affect IL-15 binding and function of the tripartite IL-15R alpha/beta/gamma membrane receptor. Our results suggest that, if naturally produced, such soluble sushi domains might be involved in the IL-15 transpresentation mechanism. Fusion proteins (RLI and ILR), in which IL-15 and IL-15R alpha-sushi are attached by a flexible linker, are even more potent than the combination of IL-15 plus sIL-15R alpha-sushi. After binding to IL-15R beta/gamma, RLI is internalized and induces a biological response very similar to the IL-15 high affinity response. Such hyper-IL-15 fusion proteins appear to constitute potent adjuvants for the expansion of lymphocyte subsets.

To detect traces of insecticides in the environment using biosensors, we engineered Drosophila acetylcholinesterase (AChE) to increase its sensitivity and its rate of phosphorylation or carbamoylation by organophosphates or carbamates. The mutants made by site-directed mutagenesis were expressed in baculovirus. Different strategies were used to obtain these mutants: (i) substitution of amino acids at positions found mutated in AChE from insects resistant to insecticide, (ii) mutations of amino acids at positions suggested by 3-D structural analysis of the active site, (iii) Ala-scan analysis of amino acids lining the active site gorge, (iv) mutagenesis at positions detected as important for sensitivity in the Ala-scan analysis and (v) combination of mutations which independently enhance sensitivity. The results highlighted the difficulty of predicting the effect of mutations; this may be due to the structure of the site, a deep gorge with the active serine at the bottom and to allosteric effects between the top and the bottom of the gorge. Nevertheless, the use of these different strategies allowed us to obtain sensitive enzymes. The greatest improvement was for the sensitivity to dichlorvos for which a mutant was 300-fold more sensitive than the Drosophila wild-type enzyme and 288 000-fold more sensitive than the electric eel enzyme, the enzyme commonly used to detect organophosphate and carbamate.

International audience; By virtual screening using a fragment-based drug design (FBDD) approach, 33 fragments were selected within small pockets around interaction hot spots on the Sec7 surface of the nucleotide exchange factor Arno, and then their ability to interfere with the Arno-catalyzed nucleotide exchange on the G-protein Arf1 was evaluated. By use of SPR, NMR, and fluorescence assays, the direct binding of three of the identified fragments to Arno Sec7 domain was demonstrated and the promiscuous aggregate behavior evaluated. Then the binding mode of one fragment and of a more active analogue was solved by X-ray crystallography. This highlighted the role of stable and transient pockets at the Sec7 domain surface in the discovery and binding of interfering compounds. These results provide structural information on how small organic compounds can interfere with the Arf1-Arno Sec7 domain interaction and may guide the rational drug design of competitive inhibitors of Arno enzymatic activity.

International audience; The GDP/GTP nucleotide exchange of Arf1 is catalyzed by nucleotide exchange factors (GEF), such as Arno, which act through their catalytic Sec7 domain. This exchange is a complex mechanism that undergoes conformational changes and intermediate complex species involving several allosteric partners such as nucleotides, Mg(2+), and Sec7 domains. Using a surface plasmon resonance approach, we characterized the kinetic binding parameters for various intermediate complexes. We first confirmed that both GDP and GTP counteract equivalently to the free-nucleotide binary Arf1-Arno complex stability and revealed that Mg(2+) potentiates by a factor of 2 the allosteric effect of GDP. Then we explored the uncompetitive inhibitory mechanism of brefeldin A (BFA) that conducts to an abortive pentameric Arf1-Mg(2+)-GDP-BFA-Sec7 complex. With BFA, the association rate of the abortive complex is drastically reduced by a factor of 42, and by contrast, the 15-fold decrease of the dissociation rate concurs to stabilize the pentameric complex. These specific kinetic signatures have allowed distinguishing the level and nature as well as the fate in real time of formed complexes according to experimental conditions. Thus, we showed that in the presence of GDP, the BFA-resistant Sec7 domain of Arno can also associate to form a pentameric complex, which suggests that the uncompetitive inhibition by BFA and the nucleotide allosteric effect combine to stabilize such abortive complex.

Background Malaria transmission occurs during the blood feeding of infected anopheline mosquitoes concomitant with a saliva injection into the vertebrate host. In sub-Saharan Africa, most malaria transmission is due to Anopheles funestus s.s and to Anopheles gambiae s.l. (mainly Anopheles gambiae s.s. and Anopheles arabiensis). Several studies have demonstrated that the immune response against salivary antigens could be used to evaluate individual exposure to mosquito bites. The aim of this study was to assess the use of secreted salivary proteins as specific biomarkers of exposure to An. gambiae and/or An. funestus bites. Methods For this purpose, salivary gland proteins 6 (SG6) and 5′nucleotidases (5′nuc) from An. gambiae (gSG6 and g-5′nuc) and An. funestus (fSG6 and f-5′nuc) were selected and produced in recombinant form. The specificity of the IgG response against these salivary proteins was tested using an ELISA with sera from individuals living in three Senegalese villages (NDiop, n = 50; Dielmo, n = 38; and Diama, n = 46) that had been exposed to distinct densities and proportions of the Anopheles species. Individuals who had not been exposed to these tropical mosquitoes were used as controls (Marseille, n = 45). Results The IgG responses against SG6 recombinant proteins from these two Anopheles species and against g-5′nucleotidase from An. gambiae, were significantly higher in Senegalese individuals compared with controls who were not exposed to specific Anopheles species. Conversely, an association was observed between the level of An. funestus exposure and the serological immune response levels against the f-5′nucleotidase protein. Conclusion This study revealed an Anopheles salivary antigenic protein that could be considered to be a promising antigenic marker to distinguish malaria vector exposure at the species level. The epidemiological interest of such species-specific antigenic markers is discussed.

We have cloned the replicative form of the Aedes albopictus parvovirus (AaPV) genome and determined the complete sequence of the viral strand. The sequence is 4176 nucleotides (nt) in length. The first 134 nt at the 3' end and the terminal 182 nt at the 5' end of the viral (minus) strand can both generate by folding and annealing of complementary sequences a typical terminal T-shaped structure although they differ in their sequence. Three large open reading frames (ORFs), each one in a different frame, are present between map units (mu) 8.0 and 87.6 on the complementary (plus) strand. The left, mid (located within the left ORF), and right ORFs have potential coding capacities of 95, 41, and 40 kDa, respectively. Two potential promoters were found upstream from the left and right ORFs, at mu 7.2 and mu 60.0, respectively. Computer search for sequence homologies suggests that the left ORF very likely encodes the nonstructural NS-1 protein since it contains the highly conserved NTP-binding amino acid (aa) domain (GKRN sequence) of all parvoviruses. Comparison with other invertebrate and vertebrate parvoviruses revealed that the AaPV genome shares 77.3% nt sequence homology and between 73 and 78% aa sequence homologies with the Aedes aegypti densonucleosis virus (Aedes DNV). Organization of both genomes was similar except that no potential ORF was found on the minus strand of AaPV. The difference of 167 nt in length between AaPV and Aedes DNV (4009 nt) genomes is due to additional noncoding sequences located between the internal coding region and the terminal palindromes in the AaPV genome. No significant homology was found between AaPV and the two other insect parvoviruses sequenced so far, the Bombyx mori DNV (BmDNV) and the Junonia coenia DNV (JcDNV).

Article Open Access Correspondance auteur: J.M. Escoubas E-mail: Jean-Michel.Escoubas@univ-montp2.fr; International audience; Background: X-tox proteins are a family of immune-related proteins only found in Lepidoptera and characterized by imperfectly conserved tandem repeats of several defensin-like motifs. Previous phylogenetic analysis of X-tox genes supported the hypothesis that X-tox have evolved from defensins in a lineage-specific gene evolution restricted to Lepidoptera. In this paper, we performed a protein study in which we asked whether X-tox proteins have conserved the antimicrobial functions of their ancestral defensins and have evolved as defensin reservoirs. Methodology/Principal Findings: We followed the outcome of Spod-11-tox, an X-tox protein characterized in Spodoptera frugiperda, in bacteria-challenged larvae using both immunochemistry and antimicrobial assays. Three hours post infection, the Spod-11-tox protein was expressed in 80% of the two main classes of circulating hemocytes (granulocytes and plasmatocytes). Located in secretory granules of hemocytes, Spod-11-tox was never observed in contact with microorganisms entrapped within phagolyzosomes showing that Spod-11-tox is not involved in intracellular pathogen killing. In fact, the Spod-11-tox protein was found to be secreted into the hemolymph of experimentally challenged larvae. In order to determine antimicrobial properties of the Spod-11-tox protein, it was consequently fractionated according to a protocol frequently used for antimicrobial peptide purification. Over the course of purification, the anti-Spod-11-tox immunoreactivity was found to be dissociated from the antimicrobial activity. This indicates that Spod-11-tox is not processed into bioactive defensins in response to a microbial challenge. Conclusions/Significance: Altogether, our results show that X-tox proteins have not evolved as defensin reservoirs and have lost the antimicrobial properties of the ancestral insect defensins. The lepidopteran X-tox protein family will provide a valuable and tractable model to improve our knowledge on the molecular evolution of defensins, a class of innate immune effectors largely distributed over the three eukaryotic kingdoms

International audience; Crystallization trials of the human retinoid X receptor alpha ligand-binding domain (RXRalpha LBD) in complex with various ligands have been carried out. Using fluorescence anisotropy, it has been found that when compared with agonists these small-molecule effectors enhance the dynamics of the RXRalpha LBD C-terminal helix H12. In some cases, the mobility of this helix could be dramatically reduced by the addition of a 13-residue co-activator fragment (CoA). In keeping with these observations, crystals have been obtained of the corresponding ternary RXRalpha LBD-ligand-CoA complexes. In contrast, attempts to crystallize complexes with a highly mobile H12 remained unsuccessful. These experimental observations substantiate the previously recognized role of co-regulator fragments in facilitating the crystallization of nuclear receptor LBDs.

We previously showed that a natural soluble form of interleukin-15 (IL-15) Ralpha corresponding to the full-length ectodomain of IL-15Ralpha behaved as a potent antagonist of IL-15 action through IL-15Ralpha/beta/gamma, whereas a recombinant soluble IL-15Ralpha sushi domain did not, but instead acted as an agonist of IL-15 action through IL-15Rbeta/gamma. In order to determine precisely the molecular basis governing these antagonistic versus agonistic actions, we compared the binding properties and biological effects of recombinant soluble IL-15Ralpha (sIL-15Ralpha) species containing the sushi domain and different remaining parts of the ectodomain. We first demonstrate that the exon-3-encoded domain and, more particularly, its N-terminal 13-amino-acid (aa) peptide are important, in addition to the adjacent exon-2-encoded sushi domain, for the stabilization of the high-affinity IL-15.IL-15Ralpha complex by slowing down its dissociation rate and by contributing to about 10-20% of the free energy of interaction. We next show that all sushi-containing sIL-15Ralpha are agonists on IL-15Rbeta/gamma, coordinately increasing IL-15 binding and IL-15-induced proliferation. Their agonistic potencies are proportional to their respective affinities for IL-15. We then show that the antagonistic effect of sIL-15Ralpha in the context of IL-15Ralpha/beta/gamma is due to the 13-aa peptide that creates a sterical constraint impeding the binding of the sIL-15Ralpha.IL-15 complex to the membrane-anchored IL-15Ralpha/beta/gamma. In the frame of the soluble IL-15Ralpha sushi domain-IL-15 fusion protein that contains the 13-aa peptide, this constraint is alleviated as a result of a conformational effect due to the covalent linking of the 13-aa peptide to the N-terminus of IL-15. The soluble IL-15Ralpha sushi domain-IL-15 fusion protein is therefore able to bind and activate both the IL-15Rbeta/gamma and the IL-15Ralpha/beta/gamma receptors.

Interleukin-15 (IL-15) is crucial for the generation of multiple lymphocyte subsets (natural killer (NK), NK-T cells, and memory CD8 T cells), and transpresentation of IL-15 by monocytes and dendritic cells has been suggested to be the dominant activating process of these lymphocytes. We have previously shown that a natural soluble form of IL-15R alpha chain corresponding to the entire extracellular domain of IL-15R alpha behaves as a high affinity IL-15 antagonist. In sharp contrast with this finding, we demonstrate in this report that a recombinant, soluble sushi domain of IL-15R alpha, which bears most of the binding affinity for IL-15, behaves as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and protection from apoptosis) through the IL-15R beta/gamma heterodimer, whereas it does not affect IL-15 binding and function of the tripartite IL-15R alpha/beta/gamma membrane receptor. Our results suggest that, if naturally produced, such soluble sushi domains might be involved in the IL-15 transpresentation mechanism. Fusion proteins (RLI and ILR), in which IL-15 and IL-15R alpha-sushi are attached by a flexible linker, are even more potent than the combination of IL-15 plus sIL-15R alpha-sushi. After binding to IL-15R beta/gamma, RLI is internalized and induces a biological response very similar to the IL-15 high affinity response. Such hyper-IL-15 fusion proteins appear to constitute potent adjuvants for the expansion of lymphocyte subsets.

Human T-cell leukemia virus (HTLV) envelope (Env) glycoproteins induce fusion, leading to rampant syncytium formation in a broad range of cell lines. Here, we identified murine, hamster, canine, and porcine cell lines that are resistant to HTLV-1 Env-induced syncytium formation. This resistance was not due to the absence of functional receptors for HTLV Env, as these cells were susceptible to infection with HTLV Env-pseudotyped virions. As murine leukemia virus (MLV) Env and HTLV Env present close structural homologies (F. J. Kim, I. Seiliez, C. Denesvre, D. Lavillette, F. L. Cosset, and M. Sitbon, J. Biol. Chem. 275: 23417-23420, 2000), and because activation of syncytium formation by MLV Env generally requires cleavage of the R peptide in the cytoplasmic domain of the Env transmembrane (TM) component, we assessed whether truncation of the cytoplasmic domain of HTLV Env would alleviate this resistance. Indeed, in all resistant cell lines, truncation of the last 8 amino acids of the HTLV Env cytoplasmic domain (HdC8) was sufficient to overcome resistance to HTLV Env-induced syncytium formation. Furthermore, HdC8-mediated cell-to-cell infection titers varied according to the target cell lines and could be significantly higher than that observed with HTLV Env on HeLa cells. These data indicate that a determinant located within the 8 carboxy-terminal cytoplasmic amino acids of TM plays a distinct role in HTLV Env-mediated cell-to-cell infection and syncytium formation.

How can enzymes be protected against denaturation and proteolysis while keeping them in a fully functional state? One solution is to encapsulate the enzymes into liposomes, which enhances their stability against denaturation and proteases. However, the permeability barrier of the lipid membrane drastically reduces the activity of enzyme entrapped in the liposome by reducing the internal concentration of the substrate. To overcome this problem, we permeabilized the wall of the liposome by reconstitution of a porin from Escherichia coli. In this way, we recovered the full functionality of the enzyme while retaining the protection against denaturation and proteolytic enzymes.

International audience; We have characterized the genomic and replicative form (RF) DNA of theAedes albopictus Parvovirus (AaPV), a virus isolated from a chronically infected C6/36 clone ofAedes albopictus cell line [22]. The genome ofAaPV virions is a single-stranded linear DNA molecule approximately 4.2 kb in length, essentially (about 90%) encapsidated as minus strand. A restriction map of the RF DNA isolated from infected C6/36 cells was established. Among the 23 restriction enzymes tested, 14 cleaved theAaPV RF DNA and 30 restriction sites were mapped and oriented with respect to the viral genomic DNA. Both viral and RF DNAs were found infectious when transfected to virus-free C6/36 cells. The asymmetrical encapsidation of the viral genome is a property common to most vertebrate autonomous parvoviruses but rather unusual among densoviruses. Both by its small size, the asymmetrical mode of encapsidation and the restriction map, theAaPV genome resembles that of theAedes Densonucleosis virus [1].

We have isolated and partially characterized from an apparently healthy C6/36 subclone of Aedes albopictus cell line a small icosahedral non-enveloped DNA virus, designated AaPV. This virus proved to be highly pathogenic for Aedes aegypti neonate larvae. Viral infection persisted for over 4 years in the cell culture without any cytopathic effect. Attempts to infect suckling mice, Drosophila melanogaster adults and Spodoptera littoralis larvae with AaPV were unsuccessful. Similarly, the AaPV failed to replicate in vertebrate and Drosophila cell lines. Virions, about 22 nm in diameter, had a buoyant density of 1.43 g/cm3 and contained three capsid polypeptides with molecular weights of 53, 41 and 40 kDa. A preliminary study of the viral genome indicated the presence of single-stranded DNA. By its biophysical and biochemical properties, this virus appears to be related to the genus Densovirus within the family Parvoviridae, but Jacks serological relationships with the other members of this genus.