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International audience; iASPP is a protein mostly known as an inhibitor of p53 pro-apoptotic activity and a predicted regulatory subunit of the PP1 phosphatase, which is often overexpressed in tumors. We report that iASPP associates with the microtubule plus-end binding protein EB1, a central regulator of microtubule dynamics, via an SxIP motif. iASPP silencing or mutation of the SxIP motif led to defective microtubule capture at the cortex of mitotic cells, leading to abnormal positioning of the mitotic spindle. These effects were recapitulated by the knockdown of the membrane-to-cortex linker Myosin-Ic (Myo1c), which we identified as a novel partner of iASPP. Moreover, iASPP or Myo1c knockdown cells failed to round up upon mitosis because of defective cortical stiffness. We propose that by increasing cortical rigidity, iASPP helps cancer cells maintain a spherical geometry suitable for proper mitotic spindle positioning and chromosome partitioning.

We isolated a new gene which shares all the features of the Ly-6/neurotoxin superfamily, from gene organization to predicted 3D structure. As it is preferentially expressed in the nervous system, we called this gene lynx2, by analogy with lynx1, a nAChR modulator. In embryonic and postnatal mouse, lynx2 is expressed in postmitotic central and peripheral neurons. These include subpopulations of motor neurons, sensory neurons, interneurons and neurons of the autonomous nervous system. In addition, lynx2 is transiently expressed around the growing nerves in the limb bud. Comparison of its spatio-temporal expression pattern with that of two other members of this family, lynx1 and ly-6h, shows that these genes are detected both in distinct and overlapping neuron populations.

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by a progressive degeneration of motoneurons in spinal cord and brainstem. The telomeric copy of a duplicated gene termed survival motor neuron (smn), which maps to chromosome 5q13, has been found to be deleted in most patients. The encoded gene product is a novel protein which recently has been shown to accumulate in specific nuclear organelles (gemini of coiled bodies, GEMS), and to play a part in the formation of the spliceosome complex. We have cloned and sequenced the rat smn cDNA. Antibodies generated against an N-terminus peptide recognized a main protein of 32 kDa in immunoblots of rat embryonic tissue extracts. Minor bands of 35 kDa, 45 kDa and, in perinatal muscle, of 24 kDa were also specifically detected, indicating that SMN is expressed as different molecular forms. Subcellular fractionation indicated that the 32 kDa form is mainly soluble, while the 35 kDa and 45 kDa products segregate to the microsomal-mitochondrial fraction. SMN protein is highly regulated during development: expression is high in embryonic tissues (central nervous system, muscle, lung and liver), and then progressively decreases to very low levels in most tissues of the adult. The demonstration of different molecular forms of SMN along with its developmental regulation may help to understand the contribution of this protein in the appearance of SMA phenotype.

Human immunodeficiency virus type 1 (HIV-1) NDK, a Zairian subtype D virus highly cytopathic for CD4-positive lymphocytes, and the prototype subtype B virus HIV-1 LAV are about 10 4 and 10 5 times more infectious, respectively, for T lymphocytes than for blood-derived macrophages (BDM). Recombinant viruses derived from HIV-1 LAV and HIV-1 NDK were used to determine the genetic control and the step of the virus/cell cycle responsible for infection of BDM with T-cell-tropic viruses. We found that recombinants bearing the envelope glycoprotein of HIV-1 NDK are able to enter more efficiently into BDM than recombinants with HIV-1 LAV envelope glycoprotein. We also found that a genetic region outside of the env gene is responsible for production of HIV-1 NDK infectious progeny from BDM. This region consists of the vif gene and the C- and N-terminal portions of pol and vpr genes, respectively. Our results suggest that productive infection of primary macrophages with T-cell-tropic strains of HIV-1 is determined by two different genetic mechanisms: one effective at the virus/cell entry, controlled by the env gene, and the second after entry, controlled by genes vif and vpr. In comparison with HIV-1 LAV, HIV-1 NDK has been able to more easily overcome both restriction mechanisms.

The human colon epithelial line HT29 represents a semipermisive cellular system for human immunodeficiency virus type 1 (HIV-1). It could be productively infected with HIV-1 NDK, a Zairian virus isolate highly cytopathic for CD4 positive lymphocytes, whereas infection with the prototype virus HIV-1 LAV was nonproductive. Recombinant viruses derived from HIV-1 LAV and HIV-1 NDK were used to determine the genetic control, step of virus/cell cycle, and molecular mechanism responsible for productive versus nonproductive infection of intestinal cells. Both parental viruses and all recombinants retrotranscribed their genomes with a similar kinetics and were able to complete HIV-1 DNA synthesis. HIV-1 LAV provirus present in preintegration complexes could be rescued by cocultivation with T-lymphocytes. However, it was aborted during prolonged cultivation of HT29 cells. Our results suggest that (i) gag/pol region of HIV-1 genome (fragment Bss HII 255 - Eco RI 4183 genetically controlled productive infection of intestinal cells and that (ii) the difference between productive and abortive infection occurred before synthesis of HIV-1 mRNA, at the integration level.

In the hindbrain, generation of the facial nucleus involves complex developmental processes that will lead to the formation of a structure composed of motor neurons, astrocytes and oligodendrocytes. The implication of LIF-related cytokines in the development of this nucleus came to light with the analysis of mice mutant for the receptor of these cytokines, LIFR beta, which exhibit a massive loss of facial branchiomotor (fbm) neurons at birth and a severe decrease in GFAP expression, a marker of astrocytes. To uncover the cellular mechanisms regulated by LIFR beta during facial nucleus development, we first analyzed its expression pattern in the hindbrain. lifr beta is first expressed at E11.5 in the hindbrain neuroepithelium. The receptor is absent during the migration of fbm post-mitotic neurons but is strongly expressed when fbm neurons have reached rhombomere 6 at E12.5, and its expression is maintained until E18.5. From the analysis of lifr beta mutant embryos, we established that LIFR beta is necessary for fbm neurons' identity determination. We also show that LIFR beta is implicated in astrocyte and oligodendrocyte differentiation, specifically within the facial nucleus.

Formation of large syncytia, rapid cell killing, and early onset of replication are characteristics of the highly cytopathic Zairian virus strain HIV1 NDK compared with the HIV1 LAV prototype. Recombinant provirus molecules derived from cloned infectious DNAs of HIV1 LAV and NDK were constructed by reciprocal exchange of genetic material using conserved restriction sites. Different regions of the HIV1 genome were responsible for variability of the direct single-cell cytotoxic and fusogenic effects. A minimal, provisionally defined portion of genetic information responsible for the higher cytotoxicity of HIV1 NDK compared to the HIV1 LAV prototype was localized in the fragment Spe l 1042 Eco RI l483 , containing the 3′-terminal half of gag and a majority of the pol gene. This region also determined the rapid replication properties of HIV1 NDK. The increased fusogenic potential of HIVI NDK was associated with the simultaneous presence of HIV1 NDK fragments Bss HII 255 Spe I 1042 and Eco RI 5278 Xho I 8401 , which contained the splicing donor, packaging sequence, pl8 gag protein, and the HIV env gene. The increase in the direct killing effect but not in the syncytium forming ability of HIVI NDK correlated with the early onset of replication and rapid spread of HIV1 NDK in cell cultures. The HIV1 NDK fragments Bss HII Spe I and Eco RI Xho I were by themselves necessary but not sufficient to induce formation of large syncytia.

The differentiation of U937 monoblastoid cells after human immunodeficiency virus type 1 (HIV-1) infection was studied using the following approaches: reverse transcriptase activity measurement, immunofluorescence labeling, and electron microscopy. For comparison, uninfected U937 cells were induced to differentiate from monocyte to macrophage by phorbol 12-myristate 13-acetate (PMA) or retinoic acid (RA) treatment. Both infected and drug-treated cells showed important and similar ultrastructural cell modifications, with a phenotype that decreased in monocyte specificity and increased in that of macrophages. When U937 cells were induced to differentiate upon HIV-1 infection, a very different pathway of viral production was observed. Production and accumulation of the virus in a vacuolar compartment of intracytoplasmic origin and escape to the antiviral lysosomal activity could explain virus persistence. This makes the cell system a good model with which to study the relationship between HIV-1 production and cell differentiation.

Phenotypic characterization of subtype B strains of human immunodeficiency virus type 1 (HIV-1) indicates that the major determinants of their cytopathogenicity and tropism are contained in the gene coding for the envelope glycoprotein gp120, namely in its variable regions V1, V2, and V3. Recombinant viruses derived from HIV-1 LAV, the subtype B prototype virus, and HIV-1 NDK, the Zairian subtype D virus highly cytopathic for CD4-positive lymphocytes, were used to elucidate genetic control of fusogenic functions in subtype D viruses. Our data demonstrate that multigenic determination of fusogenic properties is more complex in the subtype D than in clade B viruses. Variability in three regions of HIV-1 NDK genome correlated with formation of large syncytia. These regions consisted of the matrix protein, the C-terminal portion of vpr up to the C1 region of gp 120, and the V1-V3 regions of gp120. Variability in the envelope glycoprotein but not in other regions of the HIV-1 genome was related to enhanced resistance of HIV-1 NDK to treatment of target cells with OKT4-A anti-CD4 MAb. Therefore, a different genetic control affects two aspects of HIV-1 fusogenicity: (i) variability in the envelope glycoprotein itself is sufficient to influence a virus-to-cell fusion at the virus/cell entry, and (ii) a more complex genetic function including genes of matrix protein and envelope glycoprotein is related to variability of cell-to-cell fusion during formation of syncytium.

Formation of large syncytia and rapid cell killing are characteristics of the Zairian human immunodeficiency virus type 1 isolate HIV-1-NDK which is highly cytopathic for CD4-positive lymphocytes in comparison with the HIV-1-LAV prototype. Chimeric viruses containing different combinations of HIV-1-NDK genetic determinants corresponding to the splice donor the packaging signal and the coding sequence of the p18gag protein together with the HIV-1-NDK EcoRI5278-XhoI8401 fragment were obtained by polymerase chain reaction-directed recombination. Phenotypic analysis of recombinant viruses indicated that 75 amino acids from the N-terminal part of HIV-1-NDK p18gag protein together with the HIV-1-NDK envelope glycoprotein are responsible for enhanced fusogenicity of HIV-1-NDK in CD4-positive lymphocytes as well as for enhanced infectivity of HIV-1-NDK in some CD4-negative cell lines. The HIV-1-NDK splice donor/packaging sequence and the sequence encoding the gag protein p25 were not important for the variation observed in HIV-1 fusogenicity. (authors)

The prototype virus HIV-1 LAV and highly cytopathic Zairian virus HIV-1 NDK belong to the genetic subtypes B and D and represent low and highly cytopathic phenotypes, respectively. Their neutralization pattern and serotype were studied with respect to differences in their genotypes and phenotypes. Sera from HIV-1-infected persons living in four geographically distant areas, Philadelphia (USA), Ribeirao Preto (Brazil), Marseille (France) and Kinshasa (Zaire), were tested for the presence of type-specific and group-specific cross-reacting neutralizing antibodies against HIV-1 LAV and HIV-1 NDK in a continuous cell line MT4. The majority of type-specific antibodies were directed against HIV-1 LAV in Philadelphia, Ribeirao Preto and Marseille, and against HIV-1 NDK in Kinshasa. However, some sera with an HIV-1 NDK type-specific neutralization pattern were also found in Philadelphia, Ribeirao Preto and Marseille. These results indicate that strains with an HIV-1 NDK-like serotype could be found outside Africa. The presence of type-specific neutralizing antibodies against HIV-1 NDK in sera from North and South America and Europe should be taken into account during attempts to serotype HIV as well as in the course of selection of HIV-1 candidate strains for an AIDS vaccine.