Jean-Claude Lefebvre

Secreted phospholipases A2, a new class of HIV inhibitors that block virus entry into host cells

Mammalian and venom secreted phospholipases A2 (sPLA2s) have been associated with a variety of biological effects. Here we show that several sPLA2s protect human primary blood leukocytes from the replication of various macrophage and T cell–tropic HIV-1 strains. Inhibition by sPLA2s results neither from a virucidal effect nor from a cytotoxic effect on host cells, but it involves a more specific mechanism. sPLA2s have no effect on virus binding to cells nor on syncytia formation, but they prevent the intracellular release of the viral capsid protein, suggesting that sPLA2s block viral entry into cells before virion uncoating and independently of the coreceptor usage. Various inhibitors and catalytic products of sPLA2 have no effect on HIV-1 infection, suggesting that sPLA2 catalytic activity is not involved in the antiviral effect. Instead, the antiviral activity appears to involve a specific interaction of sPLA2s to host cells. Indeed, of 11 sPLA2s from venom and mammalian tissues assayed, 4 venom sPLA2s were found to be very potent HIV-1 inhibitors (ID50 < 1 nM) and also to bind specifically to host cells with high affinities (K0.5 < 1 nM). Although mammalian pancreatic group IB and inflammatory-type group IIA sPLA2s were inactive against HIV-1 replication, our results could be of physiological interest, as novel sPLA2s are being characterized in humans.

Epigenetic reprogramming of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) in HIV-1-infected CEM T cells

Sialylated glycoconjugates mediate several key lymphocyte functions. We previously reported that hyposialylation occurred in latently HIV‐1‐infected CEM T cells, despite the fully preserved catalytic activity of several sialyltransferases. We show now that these cells are affected by a down‐regulation of UDP‐N‐acetylglucosamine 2‐epimerase/N‐acetylmannosamine kinase (GNE), which leads to a dramatic decrease in the synthesis of CMP‐sialic acid, the donor substrate of all sialyltransferases. The GNE gene promoter was found to be located in a CpG island with several regulatory motifs CREB, SP1, and AP‐2. De novo hypermethylation of this promoter was observed in HIV‐1‐infected CEM cells. This phenomenon might explain some immunological disorders that persist in infected individuals despite long‐term therapeutically controlled viral replication. Indeed, an overall decrease in sialic acid engraftment can affect glycoproteins, notably those in which the sialylation status is crucial to ensure homing, recirculation, and survival of lymphocytes.

Protection of a T-cell line from human immunodeficiency virus replication by the stable expression of a short antisense RNA sequence carried by a shuttle RNA molecule.

Adenovirus VA1 gene is efficiently transcribed by RNA polymerase III and gives rise to a small highly ordered RNA. To inhibit replication of human immunodeficiency virus (HIV), a chimeric VA1 RNA molecule was designed that contained a short antisense RNA sequence complementary to a conserved region of the HIV-1 rev encoding mRNA (28 nucleotides). This sequence, which was inserted into a projecting loop of the VA1 RNA central domain, was mainly single stranded and available for binding with its complementary sequence. The chimeric VA1 antisense was abundantly expressed in human cells constituting 3% of mRNA and promoted strong and specific inhibition of HIV-1 gene replication. The stable expression of antisense RNA in human T cells (CEM) protected these cells from HIV-1 multiplication for at least 3 months. No side effects were detected because of the lack of antisense effect upon replication of the closely related HIV-2. The VA1 gene may provide a suitably compact gene cassette for the intracellular expression of short antisense RNA directed against HIV.

High Permissivity of Human HepG2 Hepatoma Cells for Influenza Viruses

ABSTRACT Human HepG2 hepatoma cells are highly permissive for influenza virus type A and type B, even without the addition of trypsin, and they exhibit a marked cytopathic effect. This property greatly facilitates the primary isolation of influenza viruses. Virus replication was significantly reduced by the plasmin(ogen)-specific inhibitor tranexamic acid, and this suggests a potential role played by the plasminogen/tissue plasminogen activator complex at the surface of HepG2 cells. This might represent a new approach for study of the interrelations of this complex with influenza viruses.

Designing a HER2/neu promoter to drive α1,3galactosyltransferase expression for targeted anti-αGal antibody-mediated tumor cell killing

IntroductionOur goal was to specifically render tumor cells susceptible to natural cytolytic anti-αGal antibodies by using a murine α1,3galactosyltransferase (mαGalT) transgene driven by a designed form of HER2/neu promoter (pNeu), the transcription of which is frequently observed to be above basal in breast tumors. Indeed, the αGalT activity that promotes Galα1,3Galβ1,4GlcNAc-R (αGal) epitope expression has been mutationally disrupted during the course of evolution, starting from Old World primates, and this has led to the counter-production of large amounts of cytotoxic anti-αGal antibodies in recent primates, including man.MethodExpression of the endogenous c-erbB-2 gene was investigated in various cell lines by northern blotting. A mαGalT cDNA was constructed into pcDNA3 vector downstream of the original CMV promoter (pCMV/mαGalT) and various forms of pNeu were prepared by PCR amplification and inserted in the pCMV/mαGalT construct upstream of the mαGalT cDNA, in the place of the CMV promoter. These constructs were transferred into HEK-293 control and breast tumor cell lines. Stably transfected cells were analyzed by northern blotting for their expression of αGalT and c-erbB-2, and by flow cytometry for their binding with fluorescein isothiocyanate-conjugated Griffonia simplicifolia/isolectin B4.ResultsWe show that expression of the mαGalT was up- or down-modulated according to the level of endogenous pNeu activity and the particular form of constructed pNeu. Among several constructs, two particular forms of the promoter, pNeu250 containing the CCAAT box and the PEA3 motif adjacent to the TATAA box, and pNeu664, which has three additional PEA3 motifs upstream of the CCAAT box, were found to promote differential αGalT expression.ConclusionOur results strengthen current concepts about the crucial role played by the proximal PEA3 motif of pNeu, and may represent a novel therapeutic approach for the development of targeted transgene expression.

Molecular Cloning of a New Secreted Sulfated Mucin-like Protein With a C-type Lectin Domain That Is Expressed in Lymphoblastic Cells

We have previously demonstrated hyposialylation of the two major CD45 and leukosialin (CD43) molecules at the surface of latently human immunodeficiency virus type 1-infected CEM T cells (CEMLAI/NP), (Lefebvre, J. C., Giordanengo, V., Doglio, A., Cagnon, L., Breittmayer, J. P., Peyron, J. F., and Lesimple, J. (1994) Virology 199, 265–274; Lefebvre, J. C., Giordanengo, V., Limouse, M., Doglio, A., Cucchiarini, M., Monpoux, F., Mariani, R., and Peyron, J. F. (1994) J. Exp. Med. 180, 1609–1617). Searching to clarify mechanism(s) of hyposialylation, we observed two sulfated secreted glycoproteins (molecular mass ∼47 and ∼40 kDa) (P47 and P40), which were differentially sulfated and/or differentially secreted in the culture supernatants of CEMLAI/NP cells when compared with parental CEM cells. A hybridoma clone (7H1) resulting from the fusion between CEMLAI/NP and human embryonic fibroblasts MRC5 cells produced very large amounts of P47 that was purified using Jacalin lectin (specific for O-glycans) and microsequenced. Cloning of P47 was achieved using a CEMLAI/NP cDNA library screened with a degenerate oligonucleotide probe based on its NH2-terminal amino acid sequence. A single open reading frame encoding a protein of 323 amino acids was deduced from the longest isolated recombinant (1.4 kilobase). P47 is a secreted sulfated protein. It carries an NH2-terminal RGD (Arg-Gly-Asp) triplet, a striking α-helical leucine zipper composed of six heptads, and a C-terminal C-type lectin domain. The NH2-terminal portion is rich in glutamic acids with a predicted pI of 3.9. In addition, a hinge region with numerous condensed potential sites forO-glycan side chains, which are also the most likely sulfation sites, is located between the RGD and leucine zipper domains. Transcripts were detected in lymphoid tissues (notably bone marrow) and abundantly in T and B lymphoblastoid but very faintly in monocytoid cell lines.

Induction by human immunodeficiency viruses types 1 and 2 of degradation of CD4 but not of a CD4 mutant unable to bind viral envelope glycoproteins.

HIV-1 appears to use a multiple gene strategy to regulate CD4 receptor expression, which emphasizes the importance of this regulation in the viral life cycle. The cytoplasmic interaction between gp160 and CD4 is probably the major event governing CD4 down-regulation, although other viral proteins, such as Nef (CD4 cell surface localization) and Vpu (CD4 degradation), are thought to participate as well. Because of the lack of vpu in HIV-2, we investigated the effects of two HIV-2 isolates (ROD 10 and EHO) on CD4 expression in the CEM T-cell line. We found that these HIV-2 strains induce CD4 degradation to a similar extent as that induced by an HIV-1 isolate (BRU). To assess the role of each viral protein involved in CD4 regulation (gp, Nef and Vpu), we developed cell lines expressing a mutated form of CD4 unable to efficiently bind gp160, in addition to their endogenous CD4. Using this system, we provide evidence that the mutated CD4 is always expressed in HIV-1-, and HIV-2-infected cells, independent of the presence of Nef, while the endogenous CD4 is completely lost. These results highlight the key role of intracytoplasmic gp-CD4 interaction, explaining in vitro the CD4 down-regulation in T-cell lines.

T cells chronically infected with HIV do not contain sufficient Nef to promote CD4 downmodulation in the absence of envelope-mediated effects.

Among HIV viral proteins, envelope glycoproteins and Nef have been both suggested to participate in CD4 downregulation during the course of HIV infection. In a previous study, we provided evidence that a mutant form of CD4 that does not bind gp120 was never downregulated in chronically HIV-1- and HIV-2-infected CEM cells. To further investigate the relative effects of Nef or glycoproteins in CD4 downregulation, recombinant vaccinia virus (VV) vectors were used to express high levels of HIV-1 viral proteins in cells expressing both wild-type and mutant CD4. It was demonstrated that during HIV infection, overexpression of Nef, achieved through the VV expression system, was necessary to induce CD4 downregulation in the mutant CD4-expressing cell model. These results are consistent with the hypothesis that Nef-mediated CD4 downregulation depends on the cellular levels of Nef expression. We concluded that during the late stage of viral replication, CD4 downregulation is mostly due to gp120 and not to Nef because of a low level of Nef expression.