Etienne Malvoisin

Identification of a conserved domain of the HIV-1 transmembrane protein gp41 which interacts with cholesteryl groups

A soluble form of the HIV-1 envelope glycoprotein gp160 devoid of the transmembrane anchor domain was found to bind to cholesteryl-hemisuccinate agarose. The external subunit gp120 failed to bind to the resin, suggesting that the site responsible for the binding to cholesterol was located in the transmembrane protein gp41. We constructed a series of maltose binding protein (MBP) fusion proteins representing overlapping fragments of the gp41 molecule and we studied their capacity to bind to cholesteryl beads. The domain responsible for binding to cholesterol was localised within the residues 668 to 684 immediately adjacent to the membrane spanning domain. We identified a short sequence (LWYIK, aa 678-683) comparable to the cholesterol interaction amino acid consensus pattern published by Li and Papadopoulos [Endocrinology 139 (1998) 4991]. We demonstrated that the sequence LWYIK synthesized fused to the MBP was able to bind to cholesteryl groups. A synthetic peptide containing the sequence LWYIK was found to inhibit the interaction between cholesteryl beads and MBP44, an MBP fusion HIV-1 envelope protein that contains the putative cholesterol binding domain. Human sera obtained from HIV-1 seropositive patients did not react in ELISA to the LWYIK sequence, suggesting that this region is not exposed to the immune system. The biological significance of the interaction between gp41 and cholesterol is discussed.

Effect of drugs which inhibit cholesterol synthesis on syncytia formation in vero cells infected with measles virus

We found that nontoxic doses of two inhibitors of cholesterol synthesis, namely W-7 and cerulenin, delayed syncytia formation in vero cells infected with measles virus. To correlate syncytia formation and lipidic membrane changes induced by these drugs, we labelled cell lipids with [14C]acetate. Measles virus infection increased the incorporation of radiolabel into fatty acids, triacylglycerol, cholesterol ester, and decreased its incorporation into cholesterol and 1,2-diacylglycerol. The ratios phosphatidylcholine/sphingomyelin and free cholesterol/lanosterol-dihydrolanosterol also decreased during the infection. W-7 and cerulenin greatly altered lipid metabolism. Both decreased the phosphatidylcholine to sphingomyelin and the cholesterol to lanosterol-dihydrolanosterol ratios. Z-D-Phe-L-Phe-L-Gly, a tripeptide which corresponds to the N-terminal sequence of the viral fusion protein (responsible for syncytia formation) and which inhibits virus-induced cell fusion without affecting virus synthesis also perturbed cholesterol metabolism. The tripeptide reversed the phosphatidylcholine to sphingomyelin ratio in infected cells. At non-toxic doses, W-7 inhibited the synthesis of infectious virus. Cerulenin which inhibited strongly the lipid synthesis did not. Finally, the well characterized inhibitors of cholesterol synthesis, mevinolin, ketoconazole and miconazole were shown to inhibit the syncytia formation. We conclude that the inhibition of syncytia by W-7 and cerulenin is associated with their capacity to alter the cholesterol metabolism, whereas the antiviral effect of W-7 does not seem related to this capacity.

Detection of IgA inhibiting the interaction between gp120 and soluble CD4 receptor in serum and saliva of HIV-1-infected patients.

Objective: To evaluate the presence of IgA directed to the CD4-binding domain of gp120 and to a conserved region of gp41 (the Kennedy epitope) in serum and parotid saliva of HIV-1-seropositive patients. Methods: IgA were separated from IgG by anion-exchange chromatography and protein G treatment. The reactivity of IgA was tested against peptides and fusion proteins of the maltose-binding protein (MBP) and the CD4-binding site (MBP24) and MBP and the Kennedy epitope (MBP42). The capacity of serum and saliva IgA to interfere with the gp120–soluble CD4 (sCD4) interaction was examined. IgA were also purified by affinity chromatography using the MBP proteins adsorbed to a resin. Results: Peptides representing the CD4-binding domain and the Kennedy epitope were recognized by serum and saliva IgA of HIV-1-seropositive patients. Of the sera and saliva samples tested, 6/26 serum IgA and 5/25 saliva IgA inhibited the gp120–sCD4 interaction by approximately 50%. The gp120–sCD4 interaction was inhibited by MBP24 affinity-purified IgA but not by MBP42 affinity-purified IgA. Conclusion: Immunogens capable of eliciting IgA antibodies that inhibit gp120–CD4 binding might be efficiently used in vaccine to prevent mucosal transmission of HIV-1.

Analysis of the human immunodeficiency virus type 1 envelope protein interaction with the CD4 host cell receptor.

A secreted form of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp160s), expressed in HeLa cells from a vaccinia virus recombinant was analysed by velocity-gradient centrifugation and chemical cross-linking. We showed that gp160s existed predominantly as a dimer, but higher forms corresponding to trimers and tetramers were also found. Soluble CD4 (sCD4) and native CD4 expressed by recombinant vaccinia viruses were analysed by sucrose-gradient sedimentation alone or after complexing with gp160s. The sCD4 sedimented in sucrose gradients as a monomer, whereas after solubilization the native CD4 was in a dimeric state. Both forms of CD4 were able to form complexes when incubated with gp160s. In the case of the sCD4, the M(r) corresponded to a (sCD4)2-(gp160s)2 complex, whereas with CD4 the complexes were of a greater order of magnitude. HIV gp120 was secreted into the medium in a monomeric state. With sCD4 it gave a one-to-one complex, whereas with the native CD4 high M(r) complexes were formed. The importance of the oligomeric state of the virus- and cell-receptor proteins are discussed regarding their avidities.

Characterization of a secreted form of measles virus haemagglutinin expressed from a vaccinia virus recombinant

The measles virus (MV) haemagglutinin (HA) is a class 2 glycoprotein by means of which the virus particle attaches to the host cell receptor. We have previously expressed this glycoprotein as a vaccinia recombinant virus and have shown that the HA glycoprotein synthesized is indistinguishable from that coded by MV. In the present study, we report that in RK13 cells a soluble form (sHA) of the HA is secreted into the medium. We show by SDS-PAGE and sucrose density gradient centrifugation that the sHA is a dimer and is smaller than the cell-associated form. Using a variety of inhibitors the production of sHA was shown to be a late event, probably occurring at the membrane; only fully glycosylated molecules were found in sHA. Finally, we demonstrate that sHA retains its antigenicity with conformation-dependent MAbs and its receptor recognition function. We conclude that sHA is a valuable tool for use in studies of the structure and function of the MV HA glycoprotein.

Antibodies purified from sera of HIV-1-infected patients by affinity on the heptad repeat region 1/heptad repeat region 2 complex of gp41 neutralize HIV-1 primary isolates.

Objective:The objective of this paper was to evaluate the presence and the neutralizing activity of antibodies directed against the complex formed between the two heptad repeat regions (HR1 and HR2) of HIV-1 gp41 in sera of HIV-1-infected patients. Research designs and methods:The HR1 region was represented by the peptide N36 and the maltose-binding protein (MBP)–HR1, the HR2 region by the peptide C34 and MBP44. Antibodies directed to the HR1/HR2 complex were purified from sera by affinity chromatography using MBP–HR1/C34 adsorbed onto a resin. Results:First, we demonstrated that human monoclonal antibodies, which are directed specifically to the HR1/HR2 complex recognized in enzyme-linked immunosorbent assay the MBP–HR1/C34 and MBP44/N36 mixtures but not the proteins or the peptides individually. We investigated the ability of 50 sera of HIV-1-infected patients to react with the MBP–HR1/C34 and MBP44/N36 complexes. We found that the majority of sera of HIV-1-infected patients recognized the HR1/HR2 complexes but not or to a lower extent the proteins or the peptides individually. Antibodies purified from sera by affinity chromatography using MBP–HR1/C34 adsorbed to a resin neutralized different primary HIV-1 isolates. Conclusion:The presence of antibodies directed to the HR1/HR2 complex in sera of HIV-infected patients highlights the immunogenic character of the complex, whereas the neutralizing activity of these antibodies suggests that immunogens representing HIV-1 HR1/HR2 complexes might be used in anti-HIV vaccine.

12-O-Tetradecanoyl phorbol 13-acetate stimulates the myristylation of an ∼ 82 kDa protein in HL-60 cells

We have studied protein acylation using [3H]myristate in the two leukemia cell lines HL‐60 and HL‐60 Blast II. The latter is a variant which does not differentiate after treatment with 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA). The acylation profiles of the two cell lines as examined by SDS‐PAGE differed. TPA induced the myristylation of an ∼ 82 kDa protein in the sensitive cells, but not in the resistant cells. Myristic acid was shown to be covalently linked to these proteins. Analysis of the cell lipids labelled with [3H]myristate showed that in contrast to observations with the proteins, the changes induced by TPA were observed in both TPA‐sensitive and TPA‐resistant cells. We conclude that the induction of myristylation may be an important step in the mechanism of differentiation.

Serum hepcidin levels in women infected with HIV-1 under antiviral therapy.

Accumulating data suggest that iron may have a role in the regulation of HIV‐infection. In the present study, we determined by radioimmunoassay the levels of hepcidin, a key regulator of iron homeostasis, in sera of 182 women infected with HIV‐1 under highly active antiretroviral therapy (HAART). In the total cohort, hepcidin levels were lower in individuals infected with HIV than in controls (3.20 ± 3.06 vs. 5.68 ± 3.66 nmol/L, P = 0.009). Serum hepcidin concentrations were strongly correlated positively with iron, ferritin, urea, and uric acid. In the total cohort of patients with abnormal viral load and CD4 cell count <500 cells/mm3, a strong positive correlation was found between hepcidin and viral load. Hepcidin level was significantly higher in HIV‐patients with high viremia than in patients with undetectable viral load. Iron level was significantly lower in HIV‐patients with high viral load compared with patients with undetectable viral load. This study suggests that hepcidin controls serum iron, especially in response of iron utilization by HIV for viral replication. The possibility of using inhibitors of hepcidin expression as adjunct therapy for HIV‐patients is discussed. J. Med. Virol. 86: 1656–1660, 2014.

Detection of AMP-activated protein kinase in human sera by immuno-isoelectric focusing.

AMPKalpha is a subunit of AMP-activated protein kinase (AMPK), a heterotrimeric enzyme that works as a fuel sensor activated in response to the depletion of cellular ATP. AMPKalpha is considered as a master switch in regulating glucose and lipid metabolism. Determining its presence in patient sera may help in diagnosing metabolic diseases. Using isoelectric focusing and Western blotting, we were able to detect AMPKalpha in human sera. Using specific antibodies, we showed that the AMPKalpha1 and alpha2 isoforms were apparently present in equal amounts in human sera. To characterize normal and abnormal AMPKalpha patterns, we used an antibody which recognized both isoforms (alpha1 and alpha2) to analyze sera of patients and healthy individuals. We also analyzed sera of HIV patients because several studies suggest that AMPK may play a role in the mechanism of lipodystrophy in HIV patients under antiretroviral therapy. We found that patients with type 2 diabetes or liver diseases presented abnormal AMPK IEF patterns. AMPK was poorly detectable in sera of patients with end-stage liver disease. Abnormal AMPK IEF patterns were more frequent in treated HIV-patients compared to those who are untreated suggesting a possible association between AMPK and the side-effects of antivirals. Our findings highlight the potential of serum AMPK as a new diagnostic biomarker and may help to study the regulation of AMPK activity in tissues.

Ability of antibodies specific to the HIV-1 envelope glycoprotein to block the fusion inhibitor T20 in a cell–cell fusion assay

The anti-HIV peptide T20 is able to inhibit the syncytia formation between CHO-WT and HeLa CD4(+)cells. We found that several sera of HIV-infected patients have the capacity to block the inhibition of fusion by T20. Suggesting that these sera may contain antibody which can block T20 access and prevent membrane fusion, we studied the ability of a panel of antibodies directed to different regions of HIV-1 envelope glycoprotein to block the inhibition of fusion by T20. We found that the C1 and V3 loop regions of gp120 and the heptad repeat 1, the immunodominant C-C region and the Kennedy epitope of gp41 located in the intracytoplasmic tail were the target for antibodies capable to block the inhibition of syncytia formation by T20. We suggest that these antibodies have the capacity to counteract the anti-fusion effect of T20 by preventing its binding to the interaction sites. Further studies are needed to determine if some of them recognize new T20 interaction sites.