Inhibition of HIV-1, HIV-2 and SIV envelope glycoprotein-mediated cell fusion by calmodulin.

Calmodulin, an EF-hand protein, inhibited the fusion between CD4+ human cells and cells stably expressing HIV-1 envelope proteins. Fusion was also inhibited when HIV-1, HIV-2 or SIV envelope glycoproteins were expressed by vaccinia virus (VV) recombinants, but calmodulin did not inhibit syncytia formation induced by measles virus glycoproteins. Calmodulin also inhibited fusion induced by vPE17, a VV-recombinant expressing a truncated form of HIV-1gp160 which lacks the two known calmodulin-binding sites located in the cytoplasmic domain of gp41. The inhibitory activity was specific to calmodulin among the EF-hand proteins. These observations may be important in understanding the mechanism of retroviral envelope glycoprotein-mediated cell fusion. Several possible mechanisms of action are discussed.

Mode of entry of morbilliviruses.

The morbilliviruses have a restricted host range. This is probably dependent on the use of specific host cell receptors. In the present article, we have reviewed our approach to identify a host cell receptor for one of the morbilliviruses, measles virus and to elucidate the interaction between viral and cellular proteins during virus entry into the host cell.

The role of N-glycosylation in cell fusion induced by a vaccinia recombinant virus expressing both measles virus glycoproteins.

We have evaluated the effect of inhibitors of N-glycosylation on syncytia formation in HeLa cell infected by a recombinant vaccinia virus expressing the measles virus hemagglutinin (HA) and fusion (F) glycoproteins (VV-HA/F). Inhibitors which block glucose trimming of oligosaccharides side chains of glycoproteins (glucosidase inhibitors) prevented syncytium formation. On the other hand, inhibitors which block trimming of mannose residues (mannosidase inhibitors) did not prevent syncytium formation. The HA and F synthesized in the presence of either glucosidase or mannosidase inhibitors oligomerized and were transported to the cell surface. Concanavalin A, a mannose-specific lectin, inhibited syncytium formation. The inhibitors did not block the cleavage of the F protein, but in fact enhanced it. These studies demonstrate the importance of protein N-glycosylation in the process of membrane fusion and suggest that trimming of glucose residues and the presence of terminal mannose residues are required for VV-HA/F syncytium induction.

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.

Measles virus glycoproteins: studies on the structure and interaction of the haemagglutinin and fusion proteins.

We have investigated the structure and interaction of the measles virus (MV) glycoproteins expressed at the cell membrane. Cross-linking studies with a variety of chemicals stabilized dimeric forms of the haemagglutinin (HA) or fusion (F) proteins, although by sucrose density gradient analysis, oligomers corresponding to tetramers and larger were observed for both proteins. In cells in which both HA and F were expressed at the surface, their close association was shown by cross-linking and co-immunoprecipitation.

A leucine zipper structure present in the measles virus fusion protein is not required for its tetramerization but is essential for fusion.

The biological role of a leucine zipper motif present in the measles virus fusion (F) protein has been investigated. This motif is present in all paramyxovirus F proteins, all coronavirus spike proteins and many if not all retrovirus envelope proteins. By analogy to its role in certain transcription factors, it has been suggested that the motif may be responsible for the oligomerization of these viral membrane proteins. In this study, one, two or four heptadic leucines in the motif were substituted using site-directed mutagenesis. We found that fusion is prevented when all four heptadic leucines present in the motif are mutated whereas cellular transport and the oligomeric state of the F protein are unaffected.

Measles virus: both the haemagglutinin and fusion glycoproteins are required for fusion.

Vaccinia-measles recombinant viruses were used to examine the contribution of the individual measles virus glycoproteins in fusion. Although vaccinia virus recombinants expressing either the haemagglutinin or fusion proteins did not induce fusion in the cell lines examined, a double recombinant expressing both measles virus glycoproteins gave extensive syncytia in cells of human and simian origin. No fusion was observed in mouse, hamster or chicken cells. The fusion induced by the double recombinant could be specifically inhibited with either anti-fusion or anti-haemagglutinin monoclonal antibodies.

Contribution of measles virus fusion protein in protective immunity: anti-F monoclonal antibodies neutralize virus infectivity and protect mice against challenge.

To study the contribution of the measles virus fusion (F) protein in the immune response, anti-F monoclonal antibodies were prepared by using a vaccinia-measles virus F recombinant. In contrast to previously described anti-F monoclonal antibodies, these antibodies not only neutralized virus infectivity and inhibited fusion but also passively protected mice. Since these monoclonal antibodies recognize a configurational epitope, presentation of the antigen during infection may play an important role in the immune response. These factors are discussed in relation to vaccination.

Relationship between membrane sterol composition and responsiveness to 12-O-tetradecanoylphorbol 13-acetate in HL-60 human promyelocytic leukaemia cell lines.

We have examined the sterol composition and metabolism of promyelocytic leukaemia cell lines (HL-60) after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). A variant cell line (Blast II cells) which is resistant to TPA was used as control. Analysis of the sterols of TPA-sensitive cells radiolabelled with [3H]leucine, [14C]acetate or [14C]pyruvate showed a high incorporation into cholesterol and a low incorporation in lanosterol + dihydrolanosterol. The inverse relationship was observed in TPA-resistant cells. Experiments with other cellular variants representing TPA-sensitive and TPA-resistant classes gave similar results. Analysis of the cellular sterol composition by gas chromatography confirmed that TPA-resistant cells are particularly rich in lanosterol/dihydrolanosterol. TPA treatment enhanced the incorporation of [14C]pyruvate into the sterol fraction of both cell types. This was accompanied by an alteration of incorporation into several lipids, particularly phospholipids. Pulse-chase studies with [14C]acetate revealed that TPA induced the release of radioactive lipids into the medium from HL-60 and Blast II cells. However this treatment released phospholipids from the TPA-sensitive cells and sterols and fatty acids from the TPA-resistant cells. We conclude that the sterol composition can regulate specific biochemical processes in the membrane and can be considered as a factor that plays a role in the responsiveness of HL-60 cells to TPA.

12-O-tetradecanoyl phorbol 13-acetate stimulates the myristylation of an approximately 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 approximately 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.

Hepatic microsomal metabolism of 1,3-butadiene.

1. In rat liver microsomes, 1,3-butadiene was metabolized to butadiene monoxide, which was subsequently transformed into 3-butene-1,2-diol by microsomal epoxide hydrolase. 2. In the metabolism of butadiene oxide in microsomes, four metabolites were detected, namely two stereoisomers of DL-diepoxybutane, and two stereoisomers of 3,4-epoxy-1,2-butanediol. No meso-diepoxybutane was detected.

Metabolic activation and mutagenicity of 4 vinylic monomers (vinyl chloride, styrene, acrylonitrile, butadiene).

The mutagenic activity and the metabolism of four vinylic monomers; vinyl chloride, styrene, acrylonitrile and butadiene are reviewed. Those chemicals are converted by the mixed function oxidases system of the endoplasmic reticulum into reactive intermediates which can interact with macromolecules within the cell. In order to examine the mutagenic activity of these compounds and their metabolites, different mutagenicity testing systems have been used: tests with S. typhimurium, E. coli, Schizosaccharomyces pombe, Saccharomyces cerevisiae, V79 Chinese Hamster cells, CHO cells, Drosophila melanogaster as well as evaluations of chromosome aberrations.

Microsomal vinyl oxide synthetase: modification of its kinetic parameters by chemical carcinogens.

The kinetic parameters of liver microsomal vinyl oxide synthetase as well as their modifications under the influence of various pretreatments have been evaluated, using 1,1,2-trichlorethylene (TCE) as a substrate. The results were correlated with those obtained using aldrin oxide synthetase and benzpyrene hydroxylase as typical cytochrome P-450 dependent enzymatic activity. The treatments of animals with chemical carcinogens selectively increase the affinity of those enzymes for their substrates.

Sensitive gas chromatographic methods for the determination of vinyl epoxide synthetase activity using trichloroethylene as a model substrate.

Two specific and very sensitive methods for the determination of vinyl epoxide synthetase activity in liver microsomes are described. Trichloroethylene, which is used as a substrate, is converted into trichloroethylene oxide by a hepatic epoxide synthetase. Chloral hydrate, the final rearrangement product of trichloroethylene oxide, is determined by electron-capture gas chromatography, either after derivatization with pentafluorophenylhydrazine or after its conversion into chloroform under alkaline conditions. The kinetic parameters of the epoxidation reaction were determined on rat hepatic microsomal suspensions.

A gas chromatographic method for the evaluation of the vinyl epoxidase activity.

Two alternative specific and very sensitive methods for determination of vinyl epoxide synthetase activity in liver microsomes are described. Trichloroethylene, which is used as a substrate, is converted into trichloroethylene oxide by a hepatic epoxide synthetase. Chloral hydrate, the final rearrangement product of trichloroethylene oxide, is evaluated by electron capture gas chromatography, either after derivatization with pentafluorophenyl-hydrazine or after its conversion into chloroform under alcaline conditions. The kinetic parameters of the epoxidative reaction have been determined on rat hepatic microsomal suspensions.