Biomedical subjects

D Gerlier

Publications and source records attributed to D Gerlier.

At least 37 records · Page 2Linked to original sources

PubMed1997

CD46 short consensus repeats III and IV enhance measles virus binding but impair soluble hemagglutinin binding.

The binding of a recombinant soluble form of the measles virus (MV) hemagglutinin (sH) to cells expressing hybrid CD46/CD4 proteins was compared to that of purified virus. For binding of both ligands, both CD46 external short consensus repeats I and II (SCR I and II) in the natural order were essential. The addition of SCR III and IV enhanced virus binding but inhibited sH binding. Accordingly, this lowered the ability of sH to compete with MV binding. Antihemagglutinin monoclonal antibodies selectively inhibited the binding of either sH or MV. Thus, sH and MV share a common binding site in SCR I and II but differ in their apparent avidity to CD46 under the influence of SCR III and IV.

Animals↗

PubMed1996

Selective expression of a subset of measles virus receptor-competent CD46 isoforms in human brain.

The human cell surface protein CD46 is the main measles virus (MV) receptor. We analyzed the CD46 isoforms expressed in the brain of three patients who died with persistent MV infections and in an unaffected brain. Complete CD46 cDNAs were produced and found to code exclusively for CD46 isoforms with cytoplasmic tail 2. Selective expression of tail 2 isoforms was shown in a second control brain by Western blots with antibodies specific for each of the cytoplasmic tails. Binding of purified MV particles and virus-dependent cell fusion were tested after transient expression of brain-derived CD46 proteins in mouse cells. All the brain-derived proteins mediated MV binding and virus-dependent fusion. Isoforms containing both serine/threonine/proline (STP)-rich domains were more active in virus binding, whereas isoforms with only one STP domain were more efficient in mediating fusion.

Amino Acid Sequence↗

PubMed1996

The ectodomain of measles virus envelope glycoprotein does not gain access to the cytosol and MHC class I presentation pathway following virus-cell fusion.

To unravel the intracellular fate of measles virus (MV) haemagglutinin (H) following fusion of the virus envelope with the cell membrane, its presentation by MHC molecules to T cells was explored. After MV infection, murine cells expressing CD46 were lysed by MHC class I-restricted CD8 CTLs specific for the ectodomain of H. In contrast, when sensitized with UV-inactivated MV, they were not lysed by these effectors, but were recognized by H-specific and class II-restricted CD4 CTLs. Thus, after MV binding and fusion, H becomes associated with plasma membrane and its ectodomain can reach the endosomal MHC-II but not the cytosolic MHC-I antigen presentation pathway. From these data and a reappraisal of previous reports, it appears that the ectodomains of both MV haemagglutinin fusion proteins, having undergone the fusion step, are not translocated into the cytosol and end up in the endosomes.

Animals↗

PubMed1996

Interactions between the ectodomains of haemagglutinin and CD46 as a primary step in measles virus entry.

Recombinant soluble forms of the ectodomains of measles virus haemagglutinin (sH) and of its receptor CD46 (sCD46) were obtained as a purified disulphide-bonded sH homodimer with an apparent molecular mass of 160 kDa and a purified sCD46 monomer with an apparent molecular mass of 60 kDa, without detectable contamination with moesin. Purified sH bound to purified and immobilized sCD46 and this binding was specifically inhibited by sCD46 in solution. sCD46 bound to wild-type H expressed on the cell surface and inhibited measles virus binding to CD46-expressing cells. Binding of sCD46 to cell surface H was increased about twofold when measles virus fusion protein was coexpressed with H. sH bound to wild-type cell surface CD46 and inhibited measles virus binding onto CD46-expressing cells. sCD46 also inhibited virus infection. Thus, the direct interaction between the ectodomains of H and CD46 is likely to be the primary event in measles virus infection.

Animals↗

PubMed1996

Transgenic mice expressing human measles virus (MV) receptor CD46 provide cells exhibiting different permissivities to MV infections.

We have generated transgenic mice ubiquitously expressing the human receptor for measles virus (MV), CD46 (membrane cofactor protein). Various cell types were isolated from these transgenic mice and analyzed for their ability to support MV replication in vitro. Although MV could enter into all CD46-expressing cells, differential susceptibilities to MV infection were detected depending on the cell type. Cell cultures obtained from transgenic lungs and kidneys were found to be permissive of MV infection, since RNA specific for MV genes was detected and viral particles were released, although at a low level. Similarly to human lymphocytes, activated T and B lymphocytes isolated from transgenic mice could support MV replication; virus could enter, transcribe viral RNA, and produce new infectious particles. When expressing viral proteins, lymphocytes down-regulated CD46 from the surface. Interestingly, while activated T lymphocytes from nontransgenic mice did not support MV infection, activated nontransgenic murine B lymphocytes replicated MV as well as transgenic B lymphocytes, suggesting the use of an alternative virus receptor for entry. In contrast to the previous cell types, murine peritoneal and bone marrow-derived macrophages, regardless of whether they were activated, could not support MV replication. Furthermore, although MV entered into macrophages and virus-specific RNA transcription occurred, no virus protein or infectious virus particles could be detected. These results show the importance of the particular cell-type-specific host factors for MV replication in murine cells which may be responsible for the differential permissivity of MV infection.

Animals↗

PubMed1996

Cell entry by measles virus: long hybrid receptors uncouple binding from membrane fusion.

The pH-independent fusion of membranes induced by measles virus (MV) requires, in addition to the fusion-competent protein F, hemagglutinin (H), and on the target membrane, the virus receptor CD46. We constructed hybrid receptors composed of different numbers and combinations of the four CD46 short consensus repeat (SCR) domains, followed by immunoglobulin-like domains of another cell surface protein, CD4. Hybrid proteins containing SCRs I and II bound MV particles and conferred fusion competence to rodent cells. SCRs III and/or IV strengthened MV binding. Increasing the distance between the MV binding site and the transmembrane domain enhanced virus binding but reduced fusion efficiency. A hybrid protein predicted to be about 120 Angstroms (12 nm) longer than the standard receptor lost fusion support function and was dominant negative over a functional receptor. These data indicate that receptor protein length influences virus binding and determines fusion efficiency.

Animals↗

PubMed1995

Quantification of measles virus by a virus receptor-dependent and haemagglutinin-specific T cell stimulation assay.

The human measles virus receptor CD46 plays a major role in the uptake of measles virus (MV) for antigen presentation by major histocompatibility complex class II molecules to T cells. On this basis, a new bioassay has been set up to quantify measles virus in a cell free tissue culture supernatant. A stable mouse B cell transfectant expressing CD46 was used as the antigen presenting cell for presentation of measles virus to a haemagglutinin-specific and class II-restricted mouse T cell hybridoma. The measles virus haemagglutinin was quantified by its ability to stimulate IL-2 secretion by the T cells. A good correlation was found between the amount of haemagglutinin measured in supernatants from infected cells using the CD46-dependent T cell stimulation assay and the number of infectious viral particles as determined in a plaque assay. When MV was purified on a discontinuous sucrose gradient, most of the infectious virus and the haemagglutinin antigen were recovered in the same fraction. These data indicate that the CD46-dependent haemagglutinin-specific T cell assay could be used to measure the production of measles virus in the supernatant of infected cells. The assay required only 48 h, was sensitive, highly specific, and did not rely on the replication of the virus. This new bioassay would be applicable for the detection of any other virus provided that antigen presenting cells expressing the corresponding virus receptor and virus envelope glycoprotein-specific T cells are available. Moreover, it would be an interesting tool to monitor the receptor binding properties of attenuated vaccine virus and envelope glycoprotein subunit vaccines.

Animals↗

PubMed1995

Formaldehyde inactivation of measles virus abolishes CD46-dependent presentation of nucleoprotein to murine class I-restricted CTLs but not to class II-restricted helper T cells.

To induce an MHC-restricted specific CTL or Th response, an antigen must be delivered into the appropriate cellular compartment. We explored the role of CD46 in the presentation of measles virus (MV) nucleoprotein (NP) to murine NP-specific and MHC Class I-restricted polyclonal CTLs and the effect of inactivating MV by uv or formaldehyde. CD46(-)- and CD46(+)-transfected murine cells were used as target cells. After MV infection, only the targets which expressed CD46 were lysed by NP-specific class I-restricted CTLs. When MV was uv-inactivated, NP presentation by MHC class I molecules was retained but could be blocked by fusion inhibitors which block virus cell entry. When MV was inactivated with formaldehyde, NP was no longer presented by MHC class I molecules, although it was still presented by MHC class II molecules to a NP-specific class II-restricted T cell hybridoma. These data show that MV binding to the CD46 molecule is a prerequisite for virus-to-cell fusion and that cytosolic delivery of NP is necessary for presentation by class I molecules. Moreover, formaldehyde inactivation of virus induces the loss of class I-restricted presentation of NP due to selective abrogation of fusion and cytosolic delivery of NP.

Amino Acid Sequence↗

PubMed1995

Deletion of a C-terminal sequence of the class II-associated invariant chain abrogates invariant chains oligomer formation and class II antigen presentation.

The MHC class II-associated invariant chain (Ii) is involved in Ag processing and presentation. Physical association of MHC class II molecules with Ii and an effect of Ii on peptide loading to class II have been demonstrated, but to date these functions have not been related to a particular region of Ii. We investigated luminal deletion mutants of Ii and their role in Ag processing and presentation. IAk-expressing L cells were transfected with deletion mutants of the Ii gene and assayed for their ability to present hen egg lysozyme to three different T cell hybridomas. It is shown that the sequence aa 131-191 of Ii is important for the presentation of native hen egg lysozyme. In addition, this C terminal region is shown to be responsible for Ii oligomer formation. It is therefore conceivable that oligomer formation of Ii is a prerequisite for class II-restricted Ag processing and presentation.

Animals↗

PubMed1995

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.

Animals↗

PubMed1995

CD46-mediated measles virus entry: a first key to host-range specificity.

Humans are the sole natural host of measles virus. The identification of CD46 as a virus receptor and of the involvement of moesin sheds some light on the molecular events occurring during virus entry into the cell. Knowledge of the key role of CD46 paves the way to creating transgenic mice sensitive to measles virus infection.

Animals↗

PubMed1994

Efficient major histocompatibility complex class II-restricted presentation of measles virus relies on hemagglutinin-mediated targeting to its cellular receptor human CD46 expressed by murine B cells.

Measles virus after binding to its cell surface human CD46 receptor fuses with the plasma membrane. This fusion results in envelope hemagglutinin (H) and fusion glycoprotein (F) incorporated into the plasma membrane and injection of the nucleocapsid made of nucleoprotein (NP) into the cytosol. The influence of targeting measles virus (MV) to CD46 in the processing and presentation of MV H and NP to antigen specific MHC class II I-E(d)- and I-A(d)-restricted T cell hybridomas was explored using murine M12-CD46 B cell transfectants. Parent M12 cells, which lack any MV receptor, were unable to present any of these two viral proteins when incubated with MV particles. Incubating M12.CD46 cells with 200 ng and 10 micrograms of MV could strongly stimulate H-specific and NP-specific T cells, respectively. Neosynthesis of MV proteins was not necessary since the efficiency of antigen presentation was similar when using ultraviolet-inactivated MV. Similar enhancing effects (more than 1,000-fold) on antigen presentation were also observed when using purified native H soluble or incorporated into liposomes whereas denaturating H glycoprotein resulted in a poor efficiency in T cell stimulation, M12.CD46 being no more potent than the parental M12 counterpart. MV H and NP presentation efficiency did not depend on MV fusion with plasma membrane as revealed by the lack of effect of specific fusion inhibitors. Both MV H and NP presentations were sensitive to chloroquine inhibition indicating that antigens from CD46-mediated captured MV were likely processed in the endosome/lysosome compartment. Altogether these data indicate that (a) MV targeting via CD46 has a strong effect on the efficiency of antigen presentation by MHC class II, (b) the effect is mediated by the binding of H to CD46, and (c) though MV does fuse with plasma membrane, endocytosis, and processing of virus particles are also occurring. Since, in humans, CD46 is expressed in almost every tissue including professional antigen-presenting cells, such a targeting is likely to play a crucial role in the CD4+ T cell-mediated primary immune response against the pathogen in vivo.

Animals↗

PubMed1994

Efficient MHC class II-restricted presentation of measles virus to T cells relies on its targeting to its cellular receptor human CD46 and involves an endosomal pathway.

The role of the measles virus (MV) receptor, human CD46, in the uptake of MV and antigen presentation by Major Histocompatibility Complex (MHC) class II molecules was investigated. Expression of CD46 in murine B cells resulted in cells highly efficient in capturing UV-inactivated MV particles and presenting both envelope hemagglutinin H and nucleoprotein N to specific T cell hybridomas. Although MV fuse with the plasma membrane of its target cells, presentation of both MV-H and -N was sensitive to inhibition by chloroquine but was not affected by a tripeptide which prevents virus-cell fusion. Whereas 50 microM of chloroquine was required to inhibit presentation of MV-H, purified H or soluble N, only a two-fold lower concentration was required to inhibit that of MV-N. This shows that some CD46-mediated captured MV particles are endocytosed, then disrupted and processed in an endosome/lysosome compartment.

Amino Acid Sequence↗

PubMed1994

Critical residue combinations dictate peptide presentation by MHC class II molecules.

Peptides encompassing the core hen egg lysozyme HEL(52-61) peptide elongated or not and substituted or not with natural and unnatural amino acids were used to find a peptide motif for binding to the major histocompatibility complex (MHC) class II I-Ak. Using a T-cell recognition functional assay, nine out of 10 positions were found to be somehow involved in the I-Ak binding, and six out of 10 residues were involved in T-cell recognition. The deleterious effect of single substitutions could be rescued by changing peptide length and/or sequence. Thus, efficient binding to MHC class II molecules requires not only few anchoring residues correctly interspaced, but a complex, nonrandom combination of residues with appropriate orientation of the peptide backbone and some crucial side chains.

Amino Acid Sequence↗

PubMed1994

Measles virus receptor properties are shared by several CD46 isoforms differing in extracellular regions and cytoplasmic tails.

Human CD46, a member of the family of regulators of complement activation, has been shown recently to act as a measles virus (MV) receptor, interacting with the virus envelope glycoprotein haemagglutinin (HA). Owing to alternative RNA splicing, several CD46 isoforms are co-expressed in all tissues except erythrocytes. The optional exons encode extracellular serine-, threonine- and proline-rich regions of CD46 (designated STP-A, -B and -C) which are located proximal to the plasma membrane, and alternatively cytoplasmic tails (CYT1 or CYT2). The ability of the BC-CYT2, B-CYT2 and BC-CYT1 CD46 isoforms, expressed in rodent Chinese hamster ovary (CHO) cells, to mediate MV infection was tested. Every isoform was recognized by a monoclonal antibody (MAb), MCI20.6, which recognizes the MV-binding site on CD46. CHO cells expressing any of these CD46 isoforms were able to bind MV, the level of binding correlating with the CD46 expression level. Likewise, MV infection induced the cell-cell fusion of all CD46-expressing CHO cells but not of the parental CHO cells. Accordingly, MV replication was observed after infection of CHO cells expressing each CD46 isoform but not after infection of parental CHO cells. Finally, cell surface expression of every isoform was decreased after infection by MV. Altogether these data showed that the specific STP regions of CD46 played no major role in HA-mediated MV binding to CD46, virus infection and virus-induced down-regulation of CD46. Moreover, the CYT1 and CYT2 cytoplasmic tails of CD46 are either functionally similar although having distinct amino acid sequences or are dispensable for interaction with HA of MV.

Animals↗

PubMed1994

Glycosyl-phosphatidylinositol-anchored and transmembrane forms of CD46 display similar measles virus receptor properties: virus binding, fusion, and replication; down-regulation by hemagglutinin; and virus uptake and endocytosis for antigen presentation by major histocompatibility complex class II molecules.

The CD46 molecule is a receptor for measles virus (MV), CD46, which protects autologous cells from complement-mediated damage, exists in several isoforms which are variably expressed in different human tissues. These isoforms differ in their cytoplasmic and transmembrane regions and in a small portion of their proximal extracytoplasmic regions. To examine the role of the cytoplasmic and transmembrane regions of CD46 in MV infection, mouse M12 B cells stably expressing a transmembrane or a chimeric glycosyl-phosphatidylinositol (GPI)-anchored form of CD46 (CD46-GPI) were used. Both the GPI-anchored and transmembrane CD46 forms were able to mediate MV binding. MV binding mediated by the GPI-anchored form but not that mediated by the transmembrane form was abolished after treatment with phosphatidylinositol phospholipase C. MV infection of both M12.CD46 and M12.CD46-GPI cells but not parental M12 cells resulted in MV replication. Expression of hemagglutinin induced cell surface down-regulation of both CD46 and CD46-GPI. Both M12.CD46 and M12.CD46-GPI cells were able to efficiently capture MV for presentation of viral antigens by major histocompatibility complex class II molecules to T cells. This presentation was blocked by chloroquine, indicating some virus endocytosis. These data imply that the extracytoplasmic region encompassing the four N-terminal invariable short consensus repeat regions of CD46 is sufficient to act as a receptor for MV and that the cytoplasmic and transmembrane regions of CD46 may not play a major role in the signal for the hemagglutinin-induced down-regulation of CD46 and/or endocytosis of MV.

Animals↗

PubMed1993

Invariant chain expression similarly controls presentation of endogenously synthesized and exogenous antigens by MHC class II molecules.

I-Ak- and I-Ed-transfected L fibroblasts were supertransfected with cDNA coding for hen egg lysozyme (HEL) or measles virus hemagglutinin (HA). Well-defined cell culture conditions allowed us to obtain L cells with either no detectable endogenous Ii mRNA or a high level of endogenous Ii mRNA induced by serum starvation. Cells supertransfected with mouse Ii chain gene stably expressing a high level of Ii chain were also used as APC in parallel experiments. Class II presentation of endogenously secreted HEL or an ER-retained form of HEL to the HEL-specific I-Ak-restricted 3A9 T cell hybridoma was found to be strongly enhanced in cell transfectants expressing Ii chain. Similar results were obtained with the presentation of transmembrane HA to the HA-specific I-Ed-restricted TH5.143 T cell hybridoma. These findings correlate with those obtained with the presentation of exogenous HEL and HA. In addition, as reported to be the case for exogenous antigen, expression of a large amount of endogenous HA by the APC supplants the requirement for Ii chain expression in the enhancement of antigen presentation. These data demonstrate that presentation by MHC class II molecules of a given antigen, either exogenously provided or endogenously synthesized, is controlled in a similar manner by the Ii chain.

Animals↗

PubMed1993

Major histocompatibility complex class II-restricted presentation of secreted and endoplasmic reticulum resident antigens requires the invariant chains and is sensitive to lysosomotropic agents.

We have tested the involvement of the invariant chains (Ii) p31 and p41 in the presentation of peptides derived from hen egg lysozyme (HEL) constructs targeted to different intracellular compartments within transfected fibroblasts. The endogenous HEL constructs were either present in the cytosol (HELc), secreted (HELs), or linked to the mammalian (KDEL C-terminal sequence that causes retention of HEL in the endoplasmic reticulum (ER)/pre-Golgi recycling compartment (HELr). Using Ii-negative antigen-presenting cells, the presentation of HELr to a HEL 46-61 specific T cell hybridoma was far less efficient than the presentation of the HELs. High levels of Ii expression enhanced drastically the presentation of the HEL 46-61 determinant derived from both HELr and HELs. HELr and HELs presentation was fully sensitive to lysosomotropic agents such as chloroquine, indicating that the formation of complexes between major histocompatibility complex (MHC) class II molecules and determinants derived from endogenous antigens entering the secretory pathway is taking place in an acidic compartment. The degradation and dissociation of Ii might be a prerequisite for the efficient presentation of endogenously derived determinants by MHC class II molecules, as for the presentation of most exogenous antigens. All our results are compatible with the notion that endogenous molecules being translocated into the lumen of the ER could be presented by class II molecules through a processing pathway involving an acidic compartment in which Ii chains dissociate from class II molecules.

Animals↗