Fabian Wild

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.

Dendritic Cells Recruitment and In Vivo Priming of CD8+ CTL Induced by a Single Topical or Transepithelial Immunization Via the Buccal Mucosa with Measles Virus Nucleoprotein

The buccal mucosa, a prototype of pluristratified mucosal epithelia, contains a network of directly accessible class II+ epithelial dendritic cells (DC), similar to skin Langerhans cells. We showed that a single buccal immunization with measles virus nucleoprotein (NP), by either topical application onto or intradermal injection in the buccal mucosa, induced in vivo priming of protective class I-restricted specific CD8+ CTL. Both routes of immunization with NP induced a rapid recruitment of DC into the mucosa, which peaked at 2 h and decreased by 24 h. Treatment of mice with Flt3 ligand resulted in an increased number of DC in the buccal mucosa and enhanced the frequency of IFN-γ-producing NP-specific effectors and the NP-specific CTL response generated after buccal immunization with NP. Finally, NP-pulsed bone marrow-derived DC induced NP-specific IFN-γ-producing cells upon adoptive transfer to naive mice. These data demonstrate that a viral protein delivered to DC of the buccal mucosa induces in vivo priming of protective anti-viral CD8+ CTL.

Mucosal and systemic immune responses to measles virus haemagglutinin in mice immunized with a recombinant vaccinia virus

The immune response to a vaccinia virus recombinant expressing the measles virus haemagglutinin (VV-HA) was compared after parenteral or mucosal immunizations in mice. Parenteral immunizations with 10(6) p.f.u. VV-HA induced HA-specific antibody-producing cells (IgG>IgA) and HA-specific class I-restricted cytotoxic T lymphocytes (CTL) in the spleen. In contrast, intranasal administrations of 10(6) p.f.u. of VV-HA induced HA-specific spot-forming cells in the spleen (IgG>IgA) and the lungs (IgA>IgG), and HA-specific CTL in the spleen. Co-immunization by the nasal route with VV-HA and cholera toxin enhanced HA-specific immune responses. Oral immunizations with 10(8) p.f.u. of VV-HA generated low numbers of HA-specific IgA-producing cells in the lamina propria of the gut, and a weak HA-specific CTL activity in the spleen and mesenteric lymph nodes. Oral co-immunization with VV-HA and cholera toxin greatly enhanced the level of HA-specific spot-forming cells in the lamina propria (IgA>IgG). Interestingly, intrajejunal immunizations with 10(8) p.f.u. VV-HA alone induced high levels of anti-HA IgG-producing cells in the spleen and anti-HA IgA-secreting cells in the lamina propria of the gut. These data show that (i) VV-HA by the nasal route is immunogenic and generates a measles-specific mucosal immune response in the lung, which represents the primary site of replication of measles virus and that (ii) VV-HA can also induce measles-specific immunity in the intestine provided that it is protected from degradation in the gastrointestinal tract, or that cholera toxin is used as an adjuvant.

Establishment and characterisation of murine cells constitutively expressing the fusion, nucleoprotein and matrix proteins of measles virus

To advance our understanding of the immunobiology of measles virus (MV) infections, we have investigated the possibility of establishing cell lines constitutively expressing the individual MV antigens. In contrast to previously published studies, we show that it is possible to establish cell lines expressing high levels of fusion (F), nucleoprotein (NP) and matrix (M) MV proteins. Once cloned, the cell lines were stable with high levels of expression for more than six months. The size and cell distribution of the NP and F proteins were similar to those observed in MV- or vaccinia-MV recombinant-infected cells. In contrast, the distribution of the M protein, although being similar to that of MV-infected cells, differed from that of Vaccinia-M recombinant virus-infected cells. Preliminary results suggest that these cell lines will be useful tools for studying the contribution of individual MV antigens to the cell-mediated immune response to this virus.

Self-association of truncated forms of HIV-1 gp120

HIV-1 gp120 and truncated forms were expressed in HeLa T4 cells by vaccinia recombinant viruses. The truncated gp120 molecules consisted of N-terminal overlapping envelope proteins of 204, 287 and 393 amino acids respectively. Immunoprecipitation with specific monoclonal antibodies and SDS-PAGE analyses of HIV-1 gp120 revealed bands corresponding to low amounts of secreted and cell-bound stable dimers. In contrast, the truncated forms of gp120 expressed larger amounts of SDS-stable putative dimers and the amounts observed were inversely proportional to their size. The shortest gp120 mutant (204 aa) was found to be secreted almost exclusively as a dimer. The processing of gp120 and its truncated forms was further investigated in the presence of inhibitors of N-glycosylation. Monomers and dimers migrated on gels with the same relative changes, confirming that the protein with the higher molecular weight is a multimer of the smaller one. The putative dimeric form of the truncated gp120s could be stabilized by chemical cross-linking. Finally, the possible existence of an association domain in the N-terminal 204 amino acids (aa) of gp120 is discussed.

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.