No abstract

We have analysed the expression and cellular localisation of the matrix protein VP40 from Ebola virus. Full-length VP40 and an N-terminal truncated construct missing the first 31 residues [VP40(31-326)] both locate to the plasma membrane of 293T cells when expressed transiently, while a C-terminal truncation of residues 213 to 326 [VP40(31-212)] shows only expression in the cytoplasm, when analysed by indirect immunofluorescence and plasma membrane preparations. In addition, we find that full-length VP40 [VP40(1-326)] and VP40(31-326) are both released into the cell culture supernatant and float up in sucrose gradients. The efficiency of their release, however, is dependent on the presence of the N-terminal 31 residues. VP40 that is released into the supernatant is resistant to trypsin digestion, a finding that is consistent with the formation of viruslike particles detected by electron microscopy. Together, these results provide strong evidence that Ebola virus VP40 is sufficient for virus assembly and budding from the plasma membrane.

contributed equally to this workThe matrix protein VP40 from Ebola virus is targeted to the plasma membrane, where it is thought to induce assembly and budding of virions through its association with the lipid bilayer. Ebola virus VP40 is expressed as a monomeric molecule in solution, consisting of two loosely associated domains. Here we show that a C-terminal truncation of seven residues destabilizes the monomeric closed conformation and induces spontaneous hexamerization in solution, as indicated by chemical cross-linking and electron microscopy. Three-dimensional reconstruction of electron microscopy images shows ring-like structures consisting of the N-terminal domain along with evidence for¯exibly attached C-terminal domains. In vitro destabilization of the monomer by urea treatment results in similar hexameric molecules in solution.In addition, we demonstrate that membrane association of wild-type VP40 also induces the conformational switch from monomeric to hexameric molecules that may form the building blocks for initiation of virus assembly and budding. Such a conformational change induced by bilayer targeting may be a common feature of many viral matrix proteins and its potential inhibition may result in new anti-viral therapies.

SummaryWe have shown recently that one of the survival strategies used by Leishmania donovani promastigotes during the establishment of infection in macrophages consists in inhibiting phagosome±endosome fusion. This inhibition requires the expression of lipophosphoglycan (LPG), the predominant surface glycoconjugate of promastigotes, as parasites expressing truncated forms of LPG reside in phagosomes that fuse extensively with endocytic organelles. In the present study, we developed a single-organelle¯uorescence analysis approach to study and analyse the intracellular traf®cking of`fusogenic' and`low-fusogenic' phagosomes induced by an LPG repeating unit-defective mutant (lpg2 KO) or by wild-type L. donovani promastigotes respectively. The results obtained indicate that phagosomes containing mutant parasites fuse extensively with endocytic organelles and transform into phagolysosomes by losing the early endosome markers EEA1 and transferrin receptor, and acquiring the late endocytic and lysosomal markers rab7 and LAMP1. In contrast, a majority of`low-fusogenic' phagosomes containing wild-type L. donovani promastigotes do not acquire rab7, wheres they acquire LAMP1 with slower kinetics. These results suggest that L. donovani parasites use LPG to restrict phagosome±endosome fusion at the onset of infection in order to prevent phagosome maturation. This is likely to permit the transformation of hydrolase-sensitive promastigotes into hydrolase-resistant amastigotes within a hospitable vacuole not displaying the harsh environment of phagolysosomes.

SummaryThe lipophosphoglycan (LPG) of Leishmania promastigotes plays key roles in parasite survival in both insect and mammalian hosts. Evidence suggests that LPG decreases phagosome fusion properties at the onset of infection in macrophages. The mechanisms of action of this molecule are, however, poorly understood. In the present study, we used a panoply of Leishmania mutants displaying modi®ed LPG structures to determine more precisely how LPG modulates phagosome±endosome fusion. Using an in vivo fusion assay measuring, at the electron microscope, the transfer of solute materials from endosomes to phagosomes, we provided further evidence that the repeating Gal(b1,4)Man(a1-PO 4 ) units of LPG are responsible for the alteration in phagosome fusion. The inhibitory effect of LPG on phagosome fusion was shown to be more potent towards late endocytic organelles and lysosomes than early endosomes, explaining how Leishmania promastigotes can avoid degradation in hydrolase-enriched compartments. The involvement of other repeating unit-containing molecules, including the secreted acid phosphatase, in the inhibition process was ruled out, as an LPG-defective mutant (lpg1 À ) which secretes repeating unit-containing glycoconjugates was present in highly fusogenic phagosomes. In L. major, oligosaccharide side-chains of LPG did not contribute to the inhibition process, as Spock, an L. major mutant lacking LPG side-chains, blocked fusion to the same extent as wild-type parasites. Finally, dead parasites internalized from the culture medium were not as ef®cient as live parasites in altering phagosome±endosome fusion, despite the presence of LPG. However, the killing of parasites with vital dyes after their sequestration in phagosomes had no effect on the fusion properties of this organelle. Collectively, these results suggest that living promastigotes displaying full-length cell surface LPG can actively in¯uence macrophages at an early stage of phagocytosis to generate phagosomes with poor fusogenic properties.