Pathogenic mycobacteria such as Mycobacterium tuberculosis and Mycobacterium avium facilitate disease by surviving intracellularly within a potentially hostile environment: the macrophage phagosome. They inhibit phagosome maturation processes, including fusion with lysosomes, acidification and, as shown here, membrane actin assembly. An in vitro assay developed for latex bead phagosomes (LBPs) provided insights into membrane signalling events that regulate phagosome actin assembly, a process linked to membrane fusion. Different lipids were found to stimulate or inhibit actin assembly by LBPs and mycobacterial phagosomes in vitro. In addition, selected lipids activated actin assembly and phagosome maturation in infected macrophages, resulting in a significant killing of M. tuberculosis and M. avium. In contrast, the polyunsaturated sigma-3 lipids behaved differently and stimulated pathogen growth. Thus, lipids can be involved in both stimulatory and inhibitory signalling networks in the phagosomal membrane.
We have studied the production and release of infectious DI-particles in vaccinia-T7-polymerase recombinant virus-infected L cells that were transfected with five different plasmids expressing the synthetic DI RNA MIDI-HD and the four structural proteins (M, N, S, and E) of the murine coronavirus MHV-A59. The DI cDNA contains the hepatitis delta ribozyme sequences to generate in the transfected cells a defined 3' end. In EM studies of transfected cells virus-like particles (VLP) were observed in vesicles. Release of the particles into the medium was studied by immunoprecipitations of proteins released into the culture supernatant. Particle release was independent of S or N, but required M and E. Coexpression of E and M was sufficient for particle release. Coexpression of the structural proteins and the MIDI-HD RNA resulted in the production and release of infectious DI-particles. Infectivity of the DI-particles was determined by adding helper virus MHV-A59 to the medium containing the VLPs and using this mixture to infect new L cells. Intracellular RNA of several subsequent undiluted passages was isolated to detect the MIDI-HD RNA. Passage of the MIDI-HD RNA was dependent on the expression of the structural proteins of MHV-A59 in the transfected cells. In the absence of either E or M, MIDI-HD RNA could not be passaged to fresh L cells. We have thus developed a system in which we can produce coronavirus-like particles and an assay to test their infectivity.
The spike (S) protein of murine coronavirus strain A59 (MHV-A59) is a type I membrane protein that induces membrane fusion. In this study we have analyzed the role of two domains in the S protein on fusion. The 180-kDa mature S protein is partially cleaved into two 90-kDa subunits during transport to the plasma membrane. We have identified several amino acids that are important for cleavage of S, and we show that cleavage is not strictly required for fusion. However, the level of cleavage seems to influence the fusion kinetics. After introduction of an arginine at position P2 to mimick the MHV-JHM cleavage site, full cleavage of the spike protein was obtained. Further, we analyzed the effect of mutations in the transmembrane (TM) domain of the S protein. Maturation and cell surface expression of the mutant proteins were not affected, and all proteins became acylated. The mutant in which the predicted transmembrane domain was shortened did not induce syncytia. From a group of mutants in which several conserved cysteines in the TM domain had been replaced by serines, one was unable to induce syncytia, another showed delayed syncytia formation, and the third mutant induced syncytia as did the wild-type protein. The potential role of the transmembrane domain in fusion is discussed.
Actin assembly on membrane surfaces is an elusive process in which several phosphoinositides (PIPs) have been implicated. We have reconstituted actin assembly using a defined membrane surface, the latex bead phagosome (LBP), and shown that the PI(4,5)P 2 -binding proteins ezrin and/or moesin were essential for this process (Defacque et al., 2000b). Here, we provide several lines of evidence that both preexisting and newly synthesized PI(4,5)P 2 , and probably PI(4)P, are essential for phagosomal actin assembly; only these PIPs were routinely synthesized from ATP during in vitro actin assembly. Treatment of LBP with phospholipase C or with adenosine, an inhibitor of type II PI 4-kinase, as well as preincubation with anti-PI(4)P or anti-PI(4,5)P 2 antibodies all inhibited this process. Incorporation of extra PI(4)P or PI(4,5)P 2 into the LBP membrane led to a fivefold increase in the number of phagosomes that assemble actin. An ezrin mutant mutated in the PI(4,5)P 2 -binding sites was less efficient in binding to LBPs and in reconstituting actin assembly than wild-type ezrin. Our data show that PI 4-and PI 5-kinase, and under some conditions also PI 3-kinase, activities are present on LBPs and can be activated by ATP, even in the absence of GTP or cytosolic components. However, PI 3-kinase activity is not required for actin assembly, because the process was not affected by PI 3-kinase inhibitors. We suggest that the ezrin-dependent actin assembly on the LBP membrane may require active turnover of D4 and D5 PIPs on the organelle membrane. INTRODUCTIONA significant fraction of the de novo nucleation of actin in cells occurs on the cytoplasmic surface of eukaryotic cell membranes, especially the plasma membrane (Tilney, 1976;Carraway and Carraway, 1989;Small et al., 1995;Mitchison and Cramer, 1996), and a role for phosphoinositides in this elusive process has been widely discussed (Divecha and Irvine, 1995;Martin, 1998;Caroni, 2001). However, the precise function of these lipids is still not clear and is likely to be quite complicated. In several cellular systems that show rapid actin assembly in response to extracellular ligands, synthesis of phosphoinositides, especially phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ], and in some cases phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P 3 ], coincides precisely with the transient burst of actin assembly (Eberle et al., 1990;Dobos et al., 1992;Apgar, 1995;Hartwig et al., 1995;Gachet et al., 1997). In addition, overexpression of phosphatidylinositol-4-phosphate [PI(4)P] 5-kinase in cells leads to a significant polymerization of actin (Shibasaki et al., 1997). However, in other systems, the synthesis of PI(4,5)P 2 as well as PI(3,4,5)P 3 coincides more with actin depolymerization, after a transient assembly of F-actin (Apgar, 1995;Gratacap et al., 1998).One important clue to the functions of phosphoinositides in actin assembly/disassembly is that these lipids can bind in vitro to an increasing number of actin-binding proteins (ABPs). Interestingly, two different...
The spike protein (S) of the murine coronavirus mouse hepatitis virus strain A59 (MHV-A59) induces both virus-to-cell fusion during infection and syncytium formation. Thus far, only syncytium formation could be studied after transient expression of S. We have recently described a system in which viral infectivity is mimicked by using virus-like particles (VLPs) and reporter defective-interfering (DI) RNAs (E. C. W. Bos, W.
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