Assembly of nucleocapsids with cytosolic and membrane-derived matrix proteins of vesicular stomatitis virus.
(41/2088)
During budding of vesicular stomatitis virus (VSV), the viral matrix (M) protein binds the viral nucleocapsid to the host plasma membrane and condenses the nucleocapsid into the tightly coiled nucleocapsid-M protein (NCM) complex observed in virions. In infected cells, the viral M protein exists mostly as a soluble molecule in the cytoplasm, and a small amount is bound to the plasma membrane. Despite the high concentrations of M protein and intracellular nucleocapsids in the cytoplasm, they are not associated with each other except at the sites of budding. The experiments presented here address the question of why M protein and nucleocapsids associate with each other only at the plasma membrane but not in the cytoplasm of infected cells. An assay for exchange of soluble M protein into NCM complexes in vitro was used to show that both cytosolic and membrane-derived M proteins bound to virion NCM complexes with affinities similar to that observed for virion M protein, indicating that both cytosolic and membrane-derived M proteins are competent for virus assembly. However, neither cytosolic nor membrane-derived M protein bound to intracellular nucleocapsids with the same high affinity observed for virion NCM complexes. Cytosolic M protein was able to bind intracellular nucleocapsids, but with an affinity approximately eightfold less than that observed in virion NCM complexes. Membrane-derived M protein exhibited little or no binding activity for intracellular nucleocapsids. These data indicate that intracellular nucleocapsids, and not intracellular M proteins, need to undergo an assembly-initiating event in order to assemble into an NCM complex. Since neither membrane-derived nor cytosolic M protein could initiate high-affinity binding to intracellular nucleocapsids, the results suggest that another viral or host factor is required for assembly of the NCM complex observed in virions. (+info)
Induction and visualization of mucosal memory CD8 T cells following systemic virus infection.
(42/2088)
Whether CD8 T cell memory exists outside secondary lymphoid organs is unclear. Using an adoptive transfer system that enables tracking of OVA-specific CD8 T cells, we explored the antigenic requirements for inducing CD8 T cell memory and identified intestinal mucosa memory cells. Although systemic immunization with soluble OVA induced clonal expansion, memory CD8 cells were not produced. In contrast, infection with virus-encoding OVA induced memory CD8 cells in the periphery and the lamina propria and intraepithelial compartments of the intestinal mucosa. Mucosal memory cells expressed a distinct array of adhesion molecules as compared with secondary lymphoid memory cells, suggesting that there may be separate mucosal and systemic memory pools. Mucosal CD8 memory cells rapidly produced IFN-gamma after Ag stimulation. Reactivation of memory cells by Ag feeding resulted in increased cell size and up-regulation of CD28 and CD11c. CD8 mucosal memory cells exhibited ex vivo lytic activity that was up-regulated dramatically following Ag reencounter in vivo. Interestingly, reactivation of memory cells did not require CD28-mediated costimulation. The ability of the intestinal mucosa to maintain CD8 memory cells provides a potential mechanism for effective mucosal vaccination. (+info)
Up-regulation of NK cytotoxic activity via IL-15 induction by different viruses: a comparative study.
(43/2088)
IL-15 is a recently identified cytokine that belongs to the four alpha-helix bundle cytokine family and possesses biological activities similar to those of IL-2. Its ability to induce effectors of NK activity suggests its involvement in innate immunity. In this study, we analyzed the effect of different viruses (HSV, EBV, respiratory syncitial virus, vesicular stomatitis virus, influenza virus, reovirus, and Sendai virus) on the up-regulation of NK activity in vitro. Exposure of human PBMC to the these viruses resulted in an immediate up-regulation of NK activity of PBMC via IL-15 induction; this effect was abrogated in the presence of mAbs to IL-15. Results of experiments conducted in parallel using mAbs to IL-15, as well as to other cytokines (IL-2, IL-12, IFN-gamma, and TNF-alpha), clearly indicated that IL-15 was specifically responsible for the observed effect. Furthermore, supernatants of virus-infected PBMC cultures significantly enhanced NK activity of uninfected PBMC in vitro. An increase of IL-15 protein levels 20 h postinfection was also confirmed in a bioassay using the IL-2-dependent cell line CTLL. Kinetic analysis of IL-15 mRNA expression using a semiquantitative RT-PCR revealed that the level of IL-15 messages peaked at different time points (up to 12 h) postinfection, depending on the nature of the virus. Taken together, these results suggest that the IL-15 response of the host to viral infection and the subsequent NK cell activation represent an important effector mechanism of the innate immune surveillance of the host against viral infections. (+info)
Antiviral and hemolytic activities of surfactin isoforms and their methyl ester derivatives.
(44/2088)
Inactivation of enveloped viruses (VSV, SFV, and SHV-1) by surfactin lipopeptides was dependent on the hydrophobicity, i.e. the number of carbon atoms of the fatty acid, and on the charge of the peptide moiety as well as on the virus species. Surfactins with fatty acid chains of 13 carbon atoms showed very low antiviral activity in comparison to C14 and C15 isoforms. C15 surfactin monomethyl ester also inactivated SFV which was resistant to the mixture of surfactin isoforms as produced by Bacillus subtilis. In contrast, the dimethyl ester showed no virus-inactivation capacity. Disintegration of viral structures as determined by electron microscopy after inactivation of VSV and SFV was comparable to the titer reduction. The effect of the surfactin isoforms and methyl esters on erythrocyte hemolysis correlated with the virus-inactivation capacity. Surfactins with a fatty acid chain moiety of 15 carbon atoms and one negative charge showed the highest antiviral activity. (+info)
TRAF2 deficiency results in hyperactivity of certain TNFR1 signals and impairment of CD40-mediated responses.
(45/2088)
Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) can interact with various members of the TNF receptor family. Previously, we reported that TRAF2-deficient mice die prematurely and have elevated serum TNF levels. In this study, we demonstrate that TRAF2-deficient macrophages produce increased amounts of nitric oxide (NO) and TNF in response to TNF stimulation. Furthermore, we could enhance the survival of TRAF2-deficient mice by eliminating either TNF or TNFR1. Using these double-knockout mice, we show that in the absence of TRAF2, the T helper-dependent antibody response, CD40-mediated proliferation, and NF-kappaB activation are defective. These data demonstrate two important roles of TRAF2, one as a negative regulator of certain TNFR1 signals and the other as a positive mediator of CD40 signaling. (+info)
Protective T cell-independent antiviral antibody responses are dependent on complement.
(46/2088)
Complement is part of the innate immune system and one of the first lines of host defense against infections. Its importance was evaluated in this study in virus infections in mice deficient either in soluble complement factors (C3(-/-), C4(-/-)) or in the complement signaling complex (complement receptor [CR]2(-/-), CD19(-/-)). The induction of the initial T cell-independent neutralizing immunoglobulin (Ig)M antibody response to vesicular stomatitis virus (VSV), poliomyelitis virus, and recombinant vaccinia virus depended on efficient antigen trapping by CR3 and -4-expressing macrophages of the splenic marginal zone. Neutralizing IgM and IgG antibody responses were largely independent of CR2-mediated stimulation of B cells when mice were infected with live virus. In contrast, immunizations with nonreplicating antigens revealed an important role of B cell stimulation via CR2 in the switch to IgG. The complement cascade was activated after infection with VSV via the classical pathway, and active complement cleavage products augmented the effector function of neutralizing IgM and IgG antibodies to VSV by a factor of 10-100. Absence of the early neutralizing antibody responses, together with the reduced efficiency of neutralizing IgM in C3(-/-) mice, led to a drastically enhanced susceptibility to disease after infection with VSV. (+info)
Analysis of 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking both 4-1BBL and CD28 reveals a role for 4-1BBL in skin allograft rejection and in the cytotoxic T cell response to influenza virus.
(47/2088)
4-1BB ligand (4-1BBL) is a member of the TNF family expressed on activated APC. 4-1BBL binds to 4-1BB (CD137) on activated CD4 and CD8 T cells and in conjunction with strong signals through the TCR provides a CD28-independent costimulatory signal leading to high level IL-2 production by primary resting T cells. Here we report the immunological characterization of mice lacking 4-1BBL and of mice lacking both 4-1BBL and CD28. 4-1BBL-/- mice mount neutralizing IgM and IgG responses to vesicular stomatitis virus that are indistinguishable from those of wild-type mice. 4-1BBL-/- mice show unimpaired CTL responses to lymphocytic choriomeningitis virus (LCMV) and exhibit normal skin allograft rejection but have a weaker CTL response to influenza virus than wild-type mice. 4-1BBL-/-CD28-/- mice retain the CTL response to LCMV, respond poorly to influenza virus, and exhibit a delay in skin allograft rejection. In agreement with these in vivo results, allogeneic CTL responses of CD28-/- but not CD28+/+ T cells to 4-1BBL-expressing APC are substantially inhibited by soluble 4-1BB receptor as is the in vitro secondary response of CD28+ T cells to influenza virus peptides. TCR-transgenic CD28-/- LCMV glycoprotein-specific T cells are insensitive to the presence of 4-1BBL when a wild-type peptide is used, but the response to a weak agonist peptide is greatly augmented by the presence of 4-1BBL. These results further substantiate the idea that different immune responses vary in their dependence on costimulation and suggest a role for 4-1BBL in augmenting suboptimal CTL responses in vivo. (+info)
Oligonucleotides tethered to a short polyguanylic acid stretch are targeted to macrophages: enhanced antiviral activity of a vesicular stomatitis virus-specific antisense oligonucleotide.
(48/2088)
The poor membrane permeability of oligonucleotides is one of the major problems of antisense technology. Here we report the construction of designer oligonucleotides for targeted delivery to macrophages. The oligonucleotides tethered to a 10-mer poly(G) sequence at their 3' ends were recognized by scavenger receptors on macrophages and were taken up about 8- to 10-fold as efficiently as those oligonucleotides that either lacked a poly(G) tail or that contained a 10-mer poly(C) tail instead of the poly(G) tail. The enhanced uptake of poly(G) constructs was inhibited in the presence of poly(G) and other known ligands of the scavenger receptor. The bioefficacy of poly(G)-mediated targeting of antisense oligonucleotides (ANS) was demonstrated by using vesicular stomatitis virus (VSV) as a model system. The ability of ANS directed against the translation initiation site of N protein mRNA of VSV to inhibit virus replication was assessed. The ANS with the 10-mer poly(G) sequences (ANS-G) brought about significant inhibition of VSV replication in J774E cells (a murine monocyte/macrophage cell line) and Chinese hamster ovary (CHO) cell transfectants expressing scavenger receptors. The ANS lacking a 10-mer poly(G) stretch were ineffective. The inhibition of VSV replication due to ANS-G was completely abrogated in the presence of 10-mer poly(G), indicating that the antisense effect of the ANS-G molecule was a consequence of scavenger receptor-mediated enhanced uptake. Importantly, antisense molecules linked exclusively by natural phosphodiester bonds were as bioeffective as those synthesized with a mixed backbone of phosphodiester and phosphorothioate. Taken together, these results suggest that macrophage-directed designer ANS against infective agents may simply be obtained by adding a short stretch of guanylic acid sequence to the desired specific ANS during solid-phase synthesis. This nucleic acid-based strategy, which utilizes homogeneous preparation of ANS, may find applications in directed manipulation of macrophage metabolism for a variety of purposes as well as in therapy of a broad spectrum of macrophage-related disorders amenable to the antisense approach. (+info)