Tyrosine phosphorylation is required for actin-based motility of vaccinia but not Listeria or Shigella.
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Studies of the actin-based motility of pathogens have provided important insights into the events occurring at the leading edge of motile cells [1] [2] [3]. To date, several actin-cytoskeleton-associated proteins have been implicated in the motility of Listeria or Shigella: vasodilator-stimulated phosphoprotein (VASP), vinculin and the actin-related protein complex of Arp2 and Arp3 [4] [5] [6] [7]. To further investigate the underlying mechanism of actin-tail assembly, we examined the localization of components of the actin cytoskeleton including Arp3, VASP, vinculin and zyxin during vaccinia, Listeria and Shigella infections. The most striking difference between the systems was that a phosphotyrosine signal was observed only at the site of vaccinia actin-tail assembly. Micro-injection experiments demonstrated that a phosphotyrosine protein plays an important role in vaccinia actin-tail formation. In addition, we observed a phosphotyrosine signal on clathrin-coated vesicles that have associated actin-tail-like structures and on endogenous vesicles in Xenopus egg extracts which are able to nucleate actin tails [8] [9]. Our observations indicate that a host phosphotyrosine protein is required for the nucleation of actin filaments by vaccinia and suggest that this phosphoprotein might be associated with cellular membranes that can nucleate actin. (+info)
Characterization of transgenic mice with targeted disruption of the catalytic domain of the double-stranded RNA-dependent protein kinase, PKR.
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The interferon-inducible, double-stranded RNA-dependent protein kinase PKR has been implicated in anti-viral, anti-tumor, and apoptotic responses. Others have attempted to examine the requirement of PKR in these roles by targeted disruption at the amino terminal-encoding region of the Pkr gene. By using a strategy that aims at disruption of the catalytic domain of PKR, we have generated mice that are genetically ablated for functional PKR. Similar to the other mouse model of Pkr disruption, we have observed no consequences of loss of PKR on tumor suppression. Anti-viral response to influenza and vaccinia also appeared to be normal in mice and in cells lacking PKR. Cytokine signaling in the type I interferon pathway is normal but may be compromised in the erythropoietin pathway in erythroid bone marrow precursors. Contrary to the amino-terminal targeted Pkr mouse, tumor necrosis factor alpha-induced apoptosis and the anti-viral apoptosis response to influenza is not impaired in catalytic domain-targeted Pkr-null cells. The observation of intact eukaryotic initiation factor-2alpha phosphorylation in these Pkr-null cells provides proof of rescue by another eukaryotic initiation factor-2alpha kinase(s). (+info)
mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase from vaccinia virions. Donor and acceptor substrate specificites.
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Characterization of the donor and acceptor specificities of mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase isolated from vaccinia virus cores has enabled us to discriminate between alternative reaction sequences leading to the formation of the 5'-terminal m7G(5')pppN-structure. The mRNA guanylyltransferase catalyzes the transfer of a residue of GMP from GTP to acceptors which possess a 5'-terminal diphosphate. A diphosphate-terminated polyribonucleotide is preferred to a mononucleoside diphosphate as an acceptor suggesting that the guanylyltransferase reaction occurs after initiation of RNA synthesis. Although all of the homopolyribonucleotides tested (pp(A)n, pp(G)n, pp(I)n, pp(U)n, and pp(C)n) are acceptors for the mRNA guanylyltransferase indicating lack of strict sequence specificity, those containing purines are preferred. Only GTP and dGTP are donors in the reaction; 7-methylguanosine (m7G) triphosphate specifically is not a donor indicating that guanylylation must precede guanine-7-methylation. The preferred acceptor of the mRNA (guanine-7-)-methyltransferase is the product of the guanylyltransferase reaction, a polyribonucleotide with the 5'-terminal sequence G(5')pppN-. The enzyme can also catalyze, but less efficiently methylation of the following: dinucleoside triphosphates with the structure G(5')pppN, GTP, dGTP, ITP, GDP, GMP, and guanosine. The enzyme will not catalyze the transfer of methyl groups to ATP, XTP, CTP, UTP, or to guanosine-containing compounds with phosphate groups in either positions 2' or 3' or in 3'-5' phosphodiester linkages. The latter specificity provides an explanation for the absence of internal 7-methylguanosine in mRNA. In the presence of PPi, the mRNA guanylyltransferase catalyzes the pyrophosphorolysis of the dinucleoside triphosphate G(5')pppA, but not of m7G(5')pppA. Since PPi is generated in the process of RNA chain elongation, stabilization of the 5'-terminal sequences of mRNA is afforded by guanine-7-methylation. (+info)
A lipid modified ubiquitin is packaged into particles of several enveloped viruses.
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An anti-ubiquitin cross-reactive protein which migrates more slowly (6.5 kDa) by SDS-PAGE than ubiquitin was identified in African swine fever virus particles. This protein was extracted into the detergent phase in Triton X-114 phase separations, showing that it is hydrophobic, and was radiolabelled with both [3H]palmitic acid and [32P]orthophosphate. This indicates that the protein has a similar structure to the membrane associated phosphatidyl ubiquitin described in baculovirus particles. A similar molecule was found in vaccinia virus and herpes simplex virus particles, suggesting that it may be a component of uninfected cell membranes, which is incorporated into membrane layers in virions during morphogenesis. (+info)
Cytotoxic T-cell responses in mice infected with influenza and vaccinia viruses vary in magnitude with H-2 genotype.
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Secondary effector T-cell populations generated by cross-priming with heterologous influenza A viruses operate only in H-2K or H-2D compatible situations, when assayed on SV40-transformed target cells infected with a range of influenza A viruses. The H2-Kb allele is associated with a total failure in the generation of influenza-immune cytotoxic T cells, though this is not seen for the primary response to vaccinia virus. In both influenza and vaccinia development of effector T cells operating at H-2Db is greatly depressed in B10.A(2R) (kkkddb) and B10.A(4R) (kkbbbb), but not in B10 (bbbbbb), mice. However, there is no defect in viral antigen expression at either H-2Kk or H-2Db in B10.A(2R) target cells. This apparently reflects some inadequacy in the stimulator environment, as (A/J X B6) F1 T cells can be induced to respond at H-2Db when exposed to vaccinia virus in an irradiated B6 but not in a B10.A(4R) recipient. The present report, together with the accompanying paper by Zinkernagel and colleagues, records the first rigorous demonstration of both a nonresponder situation and a probable Ir-gene effect for conventional infectious viruses. Possible implications for the evolution of H-2 polymorphism and mechanisms of Ir gene function are discussed. (+info)
In irradiation chimeras, K or D regions of the chimeric host, not of the donor lymphocytes, determine immune responsiveness of antiviral cytotoxic T cells.
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The H-2 haplotype of the chimeric host determines the responder phenotype of maturing T cells. Spleen cells of chimeric mice formed when (K(k) nonresponder to D(b) x K(b) responder to D(b) plus vaccinia)F(1) bone marrow cells were used to reconstitute K(b)D(b) (C57BL/6 D(b) responder) irradiated recipients generated high levels of D(b) plus vaccinia virus-specific cytotoxic T cells. The same stem cells used to reconstitute K(k)D(b) (B10.A (2R) D(b) nonresponder) irradiated recipients resulted in spleen cells that responded well to K plus vaccinia, but responsiveness to D(b) was low. A generally low response to D(k) plus vaccinia, which seems to be regulated by D(k), was confirmed in chimeras. Thus, K(d)D(d) (D(d) plus vaccinia responder) stem cells differentiating in a K(d)D(k) chimeric host failed to generate a measurable response to D(k) plus vaccinia. In contrast, stem cells from K(d)D(k) (D(k) plus vaccinia low responders) differentiating in a K(d)D(d) (K(d) and D(d) high responders to vaccinia) host do generate responsiveness to D(d) plus vaccinia. These results indicate that in chimeras, the Ir phenotype is independent of the donor T cell's Ir genotype, and that thymic selection of a T cell's restriction specificity for a particular H-2 allele of the chimeric host also defines that T cell's/r phenotype. (+info)
Induction of CD8+ T cell-mediated protective immunity against Trypanosoma cruzi.
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Trypanosoma cruzi was transformed with the Plasmodium yoelii gene encoding the circum-sporozoite (CS) protein, which contains the well-characterized CD8+ T cell epitope, SYVPSAEQI. In vivo and in vitro assays indicated that cells infected with the transformed T. cruzi could process and present this malaria parasite-derived class I MHC-restricted epitope. Immunization of mice with recombinant influenza and vaccinia viruses expressing the SYVPSAEQI epitope induced a large number of specific CD8+ T cells that strongly suppressed parasitemia and conferred complete protection against the acute T. cruzi lethal infection. CD8+ T cells mediated this immunity as indicated by the unrelenting parasitemia and high mortality observed in immunized mice treated with anti-CD8 antibody. This study demonstrated, for the first time, that vaccination of mice with vectors designed to induce CD8+ T cells is effective against T. cruzi infection. (+info)
Complementation of P37 (F13L gene) knock-out in vaccinia virus by a cell line expressing the gene constitutively.
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Vaccinia virus produces two different infectious forms, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Acquisition of the EEV envelope occurs by wrapping of IMV with vesicles of the trans-Golgi network (TGN). The most abundant protein in the envelope of EEV, P37, is a 37 kDa palmitylated protein encoded by the F13L gene. P37 is located in the inner side of the EEV envelope and accumulates in the TGN during infection. Deletion of gene F13L results in a severe defect in the wrapping process, although normal levels of IMV are produced. A cell line, derived from RK-13 cells, was obtained that stably expressed P37 (RK(P37)), and the properties of the protein were studied in the absence of other viral polypeptides. P37 produced in RK(P37) cells differed from P37 produced in vaccinia-infected cells in terms of hydrophobicity and intracellular distribution. Despite these differences, RK(P37) cells partially complemented the phenotypic defect of vaccinia virus P37- mutants. EEV production and cell-to-cell virus spread by mutant viruses were increased significantly in RK(P37) cells when compared to normal RK-13 cell cultures. Infection of RK(P37) cells with P37- virus substantially altered the hydrophobicity and the intracellular distribution of P37 in those cells. These results indicate the requirement of the infection context for determination of the normal palmitylation and intracellular localization of P37. (+info)