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G McFadden

Publications and source records attributed to G McFadden.

At least 73 records · Page 4Linked to original sources
PubMed1996

Amelioration of antigen induced arthritis in rabbits treated with a secreted viral serine proteinase inhibitor.

OBJECTIVE: To evaluate the effects of intraarticular administration of SERP-1 protein, a myxoma virus derived antiinflammatory serine protease inhibitor, in an antigen induced arthritis (AIA) model of chronic inflammation. METHODS: AIA was induced in a single joint of 15 rabbits after intraarticular (i.a.) injection of ovalbumin in animals previously immunized to the same agent administered in complete Freund's adjuvant. A 2nd i.a. injection of ovalbumin was given 2 weeks later preceded by 2 daily injections of human transforming growth factor-beta 2. SERP-1 was given by i.a. injection at 2 and 4 weeks after the last i.a. injection of ovalbumin. Three synovial specimens per joint were obtained for synovial histology. Patellar articular cartilage was assessed for collagen integrity and proteoglycan staining. RESULTS: Synovial histology revealed significant diminution in synovial lining cellular hyperplasia, chronic inflammatory infiltration, and cartilage erosion in treated animals, particularly in those receiving 2 i.a. injections of 1 ng of SERP-1. Preservation of articular cartilage was concomitantly noted in treated animals. A dose-response influence on histopathologic change was discernible. CONCLUSION: A viral derived antiinflammatory protein, SERP-1, demonstrates considerable potency in ameliorating chronic inflammation in the AIA model and warrants further investigation as a potential anti-arthritic agent.

Animals↗
PubMed1995

Species specificity of ectromelia virus and vaccinia virus interferon-gamma binding proteins.

Interferon-gamma functions within the immune system as a potent anti-viral and immunoregulatory cytokine. In order to successfully replicate within a host cell, poxviruses have evolved a number of strategies to counteract the pleiotropic effects of interferon-gamma. In particular, the leporipoxvirus myxoma virus was shown to express an extracellular soluble interferon-gamma receptor homolog, denoted M-T7, which is capable of inhibiting the anti-viral activities of rabbit interferon-gamma (C. Upton, K. Mossman, and G. McFadden, 1992, Science 258, 1369-1372). Here, we demonstrate that expression of soluble interferon-gamma receptor homologs appears to be characteristic of all poxviruses tested, including Shope fibroma virus, vaccinia virus (strains WR and IHDW), ectromelia virus, cowpox virus, and rabbitpox virus. We have cloned, sequenced, and characterized the interferon-gamma binding protein in supernatants from ectromelia virus-infected cells, and demonstrate the capability of this soluble protein to bind human, murine, and rabbit interferon-gamma with similar affinity. We also investigate the properties of the vaccinia virus interferon-gamma binding protein and demonstrate that this protein binds human and rabbit interferon-gamma with similar affinity and binds murine interferon-gamma with a significantly lower relative affinity. The implications of these studies with respect to viral pathogenesis and the evolutionary relationship between a virus and its host are discussed.

Amino Acid Sequence↗
PubMed1995

The myxoma virus-soluble interferon-gamma receptor homolog, M-T7, inhibits interferon-gamma in a species-specific manner.

The myxoma virus M-T7 protein contains significant sequence similarity to the ligand binding domain of the mammalian interferon-gamma receptors, and functions as a soluble homolog which can bind and inhibit the biological activities of rabbit interferon-gamma (Upton, C., Mossman, K., and McFadden, G. (1992) Science 258:1369-1372). M-T7, the most abundantly secreted protein from myxoma virus-infected cells, was shown to be expressed in significant biological amounts as a typical poxvirus early gene product, efficiently secreted at early times of infection to levels that exceed 5 x 10(7) molecules/cell, and function as a stable inhibitory protein in infected cell supernatants until late times of infection. M-T7 was specific in binding and inhibiting rabbit interferon-gamma, and did not bind either human or murine interferon-gamma. Scatchard analysis of rabbit interferon-gamma binding curves yielded a single high affinity binding site on M-T7, with a Kd of 1.2 x 10(-9) M, which is comparable to the affinity between soluble forms of cellular interferon-gamma receptors and their cognate ligands. In comparison, rabbit interferon-gamma was shown to bind its cellular receptor with a Kd of 5.9 x 10(-10) M, again comparable to the affinity of membrane bound forms of other mammalian interferon-gamma receptors for interferon-gamma. Thus, the myxoma virus M-T7 protein is a functional soluble interferon-gamma receptor homolog which binds and inhibits interferon-gamma with high affinity in a species-specific manner.

Amino Acid Sequence↗
PubMed1995

Myxoma virus and Shope fibroma virus encode dual-specificity tyrosine/serine phosphatases which are essential for virus viability.

Sequence analysis of the genomes of the Leporipoxviruses myxoma virus and Shope fibroma virus (SFV) led to the discovery of open reading frames homologous to the vaccinia H1L gene encoding a soluble protein phosphatase with dual tyrosine/serine specificity. These viral phosphatase genes were subsequently localized to the myxoma BamHI-I fragment and the SFV BamHI-M fragment, and the resulting encoded proteins were designated I1L and M1L, respectively. The localization and orientation of the myxoma I1L and SFV M1L open reading frames within the well conserved central core of the viral genomes closely mirror that of the Orthopoxviruses vaccinia virus and variola virus. The myxoma I1L and SFV M1L phosphatases each contain the conserved tyrosine phosphatase signature sequence motif, (I/V)HCXAGXXR(S/T)G, including the active site cysteine, found previously to be essential for phosphotyrosine dephosphorylation. The vaccinia H1L phosphatase was originally shown to have the ability to dephosphorylate phosphotyrosyl and phosphoseryl residues in vitro. To assess whether this is a common feature of poxvirus phosphatases, myxoma I1L was expressed as a GST-fusion protein, purified, and shown to dephosphorylate substrates containing tyrosine and serine phosphorylated residues, in a similar fashion to vaccinia H1L. A myxoma I1L variant, in which the active site cysteine 110 was mutated to serine, was expressed in a parallel fashion to the wild-type I1L protein and found to be completely deficient in its ability to dephosphorylate both phosphotyrosine and phosphoserine amino acids. In an attempt to ascertain the biological requirement for the myxoma I1L phosphatase, we constructed a recombinant myxoma virus containing a disrupted I1L open reading frame. This I1L mutant virus was able to successfully propagate in tissue culture only in the presence of a wild-type complementing gene, and pure virus clones containing only the disrupted allele were not viable. Thus, we conclude that the myxoma I1L dual specificity phosphatase is an essential factor for virus viability.

Amino Acid Sequence↗
PubMed1995

Interruption of cytokine networks by poxviruses: lessons from myxoma virus.

Myxoma virus is an infectious poxvirus pathogen that induces a virulent systemic disease called myxomatosis in European rabbits. The disease is rapidly and uniformly fatal to susceptible rabbits and is characterized by generalized dysfunction of cellular immunity and multiple interruptions of the host cytokine network. A number of virus genes are classified as virulence factors because virus constructs bearing targeted gene disruptions induce attenuated disease symptoms. Many of these genes encode proteins that interact directly with effector elements of the host immune system. Included among these immunosubversive viral proteins are secreted mimics of host ligands or regulators (virokines) and homologues of cellular cytokine receptors (viroceptors). Five examples of these immune modulator proteins encoded by myxoma virus are reviewed: (1) myxoma growth factor, a member of the epidermal growth factor ligand superfamily; (2) SERP-1, a secreted serine proteinase inhibitor; (3) M11L, a receptor-like surface protein; (4) T2, a tumor necrosis factor receptor homologue; and (5) T7, an interferon-gamma receptor homologue. The origin of viral strategies designed to subvert immune regulation by host cytokines is considered in the context of the biology of myxoma virus within immunocompetent hosts.

Animals↗
PubMed1995

Myxoma virus induces extensive CD4 downregulation and dissociation of p56lck in infected rabbit CD4+ T lymphocytes.

Myxoma virus is a pathogenic poxvirus that induces extensive dysregulation of cellular immunity in infected European rabbits. Infection of a rabbit CD4+ T-cell line (RL-5) with myxoma virus results in dramatic reductions of cell surface levels of CD4 as monitored by flow cytometry. The virus-induced downregulation of CD4 requires early but not late viral gene expression and could not be inhibited by staurosporine, an inhibitor of protein kinase C, which effectively blocks phorbol 12-myristate-13-acetate-induced downregulation of CD4. The decrease in total cellular levels of CD4 during myxoma virus infection could be inhibited by the lysosomotrophic agent NH4Cl, suggesting a lysosomal fate for CD4 during myxoma virus infection. Steady-state levels of the CD4-associated protein tyrosine kinase p56lck remained unchanged during myxoma virus infection, suggesting that p56lck dissociates from CD4 prior to CD4 degradation in virus infected cells. Total p56lck kinase activity was unaffected during myxoma virus infection, although the amount of p56lck physically associated with CD4 declined in parallel with the loss of CD4. Thus, myxoma virus infection of CD4+ T lymphocytes triggers CD4 downregulation via a protein kinase C-independent pathway, causing the dissociation of p56lck and the degradation of CD4 in lysosomal vesicles.

Alkaloids↗
PubMed1994

The myxoma virus TNF-receptor homologue (T2) inhibits tumor necrosis factor-alpha in a species-specific fashion.

The myxoma virus T2 protein shares extensive homology with the ligand binding domains of tumor necrosis factor (TNF) receptors. To characterize the T2-TNF interaction, myxoma T2 protein and rabbit, mouse, and human TNF alpha were expressed independently from vaccinia virus vectors. Growth of the TNF alpha-expressing viruses was significantly attenuated in TNF-hypersensitive L929-8 cells, and these cells were rapidly lysed by all three TNF alpha s. Rabbit cells showed strict species specificity in that RK-13 and SIRC cells were only sensitive to lysis by the homologous rabbit TNF alpha. Although RK-13 cells were hypersensitive to rabbit TNF alpha even in the absence of protein synthesis inhibitors, growth of T2 nonexpressing myxoma and vaccinia viruses in RK-13 cells was only modestly reduced in the presence of rabbit TNF alpha, suggesting that poxviruses possess additional anti-TNF mechanisms. When the ability of the myxoma T2 protein to inhibit biological activities of TNF alpha was assayed, T2 protein effectively protected L929-8 cells from lysis by rabbit, but not human or mouse TNF alpha. These studies show that rabbit TNF receptors may be species-specific for rabbit TNF alpha and we conclude that the myxoma T2 protein evolved to specifically inhibit TNF activities from its natural host, the South American rabbit.

Animals↗
PubMed1994

A poxvirus protein with a RING finger motif binds zinc and localizes in virus factories.

Shope fibroma virus (SFV) is a Leporipoxvirus closely related to the highly virulent myxoma virus. The DNA sequence of the BamHI N fragment of the SFV DNA genome was determined, and the single complete open reading frame (N1R) was characterized. The protein encoded by the N1R gene was found to contain a C3HC4 RING finger motif at the C terminus. This C3HC4 motif is the hallmark of a growing family of proteins, many of which are involved in regulation of gene expression, DNA repair, or DNA recombination. Complete homologs of the SFV N1R gene were also detected in variola virus, myxoma virus, and vaccinia virus strain IHD-W. In contrast, the gene is completely absent from vaccinia virus strain Copenhagen, and in vaccinia virus strain WR, the open reading frame is truncated prior to the zinc binding domain because of an 11-bp deletion, thus producing a frameshift and premature stop codon. Recombinant N1R protein from SFV was expressed in Escherichia coli and shown to bind zinc in a specific manner. Using fluorescence microscopy to visualize a peptide epitope tag (derived from ICP27 of herpes simplex virus) fused to the N terminus of the poxvirus proteins, we observed that the N1R protein of SFV and its homologs in myxoma virus and vaccinia virus IHD-W were localized primarily to the virus factories in the cytoplasm of infected cells and, to a lesser degree, the host cell nucleus. The truncated protein of vaccinia virus strain WR failed to localize in this manner but instead was observed throughout the cytoplasm.

Amino Acid Sequence↗
PubMed1993

Identification of a poxvirus gene encoding a uracil DNA glycosylase.

An open reading frame, BamHI D6R, from the central highly conserved region of the Shope fibroma virus (SFV) genome was sequenced and found to have significant homology to that of uracil DNA glycosylases from a number of organisms. Uracil DNA glycosylase catalyzes the initial step in the repair pathway that removes potentially mutagenic uracil from duplex DNA. The D6R polypeptide was expressed in reticulocyte lysates programmed with RNA transcribed from an expression vector containing the T7 RNA polymerase promoter. A highly specific ethidium bromide fluorescence assay of the in vitro translation product determined that the encoded protein does indeed possess uracil DNA glycosylase activity. Open reading frames from other poxviruses, including vaccinia virus (HindIII D4R) and fowlpox (D4), are highly homologous to D6R of SFV and are predicted to encode uracil DNA glycosylases. Identification of the SFV uracil DNA glycosylase provides evidence that this poxviral protein is involved in the repair of the viral DNA genome. Since this enzyme performs only the initial step required for the removal of uracil from DNA, creating an apyrimidinic site, we suggest that other, possibly virus-encoded, repair activities must be present in the cytoplasm of infected cells to complete the uracil excision repair pathway.

Amino Acid Sequence↗
PubMed1993

Inhibition of plasmin, urokinase, tissue plasminogen activator, and C1S by a myxoma virus serine proteinase inhibitor.

The myxoma and malignant rabbit fibroma poxviruses are lethal tumorigenic viruses of rabbits whose virulence is modulated by the production of a virus-encoded secreted serine proteinase inhibitor, SERP-1. This viral protein was detected in medium harvested from myxoma and malignant rabbit fibroma virus-infected cells, and its inhibitory profile has been characterized by gel and kinetic analysis. SERP-1 forms complexes with and inhibits the human fibrinolytic enzymes plasmin, urokinase, and two-chain tissue-type plasminogen activator (association rate constants 3.4 x 10(4), 4.3 x 10(4), and 3.6 x 10(4) M-1 s-1 respectively). It is also able to inhibit C1S, the first enzyme in the complement cascade with an association rate constant which was unaffected by the addition of heparin (1.3 x 10(3) M-1 s-1). SERP-1 acts as a substrate for and is cleaved by thrombin, porcine trypsin, human neutrophil elastase, porcine pancreatic elastase, thermolysin, subtilisin, bovine alpha-chymotrypsin, and factor Xa. Incubation with kallikrein and cathepsin G had no effect. The structure of SERP-1 has been modeled on other members of the serpin family which revealed the characteristic serpin architecture apart from the absence of the D-helix. Structural analysis and kinetic assays demonstrate that the absence of this region does not prevent inhibitory activity and furthermore allow the identification of cysteine residues involved in internal and intermolecular disulfide bonding.

Amino Acid Sequence↗
PubMed1993

Transforming growth factor alpha, Shope fibroma growth factor, and vaccinia growth factor can replace myxoma growth factor in the induction of myxomatosis in rabbits.

The epidermal growth factor (EGF) homologues encoded by vaccinia virus, myxoma virus, and malignant rabbit fibroma virus have been shown to contribute to the pathogenicity of virus infection upon inoculation of susceptible hosts. However, since the primary structures of these growth factors and the disease profiles induced by different poxvirus genera vary substantially, the degree to which the various EGF homologues perform similar roles in viral pathogenesis remains unclear. In order to determine whether different EGF-like growth factors can perform qualitatively similar functions in the induction of myxomatosis in rabbits, we created recombinant myxoma virus variants in which the native growth factor, myxoma growth factor (MGF), was disrupted and replaced with either vaccinia virus growth factor, Shope fibroma growth factor, or rat transforming growth factor alpha. Unlike the control virus containing an inactivated MGF gene, which caused marked attenuation of the disease syndrome and substantially less proliferation of the epithelial cell layers in the conjunctiva and respiratory tract, the recombinant myxoma virus strains expressing heterologous growth factors produced infections which were both clinically and histopathologically indistinguishable from wild-type myxomatosis. We conclude that these poxviral and cellular EGF-like growth factors, which are diverse with respect to primary structure and origin, have similar biological functions in the context of myxoma virus pathogenesis and are mitogenic for the same target cells.

Animals↗
PubMed1993

SERP1, a serine proteinase inhibitor encoded by myxoma virus, is a secreted glycoprotein that interferes with inflammation.

Myxoma virus is a leporipoxvirus that causes a rapidly lethal, generalized infection known as myxomatosis in the European rabbit (Oryctolagus cuniculus). A characteristic feature of myxomatosis is the specific downregulation of key pathways important for numerous host defenses against the viral infection. The SERP1 gene has significant sequence similarity to the serpin superfamily of serine proteinase inhibitors and is one of many virulence factor genes located within the terminal regions of the myxoma virus genome. Transcriptional analysis of the SERP1 gene in myxoma virus (strain Lausanne) indicates that it is expressed as a late gene and studies using a polyclonal anti-SERP1 antiserum indicate that it encodes a secreted protein with an apparent molecular weight of 55 kDa. Using myxoma virus and recombinant vaccinia virus constructs for experiments with tunicamycin and peptide N-glycosidase F, it is shown that the secreted SERP1 protein is modified by N-linked glycosylation. Mutation of both copies of the SERP1 gene in myxoma virus results in a significant attenuation of the virus, such that more than 50% of infected animals are able to recover from the otherwise lethal infection. Histological analyses of lesions taken from infected animals suggest that in the absence of the SERP1 protein, a more effective inflammatory response occurs, allowing a more rapid resolution of the infection. This suggests that SERP1 contributes to viral pathogenesis by interacting with cellular component(s) involved in the regulation of inflammation.

Animals↗
PubMed1993

A poxvirus-encoded uracil DNA glycosylase is essential for virus viability.

Infection of cultured mammalian cells with the Leporipoxvirus Shope fibroma virus (SFV) causes the induction of a novel uracil DNA glycosylase activity in the cytoplasms of the infected cells. The induction of this activity, early in infection, correlates with the early expression of the SFV BamHI D6R open reading frame which possesses significant protein sequence similarity to eukaryotic and prokaryotic uracil DNA glycosylases. The SFV BamHI D6R open reading frame and the homologous HindIII D4R open reading frame from the Orthopoxvirus vaccinia virus were cloned under the regulation of a phage T7 promoter and expressed in Escherichia coli as insoluble high-molecular-weight aggregates. During electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the E. coli-expressed proteins migrate with an apparent molecular mass of 25 kDa. The insoluble protein aggregate generated by expression in E. coli was solubilized in urea and, following a subsequent refolding step, displayed the ability to excise uracil residues from double-stranded plasmid DNA substrates, with the subsequent formation of apyrimidinic sites. The viral enzyme, like all other characterized uracil DNA glycosylases, is active in the presence of high concentrations of EDTA, is substrate inhibited by uracil, and does not display any endonuclease activity. Attempts to inactivate the HindIII D4R gene of vaccinia virus by targeted insertion of a dominant xanthine-guanine phosphoribosyltransferase selection marker or direct insertion of a frame-shifted oligonucleotide were uniformly unsuccessful demonstrating that, unlike the uracil DNA glycosylase described for herpesviruses, the poxvirus enzyme is essential for virus viability.

Animals↗
PubMed1992

Encoding of a homolog of the IFN-gamma receptor by myxoma virus.

Many poxvirus-encoded virulence factors have been identified as proteins that are secreted from infected cells. The major secreted protein (37 kilodaltons) from cells infected with myxoma virus is encoded by the M-T7 open reading frame. This protein has significant sequence similarity to the human and mouse receptors for interferon-gamma (IFN-gamma). Furthermore, the myxoma M-T7 protein specifically binds rabbit IFN-gamma and inhibits the biological activity of extracellular IFN-gamma, one of the key regulatory cytokines in the host immune response against viral infections.

Amino Acid Sequence↗
PubMed1992

Virus-induced loss of class I MHC antigens from the surface of cells infected with myxoma virus and malignant rabbit fibroma virus.

Shope fibroma virus (SFV) is a leporipoxvirus that causes localized benign fibromas in immunocompetent adult rabbits that spontaneously regress due, in part, to a cell-mediated immune response. Myxoma virus (MYX) and malignant rabbit fibroma virus (MRV) are related leporipoxviruses that induce rapidly lethal generalized infections accompanied by tumors and immunosuppression. Because only these latter two viruses are known to compromise cell-mediated antiviral responses, cell surface levels of class I MHC molecules in SFV-, MRV-, and MYX-infected cells were investigated by fluorescent activated cell sorting analysis using a variety of different anti-HLA mAb. After infection with MYX or MRV there is a rapid decrease in the levels of detectable surface class I epitopes as detected by each antibody and by 24 h postinfection class I MHC Ag levels at the cell surface approach the level of background fluorescence observed with control antibodies. In contrast, only a moderate class I decrease is seen during infection with either SFV or vaccinia virus, an orthopoxvirus that is neither tumorigenic nor immunosuppressive. Surface class I marker loss induced by MYX and MRV is not simply due to nonspecific inhibition of total cellular protein synthesis by the viruses because class I levels decrease much further than the extent measured by estimating surface marker turnover in the presence of the protein synthesis inhibitor cycloheximide. Thus the loss of cellular surface class I molecules greatly exceeds the drop in level caused by complete blockage of host cell gene expression, and must involve removal or masking of preexisting class I epitopes from the cell surface by MRV/MYX. Cell surface levels of the transferrin receptor are unaffected by MYX and MRV infection, suggesting the observed class I decrease is not a nonspecific effect on total cell surface glycoproteins. Analysis of cells infected with MRV/MYX in the presence of cycloheximide or of cytosine arabinoside, an inhibitor of poxviral DNA replication, indicates that the class I marker loss is mediated in part by one or more viral late gene products. A probable explanation is that MRV/MYX late protein(s) interact with the class I MHC complex to either physically sequester these away from the cell surface and inhibit their recycling or else induce a conformational change that precludes recognition by all class I antibodies tested. In either event, we propose that such a major perturbation of the class I MHC complex would likely downregulate the class I-mediated presentation of viral Ag required to initiate cell-mediated immunity to these viruses.

Animals↗