ABSTRACT
Infection with human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma (KS)-associated herpesvirus, is necessary for the development of KS. The HHV-8 lytic-phase gene ORF74 is related to G protein-coupled receptors, particularly interleukin-8 (IL-8) receptors. ORF74 activates the inositol phosphate/phospholipase C pathway and the downstream mitogen-activated protein kinases, JNK/SAPK and p38. We show here that ORF74 also activates NF-kappaB independent of ligand when expressed in KS-derived HHV-8-negative endothelial cells or primary vascular endothelial cells. NF-kappaB activation was enhanced by the chemokine GROalpha, but not by IL-8. Mutation of Val to Asp in the ORF74 second cytoplasmic loop did not affect ligand-independent signaling activity, but it greatly increased the response to GROalpha. ORF74 upregulated the expression of NF-kappaB-dependent inflammatory cytokines (RANTES, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor) and adhesion molecules (VCAM-1, ICAM-1, and E-selectin). Supernatants from transfected KS cells activated NF-kappaB signaling in untransfected cells and elicited the chemotaxis of monocytoid and T-lymphoid cells. Expression of ORF74 conferred on primary endothelial cells a morphology that was strikingly similar to that of spindle cells present in KS lesions. Taken together, these data, demonstrating that ORF74 activates NF-kappaB and induces the expression of proangiogenic and proinflammatory factors, suggest that expression of ORF74 in a minority of cells in KS lesions could influence uninfected cells or latently infected cells via autocrine and paracrine mechanisms, thereby contributing to KS pathogenesis.
Subject(s)
Herpesvirus 8, Human/metabolism , I-kappa B Proteins , NF-kappa B/metabolism , Paracrine Communication , Receptors, Chemokine/metabolism , Viral Proteins/metabolism , Cells, Cultured , Chemokine CCL5/biosynthesis , Chemotaxis, Leukocyte/physiology , DNA-Binding Proteins/metabolism , E-Selectin/biosynthesis , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Humans , I-kappa B Kinase , Intercellular Adhesion Molecule-1/biosynthesis , Interleukin-6/biosynthesis , Interleukin-8/biosynthesis , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Models, Biological , Monocytes/physiology , Mutagenesis , NF-KappaB Inhibitor alpha , Phosphoinositide-3 Kinase Inhibitors , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Receptors, Chemokine/genetics , Sarcoma, Kaposi , T-Lymphocytes/physiology , Vascular Cell Adhesion Molecule-1/biosynthesis , Viral Proteins/genetics , p38 Mitogen-Activated Protein KinasesABSTRACT
A bacterial endophyte was engineered for insecticidal activity against the European corn borer. The cryIA(c) gene from Bacillus thuringiensis subsp. kurstaki was introduced into the chromosome of Clavibacter xyli subsp. cynodontis by using an integrative plasmid vector. The integration vectors pCG740 and pCG741 included the replicon pGEM5Zf(+), which is maintained in Escherichia coli but not in C. xyli subsp. cynodontis; tetM as a marker for selection in C. xyli subsp. cynodontis; and a chromosomal fragment of C. xyli subsp. cynodontis to allow for homologous recombination between the vector and the bacterial chromosome. Insertion of vector DNA into the chromosome was demonstrated by DNA hybridization. Recombinant strains MDR1.583 and MDR1.586 containing the cryIA(c) gene were shown to produce the 133,000-kDa protoxin and several smaller immunoreactive proteins. Both strains were equally toxic to insect larvae in bioassays. Significant insecticidal activity was demonstrated in planta. The cryIA(c) gene and the tetM gene introduced into strain MDR1.586 were shown to be deleted from some cells, thereby giving rise to a noninsecticidal segregant population. In DNA hybridization experiments and insect bioassays, these segregants were indistinguishable from the wild-type strain. Overall, these results demonstrate the plausibility of genetically engineered bacterial endophytes for insect control.
ABSTRACT
Epstein-Barr virus (EBV) replicons which include the genetic element oriP and a functional gene for Epstein-Barr nuclear antigen (EBNA-1) can be maintained episomally in a variety of mammalian cell lines [Yates et al., Nature 313 (1985) 812-815]. We have assessed the application of an EBV replicon for foreign gene expression. Two cDNAs, human interferon-gamma (IFN-gamma) and the extracellular domain of the human epidermal growth factor receptor (EGF-Rex), cloned in an EBV replicon, were efficiently expressed and the protein was secreted into the extracellular media. Expression in human embryonic 293 cells was approximately ten-fold higher than in CV-1 cells. The expression of the human protein is dependent upon the orientation of the IFN-gamma transcriptional cassette relative to the other genetic elements within the vector.
