Vaccinia virus devoid of its E3L gene is sensitive to treatment of RK-13 cells with interferon-alpha and fails to replicate or form plaques in HeLa cells. In order to determine function of the E3L gene, vaccinia virus recombinants were constructed by inserting mutant E3L genes or a gene coding for an alternative dsRNA-binding protein into virus deleted of its wild type E3L gene. Those viruses that expressed proteins that retained dsRNA binding activity were resistant to the effects of interferon in RK-13 cells and could replicate in HeLa cells. Recombinant viruses that expressed E3L mutant proteins which were unable to bind to dsRNA were interferon sensitive in RK-13 cells and could not replicate in HeLa cells. In addition, a virus that expressed a mutant E3L protein previously characterized as having a low binding affinity for dsRNA exhibited an intermediate phenotype: it was interferon resistant in RK-13 cells but could not replicate in HeLa cells. This work suggests that the E3L gene of vaccinia virus functions primarily as a dsRNA-binding protein in allowing resistance to interferon and in promoting replication in HeLa cells.
The vaccinia virus E3L gene codes for double-stranded RNA (dsRNA) binding proteins which can prevent activation of the dsRNA-dependent, interferon-induced protein kinase PKR. Activated PKR has been shown to induce apoptosis in HeLa cells. HeLa cells infected with vaccinia virus with the E3L gene deleted have also been shown to undergo apoptosis, whereas HeLa cells infected with wild-type vaccinia virus do not. In this report, using virus recombinants expressing mutant E3L products or alternative dsRNA binding proteins, we show that suppression of induction of apoptosis correlates with functional binding of proteins to dsRNA. Infection of HeLa cells with ts23, which leads to synthesis of increased dsRNA at restrictive temperature, induced apoptosis at restrictive but not permissive temperatures. Treatment of cells with cytosine arabinoside, which blocks the late buildup of dsRNA in vaccinia virus-infected cells, prevented induction of apoptosis by vaccinia virus with E3L deleted. Cells transfected with dsRNA in the absence of virus infection also underwent apoptosis. These results suggest that dsRNA is a trigger that can initiate a suicide response in virus-infected and perhaps uninfected cells.
The alpha(IIb) and alpha(v) integrins have been shown to play a significant role in a variety of disease processes. alpha(IIb)beta(3) is a platelet-specific fibrinogen receptor that is critical for thrombosis and hemostasis. Determination of the basis of ligand recognition by alpha(IIb)beta(3) is essential for modulation of platelet function. To identify alpha(IIb) residues involved in alpha(IIb)beta(3) ligand binding function, cells expressing a constitutively active variant of alpha(IIb)beta(3) were randomly mutagenized and selected for loss of alpha(IIb)beta(3) ligand binding function. One mutant isolated in this manner contained a single amino acid substitution at position 96 in alpha(IIb) (Ser9-->Leu). Cells expressing this alpha(IIb) mutant did not bind the ligand mimetic antibody PAC1 or adhere to fibrinogen. In addition, the mutant receptor did not bind to an RGD affinity matrix. Substitution of conserved serine residues at position 1 in beta strand A of all seven repeats of alpha(IIb) similarly inhibited ligand binding to alpha(IIb)beta(3). alpha(IIb)S96 maps to the central cavity of the beta-propeller fold of the alpha(IIb) subunit immediately adjacent to a structurally important sequence at the center of the alphaand beta subunit interface. In contrast, substitution of the analogous residues in alpha(v) or alpha(4) did not disrupt the ligand binding function of alpha(v)beta(3) or alpha(4)beta(1). These data support a potential unique structural or mechanistic role for this residue in alpha(IIb)beta(3) receptor function.
Summary TP508, a 23-amino acid RGD-containing synthetic peptide representing residues 508 to 530 of human prothrombin, mitigates the effects of endothelial dysfunction in ischaemic reperfusion injury. The objective of this study was to investigate whether TP508 binds to members of the integrin family of transmembrane receptors leading to nitric oxide synthesis. Immobilised TP508 supported adhesion of endothelial cells and αvβ3-expressing human embryonic kidney cells in a dose- and RGD-dependent manner. Soluble TP508 also inhibited cell adhesion to immobilised fibrinogen. The involvement of αvβ3 was verified with function-blocking antibodies and surface plasmon resonance studies. Adhesion of the cells to immobilised TP508 resulted in an induction of phosphorylated FAK and ERK1/2. In endothelial cells, TP508 treatment resulted in an induction of nitric oxide that could be inhibited by LM609, an αvβ3-specific, function-blocking monoclonal antibody. Finally, TP508 treatment of isolated rat aorta segments enhanced carbachol-induced vasorelaxation. These results suggest that TP508 elicits a potentially therapeutic effect through an RGD-dependent interaction with integrin αvβ3.
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