Hydrodynamic, electron microscopic, and ligand-binding analysis of the Epstein-Barr virus/C3dg receptor (CR2)

The interaction of the Epstein-Barr virus/45-kDa proteolytic fragment of C3 (C3dg) receptor (CR2) with its viral ligand, the Epstein-Barr virus glycoprotein gp350/220, initiates the sequence of events leading to virus internalization and B lymphocyte transformation. Soluble recombinant receptor (rCR2) and gp350/220 as well as the natural ligand, C3dg, were subjected to a number of analytical techniques including gel permeation chromatography, density gradient ultracentrifugation, circular dichroism, and electron microscopy in order to determine their hydrodynamic, structural, and binding properties. Both rCR2 and gp350/220 were found to be highly extended proteins (f/fo = 2.1 and 2.4/2.2, respectively). C3dg, in contrast to the viral ligand, is only somewhat elongated (f/fo = 1.5). Soluble rCR2, visualized by high resolution electron microscopy, was shown to be an extended, highly flexible molecule comprised of ringlet domains, each approximately 24.1 A in length, which likely correspond to the short consensus repeat motif deduced from the CR2 cDNA nucleotide sequence. Ligand-binding studies carried out under physiological conditions indicated that gp350/220 binding to rCR2 was saturable and univalent, with a dissociation constant of 3.2 nM. In contrast, monomeric C3dg did not bind to rCR2 under physiological conditions; however, at reduced ionic strength, monomeric C3dg binding could be measured. These studies indicate that the affinity of the C3dg monomer for rCR2 under physiologic conditions is approximately 10(4)-fold less than that of the viral ligand. The molecular properties of rCR2 revealed in these studies provide essential information for future studies of the biologic functions of the Epstein-Barr virus/C3dg receptor.     

Adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells.

A major impediment to the effective use of adenovirus vectors for gene therapy is a lack of knowledge of how these vectors interact with diverse cell types in vivo. Adenovirus attachment to most human cell types is mediated by the fiber protein, which binds to an as yet unidentified cell receptor. In contrast to this, we report that adenovirus type 2 (Ad2) attachment to hematopoietic cells is facilitated by interaction of the penton base protein with members of the beta2 integrin family. Adenovirus particles were capable of binding to human monocytic cells, which lack fiber receptors, and virus binding could be blocked by a soluble penton base or by a function-blocking monoclonal antibody to integrin alphaMbeta2. To confirm the role of alphaMbeta2 integrins in Ad2 binding to hematopoietic cells, we analyzed virus attachment and gene delivery to CHO cells expressing recombinant beta2 integrins. alphaMbeta2-expressing CHO cells supported 3- to 5-fold-higher levels of Ad2 binding and 5- to 10-fold-larger amounts of gene delivery than did nontransfected CHO cells, indicating that alphaMbeta2 facilitates adenovirus attachment to and infection of hematopoietic cells. While beta2 integrins promote Ad2 attachment to hematopoietic cells, further studies demonstrated that alphav integrins were required for the next step in infection, virus internalization into cell endosomes. These studies reveal a novel pathway of Ad2 infection of hematopoietic cells mediated by distinct integrins which facilitate separate events in virus entry. They also suggest a possible strategy for selective adenovirus-mediated gene delivery to hematopoietic cells.     

Multiple adenovirus serotypes use alpha v integrins for infection.

The nucleotide sequence encoding the penton base integrin-binding domains of several human adenoviruses was obtained by homology PCR. Each of the penton base proteins contains a conserved Arg-Gly-Asp (RGD) sequence that is predicted to lie at the apex of two extended alpha helices. The penton base RGD domain promotes efficient infection of host cells by multiple adenovirus serotypes via interaction with alpha v integrins, thus indicating that alpha v integrins play a central role in the entry of adenoviruses into host cells.     

Upregulation of integrins alpha v beta 3 and alpha v beta 5 on human monocytes and T lymphocytes facilitates adenovirus-mediated gene delivery.

Entry of human adenovirus into host cells involves interaction of virus particles with two distinct receptors. The initial binding event is mediated by the fiber protein, while subsequent interaction of the penton base protein with alpha v integrins promotes virus internalization and/or penetration. Although these interactions in epithelial and endothelial cells have been well characterized, relatively little is known as to whether these events occur during virus infection of human peripheral blood mononuclear cells. We demonstrate that freshly isolated peripheral blood monocytes and T lymphocytes express very small amounts of alpha v integrins and also are resistant to adenovirus infection. Exposure of monocytes to hematopoietic growth factors granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor induced expression of cell surface alpha v integrins, promoted the binding of penton base protein, and also rendered these cells susceptible to adenovirus-mediated gene delivery. Stimulation of T cells with a mitogen, phytohemagglutinin, or a cell-activating agent, phorbol myristate acetate, induced expression of alpha v integrins and also enhanced adenovirus-mediated gene delivery. These studies further indicate that alpha v integrins play a crucial role in adenovirus infection and also provide a useful strategy for enhancing adenovirus-mediated gene delivery into human peripheral blood mononuclear cells.     

Role of the tubulin-microtubule system in lymphocyte activation

The role of the tubulin-microtubule system was examined in human peripheral blood leukocytes after activation with phytohemagglutinin (PHA). Soluble tubulin and microtubules were measured with a [(3)H]colchicine-binding assay. It was found that the tubulin content of PHA-activated lymphocytes was consistently increased relative to total protein content after 36 h of culture. There was no increase in the proportion of total tubulin synthesis which was present as microtubules at 36 h. Nevertheless, as a result of increased tubulin synthesis, there was a two-to three-fold increase in total microtubular mass. Colchicine, which disrupts microtubles, was used to assess the role of microtubule assembly in the sequence of events which follow lymphocyte activation, namely lymphokine release, protein synthesis, RNA synthesis, and DNA synthesis. Colchicine consistently inhibited DNA synthesis but did not inhibit release of the lymphokine, osteoclast activating factor (OAF). Protein and RNA syntheses were inhibited much less than DNA synthesis. The fact that some effects of PHA on lymphocytes appear to require intact microtubules and at least one does not suggest that the microtubule dependent step in PHA-stimulated lymphocyte activation occurs at a stage after propagation of the signal from the membrane to the cell interior.     

Integrin alpha(v)beta1 is an adenovirus coreceptor

The human embryonic kidney (HEK293) cell line, commonly used for recombinant adenovirus (Ad) propagation, does not express the Ad coreceptor alpha(v)beta3 or alpha(v)beta5 integrins, yet these cells are efficiently infected by Ad vectors. Here we demonstrate that Ad binds to HEK293 cells via the fiber receptor CAR and is subsequently internalized via interaction with integrin alpha(v)beta1. Function-blocking antibodies directed against alpha(v) or beta1, but not beta3, beta5, or alpha5, integrin subunits block Ad infection and viral endocytosis. Therefore, alpha(v)beta1 serves as a coreceptor for Ad infection, and the lack of beta3 and/or beta5 but the relatively high expression of alpha(v)beta1 integrins on certain tumor cell types may explain why these cells are readily transduced by Ad vectors.     

Association of p130CAS with phosphatidylinositol-3-OH kinase mediates adenovirus cell entry

The Crk-associated substrate, p130(CAS), has been implicated in the regulation of the actin cytoskeleton following ligation of cell integrins with the extracellular matrix. Integrin-mediated cell adhesion involves p130(CAS) association with focal adhesion kinase (p125(FAK)). Internalization/cell entry of type 2 and type 5 adenoviruses (Ad) is also mediated by alpha(v) integrins. However, expression of dominant negative forms of p125(FAK) does not alter virus entry, and Ad entry occurs normally in p125(FAK)-deficient fibroblasts. We now provide evidence that Ad internalization, a process which is mediated by alpha(v) integrins, also requires p130(CAS) and phosphatidylinositol-3-OH kinase (PI 3-kinase). Ad induces p130(CAS) phosphorylation and inhibition of p130(CAS) phosphorylation by tyrphostin and genistein, or expression of the substrate domain deleted p130(CAS) blocks Ad internalization. p130(CAS) was also found to associate with the p85 subunit of PI 3-kinase through its proline-rich domain during virus internalization and expression of p130(CAS) containing a deleted proline-rich domain (PRD) inhibited adenovirus cell entry. We showed further that the RPLPSPP motif in the proline-rich region of p130(CAS) interacts with the SH3 domain of p85/PI 3-kinase. These studies reveal the molecular basis by which p130(CAS) coordinates the signaling pathways involved in integrin-mediated Ad endocytosis.     

Simulated brain tumor growth dynamics using a three-dimensional cellular automaton

We have developed a novel and versatile three-dimensional cellular automaton model of brain tumor growth. We show that macroscopic tumor behavior can be realistically modeled using microscopic parameters. Using only four parameters, this model simulates Gompertzian growth for a tumor growing over nearly three orders of magnitude in radius. It also predicts the composition and dynamics of the tumor at selected time points in agreement with medical literature. We also demonstrate the flexibility of the model by showing the emergence, and eventual dominance, of a second tumor clone with a different genotype. The model incorporates several important and novel features, both in the rules governing the model and in the underlying structure of the model. Among these are a new definition of how to model proliferative and non-proliferative cells, an isotropic lattice, and an adaptive grid lattice.