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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.

Overexpression of urokinase plasminogen activator (uPA) and its receptor (uPAR) has been well documented in a wide variety of tumor cells. In breast cancer, expression of uPA/uPAR is essential for tumor cell invasion and metastasis. However, the mechanism responsible for uPA/uPAR expression in cancer cells remains unclear. In the studies reported here, we show that endogenous p38 MAPK activity correlates well with breast carcinoma cell invasiveness. Treatment of highly invasive BT549 cells with a specific p38 MAPK inhibitor SB203580 diminished both uPA/uPAR mRNA and protein expression and abrogated the ability of these cells to invade matrigel, suggesting that p38 MAPK signaling pathway is involved in the regulation of uPA/uPAR expression and breast cancer cell invasion. We also demonstrated that SB203580-induced reduction in uPA/uPAR mRNA expression resulted from the de- stabilization of uPA and uPAR mRNA. Finally, by selectively inhibiting p38alpha or p38beta MAPK isoforms, we demonstrate that p38alpha, rather than p38beta, MAPK activity is essential for uPA/uPAR expression. These studies suggest that p38alpha MAPK signaling pathway is important for the maintenance of breast cancer invasive phenotype by promoting the stabilities of uPA and uPAR mRNA.

Adenovirus, a respiratory virus with a double-stranded DNA genome, replicates in the nuclei of mammalian cells. We have developed a cytosol-dependent in vitro assay utilizing adenovirus nucleocapsids to examine the requirements for adenovirus docking to the nuclear pore complex and for DNA import into the nucleus. Our assay reveals that adenovirus DNA import is blocked by a competitive excess of classical protein nuclear localization sequences and other inhibitors of nuclear protein import and indicates that this process is dependent on hsc70. Previous work revealed that the hexon (coat) protein of adenovirus is the only major protein on the surface of the adenovirus nucleocapsid that docks at the nuclear pore complex. This, together with our finding that in vitro nuclear import of hexon is inhibited by an excess of classical nuclear localization sequences, suggests a role for the hexon protein in adenovirus DNA import. However, recombinant transport factors that are sufficient for hexon import in permeabilized cells do not support DNA import, indicating that there are other as yet unidentified factors required for this process.

Integrin alpha(V)beta(3) mediates diverse responses in vascular cells, ranging from cell adhesion, migration, and proliferation to uptake of adenoviruses. However, the extent to which alpha(V)beta(3) is regulated by changes in receptor conformation (affinity), receptor diffusion/clustering (avidity), or post-receptor events is unknown. Affinity regulation of the related integrin, alpha(IIb)beta(3), has been established using a monovalent ligand-mimetic antibody, PAC1 Fab. To determine the role of affinity modulation of alpha(V)beta(3), a novel monovalent ligand-mimetic antibody (WOW-1) was created by replacing the heavy chain hypervariable region 3 of PAC1 Fab with a single alpha(V) integrin-binding domain from multivalent adenovirus penton base. Both WOW-1 Fab and penton base bound selectively to activated alpha(V)beta(3), but not to alpha(IIb)beta(3), in receptor and cell binding assays. alpha(V)beta(3) affinity varied with the cell type. Unstimulated B-lymphoblastoid cells bound WOW-1 Fab poorly (apparent K(d) = 2.4 microM), but acute stimulation with phorbol 12-myristate 13-acetate increased receptor affinity >30-fold (K(d) = 80 nM), with no change in receptor number. In contrast, alpha(V)beta(3) in melanoma cells was constitutively active, but ligand binding could be suppressed by overexpression of beta(3) cytoplasmic tails. Up-regulation of alpha(V)beta(3) affinity had functional consequences in that it increased cell adhesion and spreading and promoted adenovirus-mediated gene transfer. These studies establish that alpha(V)beta(3) is subject to rapid regulated changes in affinity that influence the biological functions of this integrin.

Integrins mediate cell adhesion and motility on the extracellular matrix, yet they also promote viral attachment and/or entry. Evidence is presented that adenovirus internalization by alpha(v) integrins requires activation of phosphoinositide-3-OH kinase (PI3K), whereas alpha(v) integrin-mediated cell motility depends on the ERK1/ERK2 mitogen-activated protein kinase pathway. Interaction of adenovirus with alpha(v), integrins induced activation of PI3K. Pharmacologic or genetic disruption of endogenous PI3K activity blocked adenovirus internalization and virus-mediated gene delivery yet had no effect on integrin-mediated cell adhesion or motility. Therefore, integrin ligation engages distinct signaling pathways that promote viral endocytosis or cell movement.

Complementary DNA clones for complement receptor type 2 (CR2), the B-lymphocyte membrane protein that serves as the receptor for Epstein-Barr virus and the C3d complement fragment, were obtained by screening a lambda gt11 library generated from Raji B lymphoblastoid cell mRNA. A 4.2-kilobase (kb) clone, representing the entire coding sequence of the protein plus untranslated 5' and 3' nucleotide sequences was obtained and sequenced. The 4.2-kb clone, which contains all but about 500 base pairs (bp) of the 5' untranslated region of the full-length CR2 mRNA, consists of 63 bp of 5' untranslated nucleotide sequence followed successively by a start codon, a 20-amino acid hydrophobic signal peptide, 1005 amino acids having a repeating motif, a 28-amino acid probable transmembrane domain, and a 34-amino acid cytoplasmic tail. The deduced amino acid sequence of the protein indicates that the extracellular domain consists entirely of 16 tandemly arranged repeating elements, each 60-75 amino acids in length, which are identified by multiple conserved residues. This repeating motif also occurs in the C3b/C4b receptor, several complement proteins, and a number of noncomplement proteins. In CR2, the 16 repeats occur in four clusters of four repeats each. Approximately 10% of the deduced amino acid sequence, including the amino and carboxyl termini, was confirmed by amino acid sequencing of tryptic peptides derived from purified CR2. The nucleotide and derived amino acid sequence of CR2 and related studies are presented here.

The Epstein-Barr virus/C3d receptor (CR2) of human B lymphocytes was purified to homogeneity from Raji cells by immunoaffinity chromatography. The average yield of the 145-kilodalton receptor was 400 pmol (50 micrograms) per 10(10) cells, representing an approximate 75% recovery. The isolated 145-kilodalton protein was antigenically and functionally intact as it reacted with several anti-CR2 monoclonal antibodies and bound purified Epstein-Barr virus and C3d,g. These findings with the purified molecule provide an unequivocal demonstration of the dual receptor functions of this protein.

Epstein-Barr virus (EBV) is a human herpesvirus that selectively binds to and infects human B lymphocytes (B cells). In the studies presented here, we found that several phenothiazines, including trifluoperazine, chlorpromazine, prochlorpromazine, and promethazine, blocked EBV infectivity of isolated adult human B cells as measured either by outgrowth of transformed cell colonies or by [3H]thymidine incorporation. Trifluoperazine, chlorpromazine, and prochlorpromazine were equally effective with 20 microM fully inhibiting infectivity, whereas 100 microM promethazine was required for a comparable effect. Inhibition by trifluoperazine was partially reversible. Studies with radiolabeled EBV demonstrated that the inhibitors did not impair virus binding to B cells. Electron microscopic examination of B lymphocytes revealed that trifluoperazine reduced the number of large uncoated cell vacuoles and the number of membrane microvilli, indicating that this agent interfered with cell pinocytosis. This process was accompanied by inhibition of EBV endocytosis into B cells. Phenothiazines bind to and inhibit calmodulin, an intracellular calcium-binding protein that regulates several key enzymes, some of which directly affect cytoskeletal elements, although they also may interact nonspecifically with other cellular constituents. In this regard, haloperidol, a non-phenothiazine calmodulin antagonist, and R24571, a derivative of the antimycotic miconazole, which is a potent and highly specific calmodulin inhibitor, also blocked EBV infection. These studies suggest that calmodulin or a calmodulin-regulated cellular enzyme(s) is involved in normal cellular endocytic processes in B lymphocytes and thereby in the early stages of EBV infection.

The major Epstein-Barr virus (EBV) envelope glycoprotein, gp350, was purified from the B95-8 cell line and analyzed for its ability to mediate virus attachment to the isolated EBV/C3d receptor (CR2) of human B lymphocytes. Purified gp350 and EBV, but not cytomegalovirus, exhibited dose-dependent binding to purified CR2 in dot blot immunoassays. Binding was inhibited by certain monoclonal antibodies to CR2 and to gp350. Liposomes bearing incorporated gp350 bound to CR2-positive B-cell lines but not to CR2-negative lines. Liposome binding was also inhibited by the OKB7 anti-CR2 monoclonal antibody. A computer-generated comparison of the deduced gp350 amino acid sequence with that of the human C3d complement fragment revealed two regions of significant primary sequence homology, a finding which suggests that a common region on these two unrelated proteins may be involved in CR2 binding.

In pursuing studies on the early events in the infection of human B cells by Epstein-Barr virus (EBV), we examined the host cell attachment phase with a panel of B-cell-specific monoclonal antibodies. One of the monoclonal antibodies, OKB7, directly blocked the attachment of purified EBV to B lymphocytes in the absence of a second anti-immunoglobulin antibody and thereby prevented EBV infection of tonsil and peripheral blood B cells. Although earlier studies have shown a close association of the EBV and complement receptor (CR2), an anti-CR2 monoclonal antibody, anti-B2, did not directly block the binding of EBV to B cells. A comparison of the structures recognized by these monoclonal antibodies on various cell types and their functional and physiochemical properties was undertaken. Flow cytometric analysis revealed that the molecules detected by OKB7 and anti-B2 were coexpressed to the same extent on B cells but were not expressed on T-cell lines. OKB7 and anti-B2 both immunoprecipitated a 145,000-molecular-weight membrane protein with an isoelectric point of 8.2 from membrane extracts of Raji lymphoblastoid cells. OKB7 and, to a lesser extent, anti-B2 directly blocked the attachment of C3d,g-coated fluorescent microspheres and sheep erythrocytes bearing C3d to B cells, indicating that these antibodies also react with CR2. These studies indicate that the EBV-CR2 receptor is a single membrane glycoprotein which possesses multiple antigenic and functional epitopes.

Serum incubated with purified EBV was found to contain C3 cleavage fragments characteristic of C3c. Since the cofactors necessary for such cleavage of C3b by factor I are not normally present in serum, EBV was tested for factor I cofactor activity. Purified EBV from both human and marmoset EBV-producing cell lines was found to act as a cofactor for the factor I-mediated breakdown C3b to iC3b and iC3b to C3c and C3dg. EBV also acted as a cofactor for the factor I-mediated cleavage of C4b to iC4b and iC4b to C4c and C4d. EBV from both the human and marmoset cell lines accelerated the decay of the alternative pathway C3 convertase. The classical pathway C3 convertase was unaffected. Multiple lines of evidence eliminated the possibility that marmoset or human CR1 was responsible for the functional activities of EBV preparations. The spectrum of activities was different from CR1 in that EBV and EBV-expressing cell lines failed to rosette with C3b or particles bearing C3b, the primary functional assay for CR1, and EBV did not accelerate classical pathway C3 convertase decay, another property of CR1. In addition, CR1 could not be detected immunologically on marmoset or human EBV-expressing cells and mAbs to CR1 failed to alter EBV-produced decay acceleration and factor I cofactor activities, although the antibodies blocked the same CR1-dependent functional activities. The multiple complement regulatory activities exhibited by purified EBV derived from human and marmoset cells differ from those of any of the known C3 or C4 regulatory proteins. These various activities would be anticipated to provide survival value for the virus by subverting complement- and cell-dependent host defense mechanisms.