Epstein-Barr virus regulates activation and processing of the third component of complement.

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.

Adenovirus entry into host cells: a role for alpha(v) integrins.

The mechanism(s) by which nonenveloped viruses enter host cells is poorly understood. The recent identification of cell-surface alpha(v) integrins as receptors for adenovirus internalization has shed much light on this process. In addition, analysis of alpha(v) integrins as internalization receptors for adenovirus has provided further insights into the biology of integrins.

Integrin alpha v beta 5 selectively promotes adenovirus mediated cell membrane permeabilization.

Human adenovirus type 2 (Ad2) enters host cells by receptor-mediated endocytosis, an event mediated by the virus penton base binding to cell surface integrins alpha v beta 3 and alpha v beta 5. While both alpha v integrins promote virus internalization, alpha v beta 5 is involved in the subsequent event of membrane permeabilization. Cells transfected with the beta 5 or beta 3 subunit, expressing either alpha v beta 5 and alpha v beta 3, respectively, were capable of supporting Ad2 infection to varying degrees. In this case, cells expressing alpha v beta 5 were significantly more susceptible to Ad2-induced membrane permeabilization, as well as to Ad2 infection, than cells expressing alpha v beta 3. Adenovirus-mediated gene delivery was also more efficient in cells expressing alpha v beta 5. These results suggest that the interaction of alpha v beta 5 with Ad2 penton base facilitates the subsequent step of virus penetration into the cell. These studies provide evidence for the involvement of a cellular receptor in virus-mediated membrane permeabilization and suggest a novel biological role for integrin alpha v beta 5 in the infectious pathway of a human adenovirus.

Matrix valency regulates integrin-mediated lymphoid adhesion via Syk kinase.

Lymphocytes accumulate within the extracellular matrix (ECM) of tumor, wound, or inflammatory tissues. These tissues are largely comprised of polymerized adhesion proteins such as fibrin and fibronectin or their fragments. Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation. This adhesion event depends on the appropriate spacing of integrin adhesion sites. Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes. In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments. These results reveal a cooperative interaction between signals emanating from integrins and antigen receptors that can serve to regulate stable lymphoid cell adhesion and retention within a remodeling ECM.

Role of alpha(v) integrins in adenovirus cell entry and gene delivery.

Adenoviruses (Ad) are a significant cause of acute infections in humans; however, replication-defective forms of this virus are currently under investigation for human gene therapy. Approximately 20 to 25% of all the gene therapy trials (phases I to III) conducted over the past 10 years involve the use of Ad gene delivery for treatment inherited or acquired diseases. At present, the most promising applications involve the use of Ad vectors to irradicate certain nonmetastatic tumors and to promote angiogenesis in order to alleviate cardiovascular disease. While specific problems of using Ad vectors remain to be overcome (as is true for almost all viral and nonviral delivery methods), a distinct advantage of Ad is the extensive knowledge of its macromolecular structure, genome organization, sequence, and mode of replication. Moreover, significant information has also been acquired on the interaction of Ad particles with distinct host cell receptors, events which strongly affect virus tropism. This review provides an overview of the structure and function of Ad attachment (coxsackievirus and Ad receptor [CAR]) and internalization (alpha(v) integrins) receptors and discusses their precise role in virus infection and gene delivery. Recent structure studies of integrin-Ad complexes by cryoelectron microscopy are also highlighted. Finally, unanswered questions arising from the current state of knowledge of Ad-receptor interactions are presented in the context of improving Ad vectors for future human gene therapy applications.

Neutralization of Epstein-Barr virus by nonimmune human serum. Role of cross-reacting antibody to herpes simplex virus and complement.

These studies were carried out to investigate the mechanism of neutralization of purified Epstein-Barr virus (EBV) by fresh human serum from normal individuals lacking antibody to the EBV viral capsid (VCA) and nuclear antigens (EBNA). Such individuals thus lack serological evidence of immunity to EBV. Although an enzyme-linked immunosorbent assay (ELISA) with highly purified immobilized EBV detected low levels of IgG antibody reactive with EBV in these normal nonimmune sera, this antibody failed to neutralize EBV in the absence of complement. Studies with depleted sera and mixtures of purified complement proteins at physiologic concentrations showed that the IgG antibody and C1, C4, C2, and C3 of the classical pathway were able to fully neutralize EBV. Mixtures of the purified components of the alternative pathway at physiologic concentrations failed to neutralize purified EBV in the presence or absence of the antibody and the alternative pathway did not potentiate classical pathway-mediated neutralization. No evidence for a requirement for C8 was obtained, precluding lysis as the mechanism of neutralization. Since C3 deposition on the viral surface accompanied classical pathway activation, viral neutralization is most likely secondary to the accumulation of complement protein on the viral surface. A coating of protein on the virus could interfere with attachment to, or penetration of potentially susceptible cells. Experiments were undertaken to determine the specificity of the IgG antibody in the sera of EBV nonimmune individuals which, together with complement, neutralized EBV. Both purified EBV and herpes simplex I (HSV-1) absorbed the EBV ELISA reactivity and EBV-neutralizing activity of nonimmune sera, whereas another member of the herpesvirus group, cytomegalovirus, was inactive in this regard. HSV-1 was quantitatively more efficient than EBV in absorbing reactivity, a finding that indicates that the antibody has a higher affinity for HSV-1 than for EBV. Further absorption studies indicated that the cross-reaction occurred in both directions as EBV also absorbed HSV-1 reactive antibodies as tested in an HSV-1 ELISA. EBV was also less efficient than HSV-1 in absorbing reactivity with HSV-1. A serum lacking detectable antibodies to both EBV and HSV-1 failed to neutralize EBV. These studies cumulatively indicate that fresh serum from EBV nonimmune individuals neutralizes EBV by the combined action of a previously undescribed cross-reacting antibody apparently elicited by HSV-1 and C1, C4, C2, and C3 of the classical complement pathway.

Inherited deficiency of the seventh component of complement associated with nephritis. Propensity to formation of C56 and related C7-consuming activity.

A 46-yr-old female with chronic pyelonephritis was found to lack complement (C) activity by the use of hemolytic screen assays in agarose gels. These assays also revealed a propensity of patient serum to form an activated complex of the fifth and sixth components of C, C56. Each of the C component hemolytic activities was present in normal or elevated amounts with the exception of C7, which was undetectable; addition of purified C7 led to the restoration of hemolytic activity. C-dependent phagocytosis, immune adherence, and neutrophil chemotaxis were normal. Family studies demonstrated that the defect was transmitted as an autosomal codominant apparently not linked with alleles at the HLA-A or HLA-B loci. Persisting C56 was readily formed in this as compared to normal serum upon incubation with multiple C activators including zymosan, inulin, immune complexes, heat-aggregated human gamma globulin, endotoxin, and agarose. A heat-stable (56 degrees C, 30 min) activity which consumed C7 with time-and temperature-dependent kinetics was detected in plasma and serum, and seemed to be similar to a "C7 inactivator" previously described in another C7-deficient individual. However, this activity was found to have properties identical to those of C56 during low ionic strength precipitation and chromatography on Sephadex G-200, to be specifically removed upon passage through an anti-C5 immunoadsorbent column, and to be associated with a small amount of C56, suggesting that it represents an expression of small amounts of C56 rather than a new C-inhibitory activity. Thus, an individual with chronic nephritis lacking C7 is reported; the utility of a hemolytic screen assay in agarose plates for the detection of such patients is emphasized; persisting C56 is shown readily to be formed in this serum; and the presence of C7-consuming activity which is associated with and in all likelihood attributable to C56 is shown.

Cell growth and matrix invasion of EBV-immortalized human B lymphocytes is regulated by expression of alpha(v) integrins.

alpha(v) integrins have been shown to play an important role in epithelial-derived cell migration, cell growth and tumor invasion/metastasis, however their role on cells of hematopoietic origin is less clear. Epstein-Barr virus (EBV), a human herpesvirus associated with several lymphoproliferative disorders in man, induces expression of alpha(v) integrins on transformed B lymphocytes. In the studies reported here, we show that EBV infection increases alpha(v), beta3 and beta5 integrin subunit mRNAs as well as upregulates the expression of the alphavbeta3 integrin protein on human B cells. Among the nine different EBV proteins expressed in latently infected B cells (nuclear and plasma membrane-associated), only LMP1, LMP2A and EBNA2 were shown to selectively transactivate the alpha(v) integrin promoter. Treatment of EBV-transformed B cells with alpha(v) antisense oligonucleotides specifically reduced cell surface expression of alpha(v) integrins, inhibited cell growth in low serum, reduced cell invasion in matrigels and decreased expression of metalloprotease, MMP9. These studies indicate that alpha(v) integrins play a significant role in EBV-induced B-lymphocyte proliferation and invasion. Strategies to interfere with alphav integrin expression and/or function may therefore be of potential value in the treatment of EBV-associated lymphoproliferative disorders.

Recent structural solutions for antibody neutralization of viruses.

Structural studies of virus-antibody interactions can offer insights into mechanisms of virus neutralization and immune escape. Crystallography and cryoelectron microscopy can reveal the structural relationship between receptor-binding sites and neutralization epitopes, as well as whether molecular rearrangements occur upon antibody binding.

Complement effector mechanisms in health and disease.

Complement is an effector system able to mediate a number of biological activities in vitro and in vivo. Most familiar is the ability of the system to mediate the lytic destruction of numerous kinds of cells and pathogenic organisms including bacteria, viruses, and virus-infected cells. In addition, the complement system also activates neutrophils, monocytes, basophils, mast cells, and lymphocytes to perform specialized functions. While generally considered to be confined to the effector side of immune reactions, recent evidence indicates that the complement system also directly recognizes and is triggered by a number of bacteria and viruses as well as virus-infected cells in the absence of antibody. In such reactions, complement fulfills the recognition role normally associated with the antibody molecule or immune lymphocyte. The complement system may thus also function as a natural surveillance system operative prior to the induction of specific immunity. Involvement of the complement system in biological reactions has been ascertained by several techniques over the years. These include quantitation of individual complement components in human sera and demonstration of complement deposition in diseased tissues in human diseases and in experimental diseases in animals. Such techniques, however, have limitations in specificity and sensitivity. Assays which detect specific features of the complement activation process have become available in recent years. These tests detect the physical, chemical, or antigenic changes characteristic of the complement activation process. These assays are extremely specific and quantitative; furthermore, most are usable with samples from patients. Three general approaches have been utilized to develop such specific quantitative assays for complement activation. The first includes assays which quantitate activation-specific limited proteolysis of the complement components. The second type of assay includes tests which detect and quantitate new antigens or other activation-specific antigenic changes. The third category is represented by assays which detect and quantitate the protein-protein complexes characteristic of the activation process. Examples of tests presenting each of these approaches are given.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of antibody and complement in the control of viral infections.

Host defense against viral infection is extremely complex and includes both humoral and cellular immune mechanisms. This contribution examines the mechanisms by which antibody (Ab) and the complement (C) system, major constituents of the humoral immune system, inactivate viruses and block viral maturation in virus-infected cells in vitro. Ab and C may neutralize viruses by envelopment in a coating of protein, by aggregation by lysis, or by facilitating interactions with various effector cells. Ab and C molecules deposited on the surfaces of viruses may physically interfere with the ability of the virus to infect a potentially susceptible cell. This appears to be the most common mechanism by which Ab and C neutralize viruses. In rare instances, Ab and/or C may aggregate viruses; aggregation reduces the net number of infectious particles and thus is manifest as neutralization. C may lyse enveloped viruses, resulting in irreversible viral inactivation. However, this does not appear to be a major mechanism of viral neutralization. Finally, the Fc portions of bound Ab molecules as well as bound C molecules may interact with effector cells with specific receptors for these factors and thereby facilitate viral destruction. In regard to virus-infected cells, the deposition of Ab or C on the cell surface may prevent the maturation or release of viral particles and alter normal cellular functions. Ab and C may also lyse virus-infected cells, abruptly stopping further viral maturation. Such lytic events require only the F(ab')2 portion of the Ab molecule and proceed via activation of the alternative C pathway. Effector cells may also interact with Ab and/or C molecules deposited on virus-infected cells, leading to cytotoxic reactions and/or ingestion depending on the type of effector cell involved. The activated C system has the ability to produce an acute inflammatory response leading to alterations in vessel permeability, edema, changes in smooth-muscle contractility, and the influx of leukocytes. Such inflammatory responses occurring in tissues, including the skin, as a result of C activation not only retard the spread of the infection and facilitate the destruction of the infectious agent, but also in all likelihood damage normal tissues in the vicinity. In addition, C activation in tissues also has the ability to stimulate arachidonic acid metabolism and induce the release of histamine and other mediators as well as pyrogens from appropriate cell types. A number of the systemic symptoms characteristic of viral infections, such as headaches, myalgias, and fever, likely result from such processes.

CR2 complement receptor.

CR2, a membrane glycoprotein, is one of a number of cell-surface proteins which bind activation and processing fragments of the complement system. CR2, which is found on normal B lymphocytes, follicular dendritic cells in lymphoid organs, and epithelial cells, interacts preferentially with C3dg, the terminal activation/processing fragment of the third complement component. Attachment of C3dg to CR2 brings complement activators, bearing covalently bound C3dg, into direct membrane contact with CR2-bearing cells. Epstein-Barr virus, a human herpesvirus, also binds to CR2 on B lymphocytes. Attachment of EBV is followed by infection. CR2 has been purified and the binding properties of its ligands analyzed. Monoclonal antibodies have been developed and used to probe the structural correlates of CR2 functions. CR2 has been molecularly cloned and its primary amino acid sequence deduced. These data indicate that it shares characteristic structural features with a number of other complement and non-complement cell membrane and plasma proteins. Several of the complement-associated proteins in this family possess regulatory functions; they are encoded by linked genes which have been localized to band q32 on chromosome 1. CR2 has been expressed in primate and rodent cells by transfection of cDNA in antigenically and functionally intact form. It has also been expressed in soluble form and its structure, electron microscopic appearance and binding characteristics analyzed in detail. The present state of knowledge of the structure and genetics of CR2 and current understanding of its biologic functions are summarized here.

Virus receptors on lymphoid cells.

The studies described above indicate the advances made in the isolation and characterization of virus receptors of lymphoreticular cells (Table I). Although the examples of lymphotropic virus receptors cited in this chapter indicate that single membrane glycoproteins can serve as receptors, other nonlymphoid viruses such as vesicular stomatitis virus (VSV) (Table I) appear to utilize glycolipid or phospholipid components for cell attachment. These molecules may be responsible for the broad specificity of host cell attachment by these viruses. The virus-binding moiety of phospholipid/glycolipid receptors remains to be fully analyzed. It is anticipated that biochemical techniques such as the use of chemical cross-linking reagents will aid in the identification of other virus receptors such as CMV and measles which have less restricted lymphotropism than EBV. In addition, X-ray crystallographic analysis of viruses such as the recent studies of human rhinovirus and poliovirus may provide insights on the complementary structure of cellular recognition sites for viruses.

Optimization of growth methods and recombinant protein production in BTI-Tn-5B1-4 insect cells using the baculovirus expression system.

A novel insect cell line from Trichoplusia ni, BTI-Tn 5B1-4 (Tn 5), was compared to Spodoptera frugiperda, Sf 9, cells for production of two recombinant secreted proteins: truncated Epstein-Barr viral attachment protein (EBV gp105) and truncated, soluble tissue factor (sTF). Under optimum conditions for both cell lines, Tn 5 cells produced 28-fold more secreted sTF than Sf 9 cells, respectively, on a per cell basis. The total production of gp105 was similar for the two cell lines. However, Tn5 cells secreted gp105 much more efficiently, resulting in 5-fold higher levels in the extracellular medium. Despite these increases, Tn 5 cells are attachment-dependent, and protein production is sensitive to the cell density (cells/cm2), unlike the Sf9 cell line which can be easily grown and scaled up in cell suspension cultures without significantly affecting its per cell production. Thus, protein production from Tn 5 cells above 0.1 L scales was optimized with respect to cell density using standard techniques for the growth of attachment-dependent cells. Roller bottles precoated with DEAE-based microcarriers and suspension cultures employing collagen-coated microcarriers were found to be effective ways of culturing Tn 5 cells. Predetermined optimal cell densities were used to produce EBV gp105 in microcarrier-coated roller bottles or in suspension cultures using collagen-coated microcarriers at concentrations close to those observed in tissue culture flasks.

Crystallization and preliminary X-ray diffraction analysis of human adenovirus.

Replication-defective and conditionally replicating adenovirus (AdV) vectors are currently being utilized in approximately 25% of human gene transfer clinical trials. Unfortunately, progress in vector development has been hindered by a lack of accurate structural information. Here we describe the crystallization and preliminary X-ray diffraction analysis of a HAdV5 vector that displays a short flexible fiber derived from HAdV35. Crystals of Ad35F were grown in 100mM HEPES pH 7.0, 200mM Ca(OAc)(2), 14% PEG 550 MME, 15% glycerol in 100mM Tris-HCl 8.5. Freshly grown crystals diffracted well to 4.5A resolution and weakly to 3.5A at synchrotron sources. HAdV crystals belong to space group P1 with unit cell parameters a=854.03A, b=855.17A, c=865.24A, alpha=119.57 degrees , beta=91.71 degrees , gamma=118.08 degrees with a single particle in the unit cell. Self-rotation and locked-rotation function analysis allowed the determination of the particle orientation. Molecular replacement, density modification and phase-extension procedures are being employed for structure determination.

Insights into adenovirus host cell interactions from structural studies.

Human adenoviruses cause a significant number of acute respiratory, enteric and ocular infections, however they have also served as useful model systems for uncovering fundamental aspects of cell and molecular biology. In addition, replication-defective forms of adenovirus are being used in gene transfer and vaccine clinical trials. Over the past decade, steady advances in structural biology techniques have helped reveal important insights into the earliest events in the adenovirus life cycle as well as virus interactions with components of the host immune system. This review highlights the continuing use of structure-based approaches to uncover the molecular features of adenovirus-host interactions.

Isolation of Epstein Barr-virus and studies of its neutralization by human IgG and complement.

An isolation procedure for Epstein-Barr virus (EBV) that yields substantial quantities of purified infectious virus is described. The transforming strain of EBV was obtained from the marmoset lymphoma cell line B95-8 after stimulation with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Purification was achieved by dextran density gradient ultracentrifugation in the presence of bacitracin, which was included to prevent viral aggregation. When assayed in cord blood leukocytes, isolated EBV stimulated DNA synthesis and induced the formation of colonies of transformed cells. The yield of infectious virus as determined by these assays was 13 to 29%. Electron microscopic (EM) examination of negatively stained virions revealed the presence of 115-nm spherical enveloped particles containing an internal 55-nm ring-shaped nucleoid. Interactions between 3H-thymidine labeled EBV, IgG and complement (C) were examined by rate zonal ultracentrifugation. High concentrations of immune IgG aggregated the virus whereas IgG together with C induced lysis as demonstrated by release of labeled EBV nucleic acid. EM studies of the IgG and C mixtures performed in parallel revealed accumulation of protein on the viral envelope, progressive separation of the envelope from the nucleocapsid, and disintegration of the nucleoid. Approximately 25-fold less IgG was required for neutralization than for viral aggregation. Although C did not enhance the IgG dependent neutralization, physiologic concentrations of C in normal nonimmune human serum also inactivated the virus.