A model for persistent infection with Epstein-Barr virus: the stealth virus of human B cells.

Most adult humans are infected benignly and for life with the herpesvirus Epstein-Barr virus. EBV has been a focus of research because of its status as a candidate tumor virus for a number of lymphomas and carcinomas. In vitro EBV has the ability to establish a latent infection in proliferating B lymphoblasts. This is the only system available for studying human herpesvirus latency in culture and has been extremely useful for elucidating how EBV promotes cellular growth. However, to understand how EBV survives in the healthy host and what goes awry, leading to disease, it is essential to know how EBV establishes and maintains a persistent infection in vivo. Early studies on the mechanism of EBV persistence produced inconclusive and often contradictory results because the techniques available were crude and insensitive. Recent advances in PCR technology and the application of sophisticated cell fractionation techniques have now provided new insights into the behavior of the virus. Most dramatically it has been shown that EBV in vivo does not establish latency in a proliferating lymphoblast, but in a resting memory B cell. The contrasting behaviors of being able to establish a latent infection in proliferating B blasts and resting memory B cells can be resolved in terms of a model where EBV performs its complete life cycle in B lymphocytes. The virus achieves this not by disrupting normal B cell biology but by using it.     

Critical role of Epstein-Barr Virus (EBV)-encoded RNA in efficient EBV-induced B-lymphocyte growth transformation

It was demonstrated that Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) were nonessential for B-lymphocyte growth transformation. We revisited this issue by producing a large quantity of EBER-deleted EBV by using an Akata cell system. Although the EBER-deleted virus efficiently infected B lymphocytes, its 50% transforming dose was approximately 100-fold less than that of the EBER-positive EBV. We then engineered the genome of EBER-deleted virus and generated a recombinant virus with the EBER genes reconstituted at their native locus. The resultant EBER-reconstituted EBV exhibited restored transforming ability. In addition, lymphoblastoid cell lines established with the EBER-deleted EBV grew significantly more slowly than those established with wild-type or EBER-reconstituted EBV, and the difference between the growth rates was especially highlighted when the cells were plated at low cell densities. These results clearly demonstrate that EBERs significantly contribute to the efficient growth transformation of B lymphocytes by enhancing the growth potential of transformed lymphocytes.     

T cell-mediated immunoregulation of Epstein Barr virus- (EBV) induced B lymphocyte activation in EBV-seropositive and EBV-seronegative individuals

Infection with Epstein Barr virus (EBV) is accompanied by seroconversion and life-long persistence of the virus in B lymphocytes. During acute EBV-induced infectious mononucleosis, suppressor T cells become activated, which may provide an additional mechanism of host defense against the causative agent. When cultures of lymphocytes from normal adults seropositive for EBV were stimulated with the B95-8 strain of EBV, purified B cells produced increasingly higher numbers of immunoglobulin- (Ig) secreting cells, whereas in co-cultures of autologous B and T cells a profound suppressor T cell activity inhibited further B cell activation after 10 to 12 days in culture. No such T cell-mediated inhibitory effect was seen in cultures of lymphocytes obtained from normal adults seronegative for EBV, indicating a correlation between the suppressor effect with evidence of prior immunity to this virus. The T cell-mediated suppression in patients with infectious mononucleosis is characterized by an early-acting inhibitory effect on B cell differentiation that is not specific in that all polyclonal B cell activators are inhibited, whereas in EBV-seropositive normal subjects suppression is delayed in time and affects only EBV-activated cultures. These data indicate that after infection with EBV, immunoregulatory T cells are generated that are capable of inhibiting further EBV-induced activation of autologous B cells and thus may provide an additional unique mechanism of host defense against persisting EBV-infected B cells.     

Influence of Burkitt's lymphoma and primary B cells on latent gene expression by the nonimmortalizing P3J-HR-1 strain of Epstein-Barr virus.

The Epstein-Barr virus (EBV) genes expressed in B lymphocytes immortalized in vitro or in Burkitt's lymphoma (BL) cells infected in vivo have been characterized previously; however, the viral products which are essential for immortalization or for establishment of EBV latency are still not known. To approach this question, we compared the kinetics of expression of EBV nuclear antigens and the two EBV-encoded small RNAs, EBER1 and EBER2, after infection of primary B cells or EBV genome-negative BL cells with either an immortalizing EBV strain (B95-8) or the nonimmortalizing deletion mutant (HR-1). Following infection of primary cells with B95-8 virus, EBV nuclear antigen (EBNA)-2 was expressed first, followed by EBNA-1, -3, and -4 (also called leader protein [LP]) and the two small RNAs. Infection of EBV genome-negative BL cells with the same strain of virus resulted in a similar pattern of gene expression, except that the EBNAs appeared together and more rapidly. EBERs were not apparent in one BL cell line converted by B95-8. The only products detected after infection of primary B lymphocytes with the HR-1 deletion mutant were the EBNA-4 (LP) family and trace amounts of EBER1. Although HR-1 could express neither EBNA-1, EBNA-3, nor EBER2 in primary cells, all these products were expressed rapidly after HR-1 infection of EBV genome-negative BL cell lines. The results indicate that the mutation in HR-1 virus affects immortalization not only through failure to express EBNA-2, a gene which is deleted, but also indirectly by curtailing expression of several other EBV genes whose coding regions are intact in the HR-1 virus and normally expressed during latency. The pattern of latent EBV gene expression after HR-1 infection is dependent on the host cell, perhaps through products specific for the cell cycle or the state of B-cell differentiation.     

Epstein-Barr virus: exploiting the immune system

In vitro, Epstein-Barr virus (EBV) will infect any resting B cell, driving it out of the resting state to become an activated proliferating lymphoblast. Paradoxically, EBV persists in vivo in a quiescent state in resting memory B cells that circulate in the peripheral blood. How does the virus get there, and with such specificity for the memory compartment? An explanation comes from the idea that two genes encoded by the virus--LMP1 and LMP2A--allow EBV to exploit the normal pathways of B-cell differentiation so that the EBV-infected B blast can become a resting memory cell.     

Glycoprotein 110, the Epstein-Barr virus homolog of herpes simplex virus glycoprotein B, is essential for Epstein-Barr virus replication in vivo.

The Epstein-Barr virus (EBV) glycoprotein gp110 has substantial amino acid homology to gB of herpes simplex virus but localizes differently within infected cells and is essentially undetectable in virions. To investigate whether gp110, like gB, is essential for EBV infection, a selectable marker was inserted within the gp110 reading frame, BALF4, and the resulting null mutant EBV stain, B95-110HYG, was recovered in lymphoblastoid cell lines (LCLs). While LCLs infected with the parental virus B95-8 expressed the gp110 protein product following productive cycle induction, neither full-length gp110 nor the predicted gp110 truncation product was detectable in B95-110HYG LCLs. Infectious virus could not be recovered from B95-110HYG LCLs unless gp110 was provided in trans. Rescued B95-110HYG virus latently infected and growth transformed primary B lymphocytes. Thus, gp110 is required for the production of transforming virus but not for the maintenance of transformation of primary B lymphocytes by EBV.     

Biology and disease associations of Epstein-Barr virus

Epstein-Barr virus (EBV) is a human herpesvirus which infects almost all of the world's population subclinically during childhood and thereafter remains in the body for life. The virus colonizes antibody-producing (B) cells, which, as relatively long-lived resting cells, are an ideal site for long-term residence. Here EBV evades recognition and destruction by cytotoxic T cells. EBV is passed to naive hosts in saliva, but how the virus gains access to this route of transmission is not entirely clear. EBV carries a set of latent genes that, when expressed in resting B cells, induce cell proliferation and thereby increase the chances of successful virus colonization of the B-cell system during primary infection and the establishment of persistence. However, if this cell proliferation is not controlled, or if it is accompanied by additional genetic events within the infected cell, it can lead to malignancy. Thus EBV acts as a step in the evolution of an ever-increasing list of malignancies which are broadly of lymphoid or epithelial cell origin. In some of these, such as B-lymphoproliferative disease in the immunocompromised host, the role of the virus is central and well defined; in others, such as Burkitt's lymphoma, essential cofactors have been identified which act in concert with EBV in the evolution of the malignant clone. However, in several diseases in which the presence of EBV has more recently been discovered, the role of the virus is unclear. This review describes recent views on the EBV life cycle and its interlinks with normal B-cell biology, and discusses how this interrelationship may be upset and result in EBV-associated disease.     

Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death.

Epstein-Barr virus (EBV) not only induces growth transformation in human B lymphocytes, but has more recently been shown to enhance B cell survival under suboptimal conditions where growth is inhibited; both effects are mediated through the coordinate action of eight virus-coded latent proteins. The effect upon cell survival is best recognized in EBV-positive Burkitt's lymphoma cell lines where activation of full virus latent gene expression protects the cells from programmed cell death (apoptosis). Here we show by DNA transfection into human B cells that protection from apoptosis is conferred through expression of a single EBV latent protein, the latent membrane protein LMP 1. Furthermore, we demonstrate that LMP 1 mediates this effect by up-regulating expression of the cellular oncogene bcl-2. The interplay between EBV infection and expression of this cellular oncogene has important implications for virus persistence and for the pathogenesis of virus-associated malignant disease.     

Epstein-Barr Virus BARF1 Protein Is Dispensable for B-Cell Transformation and Inhibits Alpha Interferon Secretion from Mononuclear Cells

The Epstein-Barr virus (EBV) BARF1 gene encodes a soluble colony-stimulating factor 1 (CSF-1) receptor that neutralizes the effects of CSF-1 in vitro. To study the effect of BARF1 on EBV-induced transformation, we added recombinant BARF1 to B cells in the presence of EBV. BARF1 did not enhance transformation of B cells by EBV in vitro. To study the role of BARF1 in the context of EBV infection, we constructed a recombinant EBV mutant with a large deletion followed by stop codons in the BARF1 gene as well as a recombinant virus with a wild-type BARF1 gene. While BARF1 has previously been shown to act as an oncogene in several cell lines, the EBV BARF1 deletion mutant transformed B cells and initiated latent infection, and the B cells transformed with the BARF1 mutant virus induced tumors in SCID mice with an efficiency similar to that of the wild-type recombinant virus. Since human CSF-1 stimulates secretion of alpha interferon from mononuclear cells and BARF1 encodes a soluble CSF-1 receptor, we examined whether recombinant BARF1 or BARF1 derived from EBV-infected B cells could inhibit alpha interferon secretion. Recombinant BARF1 inhibited alpha interferon secretion by mononuclear cells in a dose-dependent fashion. The B cells transformed with mutant BARF1 EBV showed reduced inhibition of alpha interferon secretion by human mononuclear cells when compared with the B cells transformed with wild-type recombinant virus. These experiments indicate that BARF1 expressed from the EBV genome directly inhibits alpha interferon secretion, which may modulate the innate host response to the virus.     

Effect of activation of the Epstein-Barr virus genome on expression of B cell differentiation antigens of Burkitt's lymphoma lines

Using anti-human B cell monoclonal antibodies prepared against B1 (CD20), B2 (CD21), B4 (CD19), and BB-1 (B lymphoblast antigen-1), we compared the expression of B cell differentiation antigens on a Jijoye-P3HR-1 cell line family of Burkitt's lymphomas. The expression of BB-1 and B2 antigens was faint on P3HR-1 K cell line which is an Epstein-Barr virus (EBV) high producer. On the other hand, B1 and B4 antigens were strongly expressed on it. It was also found that BB-1 expression decreased on P3HR-1 cells after activation of intracellular EBV genes by treating chemically with tumor-promoting agent (TPA) and n-butyrate, or on Raji cells on superinfection with EBV. This decrease of BB-1 was blocked by the additional treatment with retinoic acid, an inhibitor of virus replication. Dual immunofluorescence staining analysis showed that the individual cell expressing EBV-associated antigens expressed BB-1 antigen only marginally. The relationship between the change in phenotypes of host B cells and the activation of the EBV genome is discussed.     

Model for studying virus attachment: identification and quantitation of Epstein-Barr virus-binding cells by using biotinylated virus in flow cytometry.

Epstein-Barr virus (EBV) was purified and biotinylated without significant loss of its cell-transforming activity. The use of biotinylated virus in conjunction with antibodies specific for selected cell surface molecules and flow cytometric analysis allowed for the positive identification of the virus-binding lymphocytes among a heterogeneous mononuclear cell population. Biotinylated EBV efficiently bound to all B lymphocytes, including those bearing surface mu, delta, gamma, and alpha immunoglobulin heavy chains or the surface CD5 (Leu-1) marker, but not to T lymphocytes, natural killer cells, or monocytes. By using biotinylated EBV and specific monoclonal antibodies in competitive inhibition experiments, it was also found that the virus attaches to an epitope on the CR2 molecule (the receptor for C3d and EBV), which is close to or identical with the one recognized by OKB7 monoclonal antibody, and that cell surface structures other than CR2 cannot mediate attachment of EBV. Moreover, studies on the binding of the virus to induced B lymphocytes (cells in S through G2 phase), and this was associated with the disappearance of the surface CR2 molecule and the inability of the virus to attach to these cells. The approach described here should be useful in studying the attachment of other viruses, identifying the specific cell types involved, and analyzing the effect of the cell cycle on virus binding.     

Activation and infection of B cells by Epstein-Barr virus. Role of calcium mobilization and of protein kinase C translocation

Both the activation and the transformation of human B cells by EBV were inhibited by either the Ca2+ channel blocking agent verapamil or the combination of theophylline and dibutyryl cAMP: the day 4 and day 20 peaks of [3H]TdR incorporation were abolished; the EBNA marker was not expressed by day 10; lymphoblastoid cell lines did not arise. Short term incubation of B cells with EBV or verapamil showed that the effect of verapamil was reversible and took place early in the interaction between EBV and B cells. The effect of EBV on the early metabolic events of B cell response was thus examined in the presence and in the absence of the drugs. Compared to anti-mu stimulation, supernatant of the transforming B95-8 strain as well as that of the non-transforming P3HR1 strain induced a drug sensitive increase of the free cytosolic Ca2+ concentration. This increase was associated with a protein kinase C translocation from the cytosol to a membrane bound compartment. Moreover, B95-8 supernatant induced phosphatidyl inositol metabolism by human B cells but at least four times less than that induced by anti-mu antibody. These metabolic events induced by EBV were significantly inhibited by anti-CD21 antibodies whereas anti-mu induced metabolic events were not. The infection of EBV negative Ramos cell line was prevented by verapamil or by theophylline + dibutyryl cAMP. Verapamil did not modify the density of EBV receptors but negatively interfered with the penetration of the virus into B cells. Thus B cell activation through the EBV receptor and virus penetration share a common metabolic pathway which is also used for transduction of the signal delivered through the membrane Ig.     

Important but differential roles for actin in trafficking of Epstein-Barr virus in B cells and epithelial cells

Epstein-Barr virus (EBV) uses different virus and cell proteins to enter its two major targets, B lymphocytes and epithelial cells. The routes that the virus takes into the two cell types are also different. To determine if these differences extend to movement from the cell surface to the nucleus, we examined the fate of incoming virus. Essentially all virus that entered a B cell remained stable for at least 8 h. In contrast, up to 80% of virus entering an epithelial cell was degraded in a compartment sensitive to inhibitors of components involved in autophagy. Inhibitors of actin remodeling blocked entry into a B cell but had no effect or enhanced entry into an epithelial cell. Inhibitors of the microtubule network reduced intracellular transport in both cell types, but movement to the nucleus in an epithelial cell also required involvement of the actin cytoskeleton. Deletion of the cytoplasmic tail of CR2, which in an epithelial cell interacts with the actin nucleator FHOS/FHOD when cross-linked by EBV, had no effect on infection. However, inhibitors of downstream signaling by integrins reduced intracellular transport. Cooperation of the microtubule and actin cytoskeletons, possibly activated by interaction with integrin binding proteins in the envelope of EBV, is needed for successful infection of an epithelial cell.     

Epstein-Barr virus-lymphoid cell interactions. III. Effect of concanavalin A and saccharides on Epstein-Barr virus penetration

To study some aspects of Epstein-Barr virus (EBV) penetration into target cells, the effect of concanavalin A (ConA) and various saccharides on virus infectivity and cell susceptibility to EBV infection was examined. ConA treatment of the target cells, EBV, or EBV-cell complexes was found to inhibit virus antigen expression. Several control experiments with alpha-d-methyl-mannoside elution of ConA, removal of nonfused EBV particles from the cell surface by trypsin treatment, and addition of ConA at different times postinfection were performed to define the site of ConA action on EBV infection. ConA appeared to have a dual action: (i) it inhibited EBV binding to virus receptors, and (ii) it blocked the penetration of receptor-bound virus into target cells at a trypsin-sensitive stage, thus indicating that ConA prevented the fusion of viral envelope with the target cell membrane. A high sucrose concentration (0.25 M), known to inhibit cell membrane movements, was also found to block EBV penetration at a trypsinsensitive stage, thus suggesting the implication of cell membrane movements and underlying activities (or both) in viral envelope fusion. Lower concentrations of various monosaccharides (0.12 M) did not influence EBV infection. Under conditions of ConA treatment that did not influence EBV infectivity and target cells susceptibility, ConA was able to mediate virus binding to EBV receptornegative cell lines, but no virus antigens were expressed in these cells. These observations reinforced the idea that the mere attachment of EBV to lymphoid cells is not sufficient to lead to infection. In light of the present and previously published data, we postulate the existence of a specific cellular mechanism that allows the penetration of EBV into the target (B) lymphocyte.     

A molecular link between malaria and Epstein-Barr virus reactivation

Although malaria and Epstein-Barr (EBV) infection are recognized cofactors in the genesis of endemic Burkitt lymphoma (BL), their relative contribution is not understood. BL, the most common paediatric cancer in equatorial Africa, is a high-grade B cell lymphoma characterized by c-myc translocation. EBV is a ubiquitous B lymphotropic virus that persists in a latent state after primary infection, and in Africa, most children have sero-converted by 3 y of age. Malaria infection profoundly affects the B cell compartment, inducing polyclonal activation and hyper-gammaglobulinemia. We recently identified the cystein-rich inter-domain region 1alpha (CIDR1alpha) of the Plasmodium falciparum membrane protein 1 as a polyclonal B cell activator that preferentially activates the memory compartment, where EBV is known to persist. Here, we have addressed the mechanisms of interaction between CIDR1alpha and EBV in the context of B cells. We show that CIDR1alpha binds to the EBV-positive B cell line Akata and increases the number of cells switching to the viral lytic cycle as measured by green fluorescent protein (GFP) expression driven by a lytic promoter. The virus production in CIDR1alpha-exposed cultures was directly proportional to the number of GFP-positive Akata cells (lytic EBV) and to the increased expression of the EBV lytic promoter BZLF1. Furthermore, CIDR1alpha stimulated the production of EBV in peripheral blood mononuclear cells derived from healthy donors and children with BL. Our results suggest that P. falciparum antigens such as CIDR1alpha can directly induce EBV reactivation during malaria infection that may increase the risk of BL development for children living in malaria-endemic areas. To our knowledge, this is the first report to show that a microbial protein can drive a latently infected B cell into EBV replication.     

Regulation of cell growth and death by Epstein-Barr virus

Epstein-Barr virus (EBV) efficiently induces growth of human B cells and prevents cell death. Considerable progress has been made in understanding these processes, the role of EBV in human cancer cells and the relationship of viral gene expression to virus persistence and cancer.     

Epstein-Barr virus enters B cells and epithelial cells by different routes.

Epstein-Barr virus (EBV) infects two cell types, B lymphocytes and epithelial cells. Electron microscopic studies have shown that the virus fuses with the lymphoblastoid cell line Raji but is endocytosed into thin-walled non-clathrin-coated vesicles in normal B cells before fusion takes place. To compare early interactions of EBV with epithelial cells and B cells, a fluorescence dequenching assay of fusion was employed, using virus labeled either with the pH-insensitive probe octadecyl rhodamine B chloride (R18) or with 5(N-octadecanoyl) aminofluorescein (AF), which loses emission intensity at a pH below 7.4. Fusion of virus labeled with R18 could be monitored with B cells, Raji cells, and epithelial cells. Lowering the extracellular pH or pretreatment of cells with ammonium chloride or methylamine had no effect on these measurements. In contrast, fusion of virus labeled with AF could be measured with Raji cells and epithelial cells, but not with normal B cells unless cells were previously treated with ammonium chloride. Fusion of virus with normal B cells was inhibited with chlorpromazine, chloroquine, and sodium azide, but none of these reagents had any effect on fusion with Raji or epithelial cells. These results suggest that entry of EBV into nonpolarized suspensions of epithelial cells occurs by fusion at the cell surface, that EBV may be incapable of fusing with normal B cells unless it has first been endocytosed, and that pH appears to be irrelevant to either event. A combination of the two probes, R18 and AF, may have general use for determining the sites of entry of enveloped viruses that fuse in a pH-independent manner.     

Radiobiological Inactivation of Epstein-Barr Virus

Lymphocyte transforming properties of B95-8 strain Epstein-Barr virus (EBV) are very sensitive to inactivation by either UV or X irradiation. No dose of irradiation increases the transforming capacity of EBV. The X-ray dose needed for inactivation of EBV transformation (dose that results in 37% survival, 60,000 rads) is similar to the dose required for inactivation of plaque formation by herpes simplex virus type 1 (Fischer strain). Although herpes simplex virus is more sensitive than EBV to UV irradiation, this difference is most likely due to differences in the kinetics or mechanisms of repair of UV damage to the two viruses. The results lead to the hypothesis that a large part, or perhaps all, of the EBV genome is in some way needed to initiate transformation. The abilities of EBV to stimulate host cell DNA synthesis, to induce nuclear antigen, and to immortalize are inactivated in parallel. All clones of marmoset cells transformed by irradiated virus produce extracellular transforming virus. These findings suggest that the abilities of the virus to transform and to replicate complete progeny are inactivated together. The amounts of UV and X irradiation that inactivate transformation by B95-8 virus are less than the dose needed to inactivate early antigen induction by the nontransforming P(3)HR-1 strain of EBV. Based on radiobiological inactivation, 10 to 50% of the genome is needed for early antigen induction. Inactivation of early antigen induction is influenced by the cells in which the assay is performed. Inactivation proceeds more rapidly in EBV genome-free cells than in genome carrier Raji or in P(3)HR-1 converted EBV genome-free cells clone B(1). These results indicate that the resident EBV genome participates in the early antigen induction process. Variation in radio-biological killing of B95-8 and P(3)HR-1 EBV is not attributable to variations in the repair capacities of the cells in which the viruses were assayed, since inactivation of HSV was the same in primary lymphocytes and in all lymphoid cell lines tested.     

Identification of an epitope in the major envelope protein of Epstein-Barr virus that mediates viral binding to the B lymphocyte EBV receptor (CR2)

The Epstein-Barr virus gp350/220 envelope protein mediates virus attachment to the EBV/C3dg receptor (CR2) of human B lymphocytes. Synthetic peptides corresponding to two regions in gp350/220, which have a similar amino acid sequence with the complement C3dg protein, were used to identify a receptor binding epitope. A peptide corresponding to the N terminus of gp350/220, EDPGFFNVE, bound to purified CR2 and to CR2 positive but not CR2 negative B and T lymphoblastoid cell lines. Soluble monomeric gp350/220 peptide blocked CR2 binding to immobilized EBV, while multimeric forms of the N-terminal gp350/220 peptide conjugated to albumin efficiently blocked recombinant gp350/220 and C3dg binding to B cells as well as EBV-induced B cell proliferation and transformation. These studies indicate that the N-terminal region of gp350/220 plays a crucial role in mediating the earliest stages of EBV infection of B cells and provides a molecular basis for the restricted host cell EBV tropism.