Effect on B-Cell Growth In Vitro

Infection of primary B cells with EBV in vitro results in transformation of the cells, which can then proliferate indefinitely. B-cell activation antigens, including CD23, are expressed on the surface of the EBV-transformed B cells. The supernatant of EBV-transformed B cells contains a 35-kilodalton (kDa) protein thought to represent a soluble, cleaved form of CD23. CD23 may be a growth factor for B cells and the soluble form of CD23 may have autocrine growth factor activity.² EBV infection of Burkitt lymphoma cells in vitro results in upregulation of a number of cellular proteins, including CD44, and two G protein-coupled peptide receptors.³

Gene Expression in Transformed Lymphocytes

Six different EBV nuclear proteins, two membrane proteins, and two nontranslated RNAs are known to be expressed in latently infected B lymphocytes that have been growth transformed by EBV in vitro (Table 23-1). The EBV nuclear proteins EBNA-1, EBNA-2, EBNA-LP, EBNA-3A, EBNA-3B, and EBNA-3C comprise the EBV nuclear antigen complex. EBNA-1 binds to the oriP sequence (origin of viral DNA replication) on EBV DNA and allows the virus genome to be maintained as an episome in transformed B cells.⁴ EBNA-1 also transactivates its own expression. EBNA-1 transcripts are initiated from one of four different promoters. The Cp and Wp promoters are used to express EBNA-1 in lymphoblastoid cell lines in vitro; the Qp promoter is used in tissues from Burkitt lymphoma, nasopharyngeal carcinoma, and Hodgkin disease; and the Fp promoter is used during lytic replication.⁵ Transgenic mice expressing EBNA-1 develop B-cell lymphomas.⁶ EBNA-1 inhibits its own protein degradation by proteosomes,⁷ and the reduced processing and presentation of EBNA-1 peptides to major histocompatibility complex (MHC) class I molecules may allow cells expressing this protein to avoid destruction by cytotoxic T cells.

Table 23-1

Selected EBV Genes and Their Cellular Homologs and Activities.

EBNA-2 is required for B-cell transformation by EBV. EBNA-2 transactivates expression of the EBV genes LMP-1⁸ and LMP-2,⁹ and the cellular genes CD23, CD21, c-myc, and c-fgr,^10,11 which encodes a protein tyrosine kinase and is a member of the src gene family. EBNA-2 does not bind DNA directly but interacts with several cellular proteins. EBNA-2 is targeted to the LMP-1, LMP-2, Cp EBNA, and CD23 promoters by the GTGGGAA-binding protein Jκ, and thereby activates these promoters.¹² EBNA-2 is a functional homolog of the Notch receptor, which uses Jκ to regulate gene expression during development.¹³ EBNA-2 also interacts with the DNA-binding protein PU-1 to transactivate the LMP-1 promoter¹⁴ and with AUF to transactivate the EBNA Cp promoter.¹⁵ The transactivation domain of EBNA-2 is essential for B-lymphocyte transformation.¹⁶ This domain interacts with several transcription factors, including TFIIB and the TATA-binding protein-associated factor TAF40.¹⁷ EBNA-2 is a major determinant of the type-specific transforming difference between the two naturally occurring types of EBV.

EBNA-LP enhances the ability of EBNA-2 to transactivate LMP-1 and LMP-2.¹⁸ Although EBNA-LP binds to the retinoblastoma protein, heat shock proteins, and p53 in vitro,¹⁹ the significance of these interactions is uncertain. Deletion of the carboxy terminus of EBNA-LP markedly reduces the ability of the virus to transform B lymphocytes.²⁰

EBNA-3A, EBNA-3B, and EBNA-3C are encoded by three tandem open reading frames. These three proteins are distantly related. The EBNA-3 proteins bind to Jκ preventing it from binding DNA, thereby inhibiting transactivation by EBNA-2.²¹ EBNA-3C is a regulator of transcription that upregulates expression of LMP-1 and CD21. EBNA-3C binds to Nm23-HI, a human metastasis suppressor protein, and inhibits the protein's ability to suppress migration of Burkitt lymphoma cells.²² EBNA-3A and EBNA-3C are essential for B-lymphocyte transformation in vitro, while EBNA-3B is dispensable.²³

Two latent membrane proteins, LMP-1 and LMP-2, are expressed in B cells that have been growth transformed by EBV. Both proteins are present in lipid rafts on the surface of B cells.²⁴ LMP-1 functions as a transforming oncogene; rodent cells expressing the protein form tumors in nude mice.²⁵ Expression of LMP-1 in EBV-negative Burkitt lymphoma cells results in B-cell clumping and increased villous projections. Upregulation of bcl-2, bfl-1, and A20 by LMP-1 in B cells protects the cells from apoptosis.²⁶ Expression of LMP-1 in epithelial cells inhibits differentiation of the cells.²⁷

LMP-1 interacts with several cellular proteins. LMP-1 is a functional homolog of CD40, a member of the tumor necrosis factor receptor (TNFR) family. The carboxy terminus of LMP-1 interacts with the TNFR-associated factors (TRAFs) 1, 2, 3, and 5, TRADD, RIP, and Janus-activated kinase-3 (JAK3) in vitro.^28–30 LMP-1 functions as a constitutively active form of CD40 resulting in activation of NFκB, stress-activated protein kinases, signal transducers and activators of transcription (STATs), adhesion molecules, the B7 costimulatory molecule, c-jun N-terminal kinase, and B-cell proliferation. LMP-1 can functionally substitute for CD40 in transgenic mice.³¹ LMP-1 upregulates expression of intracellular adhesion molecules, Fas, CD40, and matrix metalloproteinase-9³² in B cells and epidermal growth factor in epithelial cells.³³

LMP-1 is essential for transformation of B lymphocytes by EBV.³⁴ Expression of LMP-1 in the skin of transgenic mice induces epithelial hyperplasia with increased expression of keratin 6.³⁵ Expression of LMP-1 in lymphocytes of transgenic mice results in the development of B-cell lymphomas; these tumors contain elevated levels of the antiapoptotic proteins Bcl-2 and A20.³⁶ Analysis of EBV-containing human lymphomas shows that LMP-1 localizes with TRAF-1, TRAF-3, and that activated NFκB is present, suggesting that these activities may have an important role in oncogenesis.³⁷

LMP-2 is dispensable for transformation of B cells,³⁸ but induces a transforming phenotype in epithelial cells.³⁹ Two forms of LMP-2 (LMP-2A and LMP-2B), which differ only in their first exon, are expressed in latently infected B cells. LMP-2 prevents lytic reactivation of EBV-infected primary B cells and calcium mobilization in response to activation of the B-cell receptor complex by cross-linking of surface immunoglobulin. LMP-2 is tyrosine phosphorylated and associates with the src family and syk protein-tyrosine kinases that are coupled to the B-cell receptor complex.⁴⁰ Binding of LMP-2 to these proteins results in their constitutive phosphorylation, which inhibits their ability to mediate signaling for virus reactivation.^40,41 B cells from transgenic mice expressing LMP-2 survive even without normal B-cell receptor signaling activity.⁴²

The two EBV-encoded RNAs, EBER-1 and EBER-2, are the most abundant EBV RNAs in latently infected B cells; however, they are not required for latent or lytic EBV infection or cell transformation in vitro.⁴³ The EBERs upregulate expression of bcl-2 and IL-10.⁴⁴ These RNAs are not polyadenylated and interact with the nuclear antigens La and EAP, ribosome protein L22, the double-stranded RNA-activated protein kinase, and interferon-inducible oligoadenylate synthetase.^45,46

Epstein-Barr Virus Genes Expressed During Productive Infection

Infection of epithelial cells with EBV results in productive infection, with replication of virus and lysis of infected cells. Immediate-early genes encode regulators of virus gene expression, including the BZLF1 and BRLF1 proteins, which act as switches to initiate lytic infection. The BZLF1 protein inhibits the expression of the interferon-γ receptor and the activity of interferon-γ.⁴⁷ Early genes encode proteins that are involved in viral DNA synthesis, such as the viral DNA polymerase and thymidine kinase. Late genes encode structural proteins of the virus, including the viral capsid antigen and the major envelope glycoprotein gp350.

Three viral genes expressed during productive infection are functional homologs of cellular genes and are important for the survival of EBV-infected B cells. The EBV BCRF-1 protein is highly homologous to interleukin-10 and has interleukin-10 activity.⁴⁸ Recombinant BCRF-1 and BCRF-1 secreted from EBV-infected cells inhibit interferon-γ release from activated human peripheral blood mononuclear cells and secretion of interleukin-12 from macrophages. Because interferon-γ is important for the activity of T cells, expression of BCRF-1 during lytic infection may inhibit the ability of cytotoxic T cells to destroy EBV-infected cells. BCRF-1 protein also acts as a B-cell growth factor.

The EBV BARF-1 protein acts as a soluble receptor for colony stimulating factor 1.⁴⁹ BARF-1 inhibits interferon-α secretion by human monocytes. Because interferon-α inhibits outgrowth of EBV-infected B cells in vitro, BARF-1 may act in concert with BCRF-1 to inhibit interferon and promote increased survival of EBV-infected cells.

The EBV BHRF-1 protein is homologous to Bcl-2, a cellular protein that is activated in follicular lymphomas and protects cells from apoptosis. BHRF-1 colocalizes with bcl-2 in the cytoplasm and protects Burkitt lymphoma cells from apoptosis.⁵⁰ EBV BALF1 is also homologous to bcl-2 and inhibits apoptosis.⁵¹

Animal Models

Several animal models have been used to study EBV oncogenesis. First, EBV-infected cell lines produce B-cell tumors when inoculated intracerebrally into nude mice. Second, inoculation of peripheral blood leukocytes from EBV-seropositive humans into mice with severe combined immunodeficiency results in development of human B-cell lymphomas in the animals. Inoculation of these mice with peripheral blood leukocytes from EBV-seronegative humans results in engraftment of a human immune system, and if these latter mice are subsequently injected with cell-free EBV, the animals develop immunoblastic lymphomas. These B cell tumors express the full complement of EBNA and LMP genes characteristic of latently infected, growth transformed cell lines.⁵² In the third model, cotton-top tamarins inoculated with a large dose of cell-free EBV develop multifocal large cell lymphomas over the ensuing few weeks. These tumors express EBNA-1, EBNA-2, EBNA-LP, and LMP-1⁵³ and are monoclonal or oligoclonal in origin.

Clinical Aspects

EBV infection is usually spread by saliva. The virus infects B cells directly, or oropharyngeal epithelial cells and then spreads to subepithelial B cells.⁵⁴ During primary infection, up to a few percent of the peripheral blood B lymphocytes are infected with EBV and have the capacity to proliferate indefinitely in vitro. Natural killer (NK) cells, suppressor T cells, and HLA- and EBNA- or LMP-restricted cytotoxic T cells control the latently infected B lymphocytes. T- and B-cell interactions release lymphokines and cytokines, giving rise to many of the clinical manifestations of acute infectious mononucleosis. After recovery, the fraction of B cells latently infected with EBV in the peripheral blood remains at 1 in 10⁵ to 1 in 10⁶. These lymphocytes are the primary site of EBV persistence and a source of virus for persistent infection of epithelial surfaces.

The B-cell tumors that occur early after EBV infection are usually lymphoproliferative processes in which latent virus infection in B cells is the principal cause of proliferation. Oral hairy leukoplakia may be the epithelial counterpart. In contrast, Burkitt lymphoma and nasopharyngeal carcinoma occur long after primary EBV infection; although etiologically related to EBV, viral gene expression may not be important to the growth of the clinically evident malignant cells.

Lymphoproliferative Disease

EBV is associated with B-cell lymphoproliferative disease in patients with congenital immunodeficiency. X-linked lymphoproliferative syndrome is an inherited immunodeficiency of males who have apparently normal cellular and humoral immune responses before infection with EBV. With EBV infection, most of the patients die of a fatal lymphoproliferative disorder or fulminant hepatitis, but some survive with hypogammaglobulinemia. EBV nuclear antigens have been detected in lesions from these patients. The gene mutated in X-linked lymphoproliferative syndrome has been identified as SAP,⁵⁵ also termed SH2D1A or DSHP. SAP encodes an SH2-containing protein that interacts with the signaling lymphocyte-activation molecule (SLAM) on B and T cells, and with 2B4 on NK and T cells.

EBV is also associated with fatal infectious mononucleosis in persons with no known underlying genetic predisposition or in patients with congenital immunodeficiencies, such as severe combined immunodeficiency. EBV lymphoproliferative disease occurs in patients who are immunosuppressed as a result of transplantation or acquired immunodeficiency syndrome (AIDS).^56,57 Risk factors for development of lymphoproliferative disease include EBV-seronegativity prior to transplant, receipt of T-cell depleted bone marrow or antilymphocyte antibody, and concurrent cytomegalovirus disease. Lymphoproliferative lesions are most commonly seen in the lymph nodes, liver, lungs, kidney, bone marrow, or small intestine. Tumors in transplant patients are usually classified as lymphomas or immunoblastic sarcomas; some patients have hyperplastic lesions. The proliferating lymphocytes in these tumors generally do not have chromosomal translocations.

AIDs-related lymphomas may be systemic (nodal or extranodal) lymphomas, primary central nervous system lymphomas, or primary effusion lymphomas. Primary effusion lymphomas often contain EBV in addition to HHV-8. While most B-cell tumors in transplant recipients and central nervous system lymphomas in AIDS patients contain EBV, about 50% of other lymphomas in AIDS patients contain EBV. Tumors in patients with AIDS are usually either immunoblastic lymphomas or Burkitt lymphomas; most of the latter have c-myc translocations.

Tissues from transplant recipients or AIDS patients with EBV lymphoproliferative disease show expression of EBERs, EBNA-1, EBNA-2, and LMP-1 (Table 23-2). The EBV viral load in the peripheral blood has been used to predict development of disease and to follow patients after therapy. The expression of these EBV genes, which are targets for cytotoxic T cells, has important implications for therapy. Infusion of EBV-specific cytotoxic T cells or nonirradiated donor leukocytes has been effective in some cases for treatment of EBV lymphoproliferative disease.^58,59

Table 23-2

Diseases Associated with EBV Latent Gene Expression.