EBV gene expression in transformed lymphocytes

Infection of primary B cells with EBV in vitro results in transformation of the cells, which can then proliferate indefinitely. Eight EBV proteins and several nontranslated RNAs are expressed in latently infected B lymphocytes that have been growth transformed by EBV in vitro (Table 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 lymphoma; and the Fp promoter is used during lytic replication.⁵ EBNA-1 inhibits its own protein degradation by proteasomes⁶ and limits its own translation,⁷ both of which may reduce its presentation to CD8+ cytotoxic T cells; however, EBNA-1 remains a target for CD4+ cells.^8–11 EBNA-1 inhibits apoptosis induced by expression of p53.¹²

EBNA-2 transactivates expression of EBV LMP1¹³ and LMP2,¹⁴ and the cellular genes CD21, CD23, c-myc, and c-fgr.^{15, 16} EBNA-2 is targeted to the LMP1, LMP2, Cp EBNA, and CD23 promoters by the GTGGGAA-binding protein Jκ, and thereby activates these promoters.¹⁷ EBNA-2 is a functional homolog of the Notch receptor, which uses Jκ to regulate gene expression during development.¹⁸ EBNA-2 also interacts with the DNA-binding protein PU-1 to transactivate the LMP1 promoter¹⁹ and with AUF to transactivate the EBNA Cp promoter.²⁰ The transactivation domain of EBNA-2 is essential for B-lymphocyte transformation.²¹ This domain interacts with transcription factors TFIIB and the TATA-binding protein-associated factor TAF40.²² EBNA-2 inhibits apoptosis mediated by Nur77.²³

EBNA-LP interacts with and enhances the ability of EBNA-2 to transactivate LMP1 and LMP2.²⁴ Although EBNA-LP binds to Rb and p53 in vitro,²⁵ 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.²⁶

EBNA-3A, EBNA-3B, and EBNA-3C are distantly related to each other. The EBNA-3 proteins bind to Jκ preventing it from binding DNA, thereby inhibiting transactivation by EBNA-2.²⁷ EBNA-3C upregulates expression of LMP1 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.²⁸ EBNA-3C degrades Rb and enhances kinase activity by disrupting p27.^{29, 30} EBNA-3A and EBNA-3C are essential for B-lymphocyte transformation in vitro, while EBNA-3B is dispensable.^{31, 32}

LMP1 functions as a transforming oncogene in nude mice.³³ Expression of LMP1 in EBV-negative Burkitt lymphoma cells results in B-cell clumping and increased villous projections. Upregulation of bcl-2, bfl-1, and A20, and inhibition of Bax, by LMP1 in B cells protects the cells from apoptosis.^{34, 35} Expression of LMP1 in epithelial cells inhibits differentiation of the cells.³⁶

LMP1 is a functional homolog of CD40, a member of the tumor necrosis factor receptor (TNFR) family.³⁷ The carboxy terminus of LMP1 interacts with the TNFR-associated factors (TRAFs) 1, 2, 3, and 5, TRADD, RIP, and JAK3 in vitro.³⁸ LMP1 functions as a constitutively active form of CD40 resulting in activation of NF-κB, stress-activated protein kinases, STATs, adhesion molecules, the B7 co-stimulatory molecule, JNK, and B-cell proliferation.³⁹ LMP1 upregulates expression of intracellular adhesion molecules, Fas, CD40, and MMP-9⁴⁰ in B cells and EGF in epithelial cells.⁴¹ LMP1 inhibits phosphorylation of Tyk2 resulting in inhibition of interferon (IFN)-α signaling.⁴² LMP1 is essential for transformation of B lymphocytes by EBV.⁴³ Analysis of EBV-containing human lymphomas shows that LMP1 localizes with TRAF-1, TRAF-3, and that activated NF-κB is present.⁴⁴

LMP2 is dispensable for transformation of B cells,⁴⁵ but induces a transforming phenotype in epithelial cells and promotes their motility.^{46, 47} LMP2 prevents lytic reactivation of EBV-infected primary B cells in response to activation of the B-cell receptor complex by cross-linking of surface immunoglobulin. LMP2 associates with the src family and syk protein-tyrosine kinases that are coupled to the B-cell receptor complex.⁴⁸ Binding of LMP2 to these proteins results in their constitutive phosphorylation, which inhibits their ability to mediate signaling for virus reactivation.^{48, 49} B cells from transgenic mice expressing LMP2 survive even without normal B-cell receptor signaling activity.⁵⁰ LMP2 activates β-catenin and Ras/PI3K/Akt signaling pathways in epithelial cells resulting in transformation of the cells.^{51, 52} LMP2 also activates mTOR and increases c-myc expression.⁵³

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, but may contribute to B-cell transformation.^{54, 55} The EBERs upregulate expression of bcl-2 and IL-10⁵⁶ and interact with the double-stranded RNA-activated protein kinase, and IFN-inducible oligoadenylate synthetase.^{57, 58}

EBV encodes at least 44 miRNAs from the BART and BHRF1 regions of the genome; both BART and BHRF1 microRNAs downregulate cellular genes and inhibit apoptosis and enhance B-cell proliferation.⁵⁹ EBV also encodes cellular RNAs that enhance transformation.⁶⁰ Both EBV microRNAs and LMP1 are secreted from infected cells in exosomes, which may enhance tumorigenicty.^{61, 62}

EBV 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 TNF-α signaling⁶³ and helps the virus evade T-cell responses.⁶⁴ BZLF1 protein also inhibits the expression of the IFN-γ receptor⁶⁵ and inhibits the function of p53.⁶⁶ Early genes encode proteins involved in viral DNA synthesis, while late genes encode structural proteins.

Three viral genes expressed during productive infection are functional homologs of cellular genes and are important for the survival of EBV-infected B cells.^{67, 68} The EBV BCRF-1 protein is homologous to IL-10 and has IL-10 activity.⁶⁹ BCRF-1 is a B-cell growth factor and inhibits IFN-γ release from activated peripheral blood mononuclear cells and secretion of IL-12 from macrophages.

The EBV BARF-1 protein acts as a soluble receptor for CSF-1.⁷⁰ BARF-1 inhibits IFN-α secretion by human monocytes. The EBV BNLF2a protein interacts with the TAP complex to block antigen-specific CD8 T-cell recognition.⁷¹ The EBV BHRF1 protein is homologous to bcl-2 and protects cells from apoptosis.⁷² EBV BALF1 is also homologous to bcl-2 and antagonizes the antiapoptotic effect of BHRF1.⁷³

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.⁷⁴ During primary infection, up to a few percent of peripheral blood B lymphocytes are infected with EBV and have the capacity to proliferate indefinitely in vitro. Natural killer (NK) cells, CD4 T cells, and HLA- and EBNA- or LMP-restricted cytotoxic T cells control the latently infected B lymphocytes.⁸ 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.

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. In contrast, nasopharyngeal carcinoma occurs long after primary EBV infection and viral gene expression is less important to the growth of the malignant cells.

Viral proteins

Several KSHV proteins are important for transformation, establishing latency, and modulating the immune response to the virus.^108–110 KSHV encodes a large number of cellular homologs (Table 3) that have been grouped into different classes, depending on when they are expressed in primary effusion lymphoma cell lines.¹¹¹ Expression of the KSHV K1 gene in rodent fibroblasts results in transformation of the cells.¹¹² The K1 protein induces tyrosine phosphorylation in cells¹¹³ and activates the Akt signaling pathway.¹¹⁴ The K1 protein inhibits apoptosis¹¹⁵ and results in constitutive calcium-dependent signal activation in B cells.¹¹⁶

Expression class I = latent gene, expressed in uninduced primary effusion lymphoma cells, not induced by phorbol ester; class II = expressed in uninduced cells, induced by TPA; class III = lytic gene, expressed only after induction by TPA (includes many structural proteins and DNA replication enzymes). FLIP = FLICE inhibitory protein; GPCR = G-protein-coupled receptor; IRF = interferon regulatory factor; ITAM = immunoreceptor tyrosine-based activation motif; MHC = major histocompatibility complex; MIP = macrophage inflammatory protein; NK = natural killer; Rb = retinoblastoma protein; TRAFs = TNFR-associated factors.

The KSHV K2 gene encodes an IL-6 homolog (vIL-6). IL-6 is a B-cell growth factor and acts as an autocrine growth factor for lymphoid tumors resulting in proliferation.¹¹⁷ vIL-6 prevents death of IL-6-dependent B9 cells in vitro,¹¹⁸ promotes hematopoiesis, and induces VEGF to promote angiogenesis.¹¹⁹ The K3 [modulator of immune recognition 1 (MIR1)] and K5 (MIR2) proteins induce rapid endocytosis of MHC (major histocompatibility complex) class I molecules and IFN-γ receptor 1 from the surface of cells by ubiquitination of these proteins.^120–122 The K5 protein also downregulates ICAM-1 and B7.2, which results in inhibition of NK-cell-mediated cytotoxicity,¹²³ and removes CD31 from the surface of endothelial cells.¹²⁴

The KSHV K4, K4.1, and K6 genes encode three chemokines: the viral macrophage inflammatory proteins (MIP)-II, -III, and -I, respectively. vMIP-I inhibits replication of HIV strains dependent on CCR5.¹¹⁸ vMIP-I and vMIP-III are chemokine receptor agonists for CCR8¹²⁵ and CCR4,¹²⁶ respectively, while vMIP-II is a broad-spectrum chemokine receptor antagonist. vMIP-II is a chemoattractant for eosinophils,¹²⁷ binds to both CC and CXC chemokines, and blocks calcium mobilization induced by chemokines.¹²⁸

The KSHV K9, K11.1, and K10.5 proteins are referred to as viral IFN regulatory factor (vIRF)-1, -2, and -3, respectively. Each of these proteins inhibits virus-mediated activation of the IFN-α promoter.¹²⁹ vIRF1 inhibits MHC-1 transcription and surface expression.¹³⁰ vIRF1 and vIRF3 inhibit p53-mediated apoptosis^{131, 132} and transform NIH 3T3 cells.¹³³ vIRF3 (also called LANA2) protects cells from p53-induced apoptosis.^{134, 135} KSHV ORF45 blocks the activity of IRF7 and inhibits the activation of IFN-α and -β.¹³⁶

The KSHV K12 locus encodes several proteins termed kaposins. Kaposin A induces transformation of cells.¹³⁷ Kaposin B increases expression of cytokines by activating MAP-kinase-associated protein kinase 2 and inhibiting the degradation of cytokine mRNA.^{138, 139} The KSHV K14 protein, a homolog of the cellular OX2 protein, stimulates monocytes to produce proinflammatory cytokines such as TNF-α, IL-1β, and IL-6.¹⁴⁰ KSHV ORF4 protein inhibits the complement system.¹⁴¹ KSHV ORF16 encodes a homolog of bcl-2 and inhibits bax-induced apoptosis.¹⁴² The ORF71 gene encodes a homolog of cellular FLIP that blocks apoptosis. ORF71 binds to Atg3 and protects cells from autophagy. KSHV ORF71 activates NF-κB, promotes tumor growth, and is required for survival of KSHV-infected lymphoma cells.^143–147 KSHV ORF72 encodes a cyclin D homolog that binds to and activates cdk6 and phosphorylates p27, and stimulates cell-cycle progression in normally quiescent fibroblasts.^{148, 149} The viral cyclin protein phosphorylates and thereby inactivates Rb.¹⁵⁰

KSHV ORF73 encodes LANA1 that localizes with viral DNA episomes and tethers them to chromosomes during cell division.¹⁵¹ KSHV LANA1 is required for persistence of the episome in dividing cells and transactivates its own promoter. In addition, LANA1 inhibits the activity of both p53 and Rb,^{152, 153} upregulates expression and stabilizes β-catenin,¹⁵⁴ upregulates and activates survivin,¹⁵⁵ induces nuclear accumulation of HIF,¹⁵⁶ and activates c-myc.¹⁵⁷ Expression of LANA in transgenic mice results in development of lymphomas.¹⁵⁸ LANA inhibits TGF-β signaling.¹⁵⁹ KSHV ORF74 encodes a G-protein-coupled receptor that is homologous to the cellular IL-8 receptor; unlike the latter protein, however, the KSHV receptor is constitutively active and induces cellular proliferation.¹⁶⁰ ORF74 protein induces angiogenesis,¹⁶¹ activates the Akt signaling pathway,¹⁶² and induces proliferation and vascular permeability of endothelial cells.^163–165 ORF74 activates NF-κB and JNK, and upregulates IL-1, IL-8, TNF-α, and FGF, and inhibits viral lytic gene expression.¹⁶⁶

KSHV K15 encodes the latency-associated membrane protein (LAMP) and interacts with TRAFs 1, 2, and 3.¹⁶⁷ K15 suppresses tyrosine phosphorylation and intracellular calcium mobilization, inhibiting B-cell receptor signaling.¹⁶⁸ KSHV encodes several microRNAs located between K12 and ORF71 or within K12 that are expressed during latency and detected in patient plasma.¹⁶⁹ These microRNAs are important for NF-κB activation and blocking cell-cycle arrest of latently infected cells.^{109, 170} KSHV microRNAs also target cellular genes.¹⁷¹

Clinical aspects

Seroprevalence rates for KSHV vary from <5% in normal blood donors in the United States or United Kingdom to 30–35% in HIV-positive homosexual men.¹⁷² Antibody to KSHV is more common in African and Mediterranean populations. At least 85% of patients with Kaposi sarcoma have antibodies to KSHV.¹⁷³ The prevalence of Kaposi sarcoma is lower in women than in men, and HIV-seropositive women have a much lower incidence of antibody to KSHV than do seropositive men. KSHV seropositivity in HIV-positive homosexual men is predictive of subsequent development of Kaposi sarcoma.¹⁷⁴ Levels of KSHV DNA are higher in patients with active Kaposi sarcoma or multicentric Castleman disease, than in those in remission, and are also elevated in patients with primary effusion lymphoma.¹⁷⁵ The virus is not thought to be pathogenic in most healthy individuals and can persist in a latent phase for life; however, in immunocompromised persons, it is strongly associated with Kaposi sarcoma. Thus, although infection with KSHV appears to be required for development of Kaposi sarcoma, it is probably not sufficient by itself and other cofactors, such as HIV and impaired cellular immunity, are important. KSHV is thought to be sexually transmitted in homosexual men¹⁷² and has been associated with sexual transmission and intravenous drug use in women.¹⁷⁶ In endemic populations (e.g., Africa), KSHV may be transmitted vertically from mother to child and between siblings. KSHV has been transmitted by renal allografts.^{177, 178}

Multicentric Castleman disease

KSHV has also been detected in biopsies from some patients with multicentric Castleman disease, especially in the variant known as the plasma cell type.^{180, 181, 192–194} This disease is usually polyclonal and presents as generalized lymphadenopathy, fever, and hypergammaglobulinemia. Symptoms are thought to be due to increased levels of IL-6 and vIL-6.¹⁹⁵ KSHV is detected more frequently in biopsies from HIV-positive patients than in biopsies from those patients without HIV. KSHV is present in the immunoblastic B cells of the mantle zone of the lesions. Zidovudine plus valganciclovir showed activity in some patients with KSHV-associated Castleman disease.¹⁹⁶

Summary

• Two herpesviruses, Epstein–Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV), are associated with human tumors.
  
• EBV is associated with Burkitt lymphoma, Hodgkin and non-Hodgkin lymphoma, post-transplant lymphoproliferative disease, T-cell lymphoma, nasopharyngeal carcinoma, and certain types of gastric carcinoma.
  
• EBV transformed B cells and EBV post-transplant lymphoproliferative lesions express EBNA-1, -2, -3s, and -LP and LMP1 and LMP2 and RNAs including EBERs and BARTs in culture. Burkitt lymphomas express only EBNA-1, EBERs, and BARTs. Hodgkin lymphomas and nasopharyngeal carcinomas express EBNA-1, LMP1, LMP2, EBERs, and BARTs.
  
• EBV EBNA-1 is important for maintaining the viral episome during replication of the cells. EBNA-2 transactivates several virus and cellular promoters and is a functional homolog of the Notch receptor. EBNA-3’s regulate the activity of EBNA-2 and also transactivate viral genes. LMP1 is a functional homolog of CD40 and binds to TNFR-associated factors to upregulate NF-κB, STATs, JNK, and stress-activated protein kinases.
  
• KSHV is associated with primary effusion lymphoma, Kaposi sarcoma, and Castleman disease.
  
• Each of the three KSHV-associated tumors express LANA (ORF73) and v-FLIP (ORF71). Other KSHV proteins, including Kaposin (K12), v-cyclin (ORF72), a G-protein-coupled receptor (GPCR, ORF74), v-IRF3 (K10.5), v-IRF1 (K9), and v-IL-6 (K2) are expressed in some, both not all virus-associated malignancies.
  
• LANA maintains the viral episome during replication and inhibits the activity of Rb and p53. KSHV v-FLIP is a homolog of cellular FLIP and inhibits apoptosis and activates NF-κB. Kaposin increases the activity of cytokines, v-cyclin inhibits Rb and helps virus-infected cells to progress through the cell cycle, and the viral GPCR contributes to proliferation of KSHV-infected cells. v-IRF1 and v-IRF3 inhibit the activity of interferon and p53, while v-IL-6 functions as a B-cell growth factor.

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