Interferon regulatory factor 7 regulates expression of Epstein-Barr virus latent membrane protein 1: a regulatory circuit

We have shown previously that interferon regulatory factor 7 (IRF7), a multifunctional protein intimately involved in latent Epstein-Barr virus (EBV) infection, is induced as well as activated by EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein. Since the LMP1 promoter (LMP1p) contains an interferon-stimulated response element (ISRE), we hypothesized that IRF7 might be able to regulate LMP1 expression and thus participate in a regulatory circuit between these two genes. In this study, IRF7 was shown first to activate LMP1p in transient transfection assays. Compared with EBV nuclear antigen 2 (EBNA2), the most potent viral transactivator of LMP1p, IRF7 has a lesser effect (approximately 10% that of EBNA2) on induction of LMP1p. Study with IRF7 deletion mutants showed that IRF7 functional domains have similar effects on both the beta interferon (IFN-beta) and LMP1 promoters in BJAB and 293 cells, and study with IRF7 phosphomimetic mutants showed that IRF7 phosphorylation may be involved in the activation of these two promoters. Further, the ISRE in LMP1p responds to IRF7 induction and IRF7 binds to this element. In the EBV-positive cell line P3HR1, which lacks the complete EBNA2 and EBV-encoded leader protein genes and hence expresses low-level LMP1, IRF7 alone can notably increase the endogenous LMP1 mRNA and protein levels. These results indicate that LMP1 is regulated by this host cell gene in addition to the viral factor, EBNA2, and may help to explain how LMP1 is expressed in type II latency in the absence of EBNA2. Moreover, IRF7 can regulate a viral gene in addition to a host cellular gene such as the IFN-beta gene. Together with the previous data that LMP1 can induce IRF7 expression and facilitate IRF7 phosphorylation and nuclear translocation, these results suggest a positive regulatory circuit between IRF7 and LMP1.     

A retrospective analysis of EBV‐DNA status with the prognosis of lymphoma

Epstein–Barr virus (EBV) infection is proved to be associated with clinicopathology of lymphoma. However, little is known about the relationship between EBV‐DNA status after treatment and prognosis. In this study, real‐time polymerase chain reaction (PCR) was used for quantitative detection of EBV‐DNA load in peripheral blood of all 26,527 patients with lymphoma, and the clinical characteristics and prognosis of 202 patients were retrospectively analysed, including 100 patients with positive EBV‐DNA and 102 randomly selected patients with negative EBV‐DNA. We found that the average rate of EBV‐DNA positivity in lymphomas was 0.376%, and EBV‐DNA‐positive patients presented higher risk with elevated lactate dehydrogenase (LDH) and β2‐MG level, B symptoms, secondary hemophagocytic syndrome and lower objective response rate compared to EBV‐DNA‐negative patients. Multivariate analysis revealed EBV‐DNA‐positive patients had inferior progression‐free survival (PFS) and overall survival (OS) and EBV‐DNA level before treatment was related to PFS but not OS of T/NK cell lymphoma. In T/NK cell lymphoma, EBV‐DNA converting negative after treatment was correlated with better PFS but not OS, and second‐line therapy could induce more EBV‐DNA‐negative conversion compared to CHOP‐based therapy. In all, EBV‐DNA positivity before treatment can be a biomarker representing the tumour burden and an independent prognostic factor. EBV‐DNA‐negative conversion after treatment is a good prognostic factor for T/NK cell lymphomas.     

Latent membrane protein 2B regulates susceptibility to induction of lytic Epstein-Barr virus infection

The B-lymphotropic Epstein-Barr virus (EBV) encodes two isoforms of latent membrane protein 2 (LMP2), LMP2A and LMP2B, which are expressed during latency in B cells. The function of LMP2B is largely unknown, whereas LMP2A blocks B-cell receptor (BCR) signaling transduction and induction of lytic EBV infection, thereby promoting B-cell survival. Transfection experiments on LMP2B in EBV-negative B cells and the silencing of LMP2B in EBV-harboring Burkitt's lymphoma-derived Akata cells suggest that LMP2B interferes with the function of LMP2A, but the role of LMP2B in the presence of functional EBV has not been established. Here, LMP2B, LMP2A, or both were overexpressed in EBV-harboring Akata cells to study the function of LMP2B. The overexpression of LMP2B increased the magnitude of EBV switching from its latent to its lytic form upon BCR cross-linking, as indicated by a more-enhanced upregulation and expression of EBV lytic genes and significantly increased production of transforming EBV compared to Akata vector control cells or LMP2A-overexpressing cells. Moreover, LMP2B lowered the degree of BCR cross-linking required to induce lytic EBV infection. Finally, LMP2B colocalized with LMP2A as demonstrated by immunoprecipitation and immunofluorescence and restored calcium mobilization upon BCR cross-linking, a signaling process inhibited by LMP2A. Thus, our findings suggest that LMP2B negatively regulates the function of LMP2A in preventing the switch from latent to lytic EBV replication.     

Role of latent membrane protein 2 isoforms in Epstein-Barr virus latency

The oncogenic Epstein-Barr virus (EBV) infects the majority of the human population without doing harm and establishes a latent infection in the memory B-cell compartment. To accomplish this, EBV hijacks B-cell differentiation pathways and uses its own viral genes to interfere with B-cell signalling to achieve life-long persistence. EBV latent membrane protein 2A (LMP2A) provides a surrogate B-cell receptor signal essential for cell survival and is believed to have a crucial role in the maintenance of latency by blocking B-cell activation which would otherwise lead to lytic EBV infection. These two functions demand tight control of LMP2A activity and expression levels. Based on recent insights in the function of LMP2B, an isoform of LMP2A, we propose a model for how LMP2B modulates the activity of LMP2A contributing to maintenance of EBV latency.     

Immune activation suppresses initiation of lytic Epstein-Barr virus infection

Primary infection with Epstein-Barr virus (EBV) is asymptomatic in children with immature immune systems but may manifest as infectious mononucleosis, a vigorous immune activation, in adolescents or adults with mature immune systems. Infectious mononucleosis and chronic immune activation are linked to increased risk for EBV-associated lymphoma. Here we show that EBV initiates progressive lytic infection by expression of BZLF-1 and the late lytic genes gp85 and gp350/220 in cord blood mononuclear cells (CBMC) but not in peripheral blood mononuclear cells (PBMC) from EBV-naive adults after EBV infection ex vivo. Lower levels of proinflammatory cytokines in CBMC, used to model a state of minimal immune activation and immature immunity, than in PBMC were associated with lytic EBV infection. Triggering the innate immunity specifically via Toll-like receptor-9 of B cells substantially suppressed BZLF-1 mRNA expression in acute EBV infection ex vivo and in anti-IgG-stimulated chronically latently EBV-infected Akata Burkitt lymphoma cells. This was mediated in part by IL-12 and IFN-gamma. These results identify immune activation as critical factor for the suppression of initiation of lytic EBV infection. We hypothesize that immune activation contributes to EBV-associated lymphomagenesis by suppressing lytic EBV and in turn promotes latent EBV with transformation potential.     

Three pathways of Epstein-Barr virus gene activation from EBNA1-positive latency in B lymphocytes.

Previous studies on Epstein-Barr virus (EBV)-positive B-cell lines have identified two distinct forms of virus latency. Lymphoblastoid cell lines generated by virus-induced transformation of normal B cells in vitro, express the full spectrum of six EBNAs and three latent membrane proteins (LMP1, LMP2A, and LMP2B); furthermore, these lines often contain a small fraction of cells spontaneously entering the lytic cycle. In contrast, Burkitt's lymphoma-derived cell lines retaining the tumor biopsy cell phenotype express only one of the latent proteins, the nuclear antigen EBNA1; such cells do not enter the lytic cycle spontaneously but may be induced to do so by treatment with such agents as tetradecanoyl phorbol acetate and anti-immunoglobulin. The present study set out to determine whether activation of full virus latent-gene expression was a necessary accompaniment to induction of the lytic cycle in Burkitt's lymphoma lines. Detailed analysis of Burkitt's lymphoma lines responding to anti-immunoglobulin treatment revealed three response pathways of EBV gene activation from EBNA1-positive latency. A first, rapid response pathway involves direct entry of cells into the lytic cycle without broadening of the pattern of latent gene expression; thereafter, the three "latent" LMPs are expressed as early lytic cycle antigens. A second, delayed response pathway in another cell subpopulation involves the activation of full latent gene expression and conversion to a lymphoblastoidlike cell phenotype. A third response pathway in yet another subpopulation involves the selective activation of LMPs, with no induction of the lytic cycle and with EBNA expression still restricted to EBNA1; this type of latent infection in B lymphocytes has hitherto not been described. Interestingly, the EBNA1+ LMP+ cells displayed some but not all of the phenotypic changes normally induced by LMP1 expression in a B-cell environment. These studies highlight the existence of four different types of EBV infection in B cells, including three distinct forms of latency, which we now term latency I, latency II, and latency III.     

Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.     

Epstein-Barr virus transforming protein LMP1 plays a critical role in virus production

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), which is critical for EBV-induced B-cell transformation, is also abundantly expressed during the lytic cycle of viral replication. However, the biological significance of this strong LMP1 induction remains unknown. We engineered a bacterial artificial chromosome clone containing the entire genome of Akata strain EBV to specifically disrupt the LMP1 gene. Akata cell clones harboring the episomes of LMP1-deleted EBV were established, and the effect of LMP1 loss on virus production was investigated. We found that the degree of viral DNA amplification and the expression levels of viral late gene products were unaffected by LMP1 loss, demonstrating that the LMP1-deleted EBV entered the lytic replication cycle as efficiently as the wild-type counterpart. This was confirmed by our electron microscopic observation that nucleocapsid formation inside nuclei occurred even in the absence of LMP1. By contrast, loss of LMP1 severely impaired virus release into culture supernatants, resulting in poor infection efficiency. The expression of truncated LMP1 in Akata cells harboring LMP1-deleted EBV rescued the virus release into the culture supernatant and the infectivity, and full-length LMP1 partially rescued the infectivity. These results indicate that inducible expression of LMP1 during the viral lytic cycle plays a critical role in virus production.     

EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

Nasopharyngeal carcinoma (NPC) is an Epstein‐Barr virus (EBV)‐associated epithelial malignancy. The high expression of BART‐miRNAs (miR‐BARTs) during latent EBV infection in NPC strongly supports their pathological importance in cancer progression. Recently, we found that several BART‐miRNAs work co‐operatively to modulate the DNA damage response (DDR) by reducing Ataxia‐telangiectasia‐mutated (ATM) activity. In this study, we further investigated the role of miR‐BARTs on DDR. The immunohistochemical study showed that the DNA repair gene, BRCA1, is consistently down‐regulated in primary NPCs. Using computer prediction programs and a series of reporter assays, we subsequently identified the negative regulatory role of BART2‐3p, BART12, BART17‐5p and BART19‐3p in BRCA1 expression. The ectopic expression of these four miR‐BARTs suppressed endogenous BRCA1 expression in EBV‐negative epithelial cell lines, whereas BRCA1 expression was enhanced by repressing endogenous miR‐BARTs activities in C666‐1 cells. More importantly, suppressing BRCA1 expression in nasopharyngeal epithelial cell lines using miR‐BART17‐5p and miR‐BART19‐3p mimics reduced the DNA repair capability and increased the cell sensitivity to the DNA‐damaging chemotherapeutic drugs, cisplatin and doxorubicin. Our findings suggest that miR‐BARTs play a novel role in DDR and may facilitate the development of effective NPC therapies.     

Epigenetic and transcriptional changes which follow Epstein-Barr virus infection of germinal center B cells and their relevance to the pathogenesis of Hodgkin's lymphoma

Although Epstein-Barr virus (EBV) usually establishes an asymptomatic lifelong infection, it is also implicated in the development of germinal center (GC) B-cell-derived malignancies, including Hodgkin's lymphoma (HL). Following primary infection, EBV remains latent in the memory B-cell population, where host-driven methylation of viral DNA contributes to the repression of viral gene expression. However, it is still unclear how EBV harnesses the cell's methylation machinery in B cells, how this contributes to viral persistence, and what impact this has on the methylation of cellular genes. We show that EBV infection of GC B cells is followed by upregulation of the DNA methyltransferase DNMT3A and downregulation of DNMT3B and DNMT1. We show that the EBV latent membrane protein 1 (LMP1) oncogene downregulates DNMT1 and that DNMT3A binds to the viral promoter Wp. Genome-wide promoter arrays performed with these cells showed that EBV-associated methylation changes in cellular genes were not randomly distributed across the genome but clustered at chromosomal locations, consistent with an instructive pattern of methylation, and were in part determined by promoter CpG content. Both DNMT3B and DNMT1 were downregulated and DNMT3A was upregulated in HL cell lines, recapitulating the pattern of expression observed following EBV infection of GC B cells. We also found, by using gene expression profiling, that genes differentially expressed following EBV infection of GC B cells were significantly enriched for those reported to be differentially expressed in HL. These observations suggest that EBV-infected GC B cells are a useful model for studying virus-associated changes contributing to the pathogenesis of HL.     

The lytic cycle of Epstein-Barr virus is associated with decreased expression of cell surface major histocompatibility complex class I and class II molecules

Human herpesviruses utilize an impressive range of strategies to evade the immune system during their lytic replicative cycle, including reducing the expression of cell surface major histocompatibility complex (MHC) and immunostimulatory molecules required for recognition and lysis by virus-specific cytotoxic T cells. Study of possible immune evasion strategies by Epstein-Barr virus (EBV) in lytically infected cells has been hampered by the lack of an appropriate permissive culture model. Using two-color immunofluorescence staining of cell surface antigens and EBV-encoded lytic cycle antigens, we examined EBV-transformed B-cell lines in which a small subpopulation of cells had spontaneously entered the lytic cycle. Cells in the lytic cycle showed a four- to fivefold decrease in cell surface expression of MHC class I molecules relative to that in latently infected cells. Expression of MHC class II molecules, CD40, and CD54 was reduced by 40 to 50% on cells in the lytic cycle, while no decrease was observed in cell surface expression of CD19, CD80, and CD86. Downregulation of MHC class I expression was found to be an early-lytic-cycle event, since it was observed when progress through late lytic cycle was blocked by treatment with acyclovir. The immediate-early transactivator of the EBV lytic cycle, BZLF1, did not directly affect expression of MHC class I molecules. However, BZLF1 completely inhibited the upregulation of MHC class I expression mediated by the EBV cell-transforming protein, LMP1. This novel function of BZLF1 elucidates the paradox of how MHC class I expression can be downregulated when LMP1, which upregulates MHC class I expression in latent infection, remains expressed in the lytic cycle.     

Hairy leukoplakia: an unusual combination of transforming and permissive Epstein-Barr virus infections

Human herpesviruses are characterized by distinct states of infection. Typically in permissive herpesvirus infection, abundant virus production results in cell lysis. In latent transforming Epstein-Barr virus (EBV) infection, viral proteins that induce cell growth are expressed. The immunodeficiency-associated hairy leukoplakia (HLP) lesion is the only pathologic manifestation of permissive EBV infection; however, within HLP, viral proteins characteristic of latent infection have also been detected. In this study, we further analyzed expression of EBV latent genes and investigated their contribution to the unique histologic phenotype of HLP. Coexpression of lytic and transforming viral proteins was detected simultaneously within individual HLP keratinocytes. LMP1 has now been shown to be uniformly expressed in the affected tissue, and it is associated and colocalizes with tumor necrosis factor receptor-associated factor (TRAF) signaling molecules. Effects induced by activated TRAF signaling that were detected in HLP included activation of NF-kappaB and c-Jun terminal kinase 1 (JNK1) and upregulated expression of epidermal growth factor receptor (EGFR), CD40, A20, and TRAFs. This study identifies a novel state of EBV infection with concurrent expression of replicative and transforming proteins. It is probable that both replicative and latent proteins contribute to HLP development and induce many of the histologic features of HLP, such as acanthosis and hyperproliferation. In contrast to other permissive herpesvirus infections, expression of EBV transforming proteins within the permissively infected HLP tissue enables epithelial cell survival and may enhance viral replication.