Epithelial cell retention of transcriptionally active, P3HR-1-derived heterogeneous Epstein-Barr virus DNA with concurrent loss of parental virus.

Deleted, rearranged, heterogeneous (het) Epstein-Barr virus (EBV) DNA with the distinctive capability of disrupting EBV latency has been reported in biopsy samples of EBV-associated tumors whose onset in immunocompetent hosts is characteristically preceded by an antibody response indicative of EBV reactivation. Using the EBV P3HR-1 strain, we have reproduced in long-term culture of SVK epithelial cells an unusual pattern of infection previously observed in a subset of tumor biopsy samples: the persistence of het DNA in the absence of the parental helper virus. Fluorescence in situ hybridization (FISH) of infected cell subclones indicated the retention of het DNA in an integrated form. Incorporation of an intact het DNA molecule was confirmed by PCR, using primers that framed junctions of the four rearranged EBV DNA segments comprising P3HR-1-derived het DNA. Structural analysis of EBV terminal repeats revealed a banding pattern consistent with the integration of het DNA as a concatemer. Linkage of concatemeric monomers was defined at a nucleotide level, and that junctional sequence was detected in cell-free P3HR-1 virion DNA, confirming that subgenomic het DNA was packaged into infectious particles in a concatemeric configuration. Stable integration into cells having lost the standard viral genome allowed the unambiguous designation of het DNA as the source for viral gene products potentially encoded by both. Continuous expression of the latency-to-lytic switch protein Zta and detection of the BALF4 gene product gB, known to expand the target cell range of standard virus when incorporated at augmented levels into infectious progeny, add to a presumption of het DNA-enhanced pathogenesis in diseases of EBV reactivation.

Augmented latent membrane protein 1 expression from Epstein-Barr virus episomes with minimal terminal repeats.

The major oncogene of the Epstein-Barr virus (EBV), latent membrane protein 1 (LMP1), can be expressed from either of two promoters, ED-L1 or L1-TR, producing mRNAs of 2.8 kb or 3.5 kb, respectively. L1-TR, active in nasopharyngeal carcinoma and Hodgkin's lymphoma, is located within the first of a highly variable reiteration of terminal repeat (TR) sequences that are joined by random recombination upon circularization of the linear genome at entry into cells. To determine whether the resultant TR number affects LMP1 promoter activity, we isolated single-cell clones bearing episomes of distinct TR numbers (6TR to 12TR) from epithelial cells newly infected with EBV. LMP1 mRNA levels correlated directly with the quantity of LMP1 protein expressed but varied inversely to TR number. Unexpectedly, the 3.5-kb transcript predominated only at lower TR reiterations. Diminished L1-TR activity in the context of a higher TR count was confirmed with a green fluorescent protein (GFP) reporter construct driven by L1-TR. Various levels of LMP1, expressed from virus isogenic in all but TR number, produced divergent morphological and growth phenotypes in each cell clone. Abundant LMP1 in 6TR cells yielded a relatively cytostatic state compared to the proliferative one produced by intermediate and smaller amounts in 8TR and 12TR clones. These findings suggest that the diversification of TR number, inherent in a round of EBV reactivation and reinfection, may itself be a component of the oncogenic process. The replicative burst preceding onset of many EBV-linked cancers may increase the likelihood that LMP1 levels compatible with clonal outgrowth are achieved in a subset of infected cells.

Points of recombination in Epstein-Barr virus (EBV) strain P3HR-1-derived heterogeneous DNA as indexes to EBV DNA recombinogenic events in vivo.

Deletions and rearrangements in the genome of Epstein-Barr virus (EBV) strain P3HR-1 generate subgenomic infectious particles that, unlike defective interfering particles in other viral systems, enhance rather than restrict EBV replication in vitro. Reports of comparable heterogeneous (het) DNA in EBV-linked human diseases, based on detection of an abnormal juxtaposition of EBV DNA fragments BamHI W and BamHI Z that disrupts viral latency, prompted us to determine at the nucleotide level all remaining recombination joints formed by the four constituent segments of P3HR-1-derived het DNA. Guided by endonuclease restriction maps, we chose PCR primer pairs that approximated and framed junctions creating the unique BamHI M/B1 and E/S fusion fragments. Sequencing of PCR products revealed points of recombination that lacked regions of extensive homology between constituent fragments. Identical recombination junctions were detected by PCR in EBV-positive salivary samples from human immunodeficiency virus-infected donors, although the W/Z rearrangement that induces EBV reactivation was frequently found in the absence of the other two. In vitro infection of lymphoid cells similarly indicated that not all three het DNA rearrangements need to reside on a composite molecule. These results connote a precision in the recombination process that dictates both composition and regulation of gene segments altered by genomic rearrangement. Moreover, the apparent frequency of het DNA at sites of EBV replication in vivo is consistent with a likely contribution to the pathogenesis of EBV reactivation.

A replication-defective gammaherpesvirus efficiently establishes long-term latency in macrophages but not in B cells in vivo.

Murine gammaherpesvirus 68 (gammaHV68 or MHV68) is genetically related to the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), providing a useful system for in vivo studies of the virus-host relationship. To begin to address fundamental questions about the mechanisms of the establishment of gammaherpesvirus latency, we previously generated a replication-defective gammaHV68 lacking the expression of the single-stranded DNA binding protein encoded by orf6. In work presented here, we demonstrate that this mutant virus established a long-term infection in vivo that was molecularly identical to wild-type virus latency. Thus, despite the absence of an acute phase of lytic replication, the mutant virus established a chronic infection in which the viral genome (i) was maintained as an episome and (ii) expressed latency-associated, but not lytic replication-associated, genes. Macrophages purified from mice infected with the replication-defective virus harbored viral genome at a frequency that was nearly identical to that of wild-type gammaHV68; however, the frequency of B cells harboring viral genome was greatly reduced in the absence of lytic replication. Thus, this replication-defective gammaherpesvirus efficiently established in vivo infection in macrophages that was molecularly indistinguishable from wild-type virus latency. These data point to a critical role for lytic replication or reactivation in the establishment or maintenance of latent infection in B cells.

Modulation of the cell growth regulator mTOR by Epstein-Barr virus-encoded LMP2A.

Control of translation initiation is one means by which cells regulate growth and proliferation, with components of the protein-synthesizing machinery having oncogenic potential. Expression of latency protein LMP2A by the human tumor virus Epstein-Barr virus (EBV) activates phosphatidylinositol 3-kinase/Akt located upstream of an essential mediator of growth signals, mTOR (mammalian target of rapamycin). We show that mTOR is activated by expression of LMP2A in carcinoma cells, leading to wortmannin- and rapamycin-sensitive inhibition of the negative regulator of translation, eukaryotic initiation factor 4E-binding protein 1, and increased c-Myc protein translation. Intervention by this DNA tumor virus in cellular translational controls is likely to be an integral component of EBV tumorigenesis.

Reactive oxygen signaling and MAPK activation distinguish Epstein-Barr Virus (EBV)-positive versus EBV-negative Burkitt's lymphoma.

Burkitt's lymphoma (BL) is an aggressive B cell neoplasm, which is one of the most common neoplasms of childhood. It is highly widespread in East Africa, where it appears in endemic form associated with Epstein-Barr virus (EBV) infection, and around the world in a sporadic form in which EBV infection is much less common. In addition to being the first human neoplasm to be associated with EBV, BL is associated with a characteristic translocation, in which the Ig promoter is translocated to constitutively activate the c-myc oncogene. Although many BLs respond well to chemotherapy, a significant fraction fails to respond to therapy, leading to death. In this article, we demonstrate that EBV-positive BL expresses high levels of activated mitogen-activated protein kinase and reactive oxygen species (ROS), and that ROS directly regulate NF-kappaB activation. EBV-negative BLs exhibit activation of phosphoinositol 3-kinase, but do not have elevated levels of ROS. Elevated reactive oxygen may play a role in diverse forms of viral carcinogenesis in humans, including cancers caused by EBV, hepatitis B, C, and human T cell lymphotropic virus. Our findings imply that inhibition of ROS may be useful in the treatment of EBV-induced neoplasia.

Cytotoxic T lymphocyte therapy for Epstein-Barr virus+ Hodgkin's disease.

Epstein Barr virus (EBV)+ Hodgkin's disease (HD) expresses clearly identified tumor antigens derived from the virus and could, in principle, be a target for adoptive immunotherapy with viral antigen-specific T cells. However, like most tumor-associated antigens in immunocompetent hosts, these potential targets are only weakly immunogenic, consisting primarily of the latent membrane protein (LMP)1 and LMP2 antigens. Moreover, Hodgkin tumors possess a range of tumor evasion strategies. Therefore, the likely value of immunotherapy with EBV-specific cytotoxic effector cells has been questioned. We have now used a combination of gene marking, tetramer, and functional analyses to track the fate and assess the activity of EBV cytotoxic T lymphocyte (CTL) lines administered to 14 patients treated for relapsed EBV+ HD. Gene marking studies showed that infused effector cells could further expand by several logs in vivo, contribute to the memory pool (persisting up to 12 mo), and traffic to tumor sites. Tetramer and functional analyses showed that T cells reactive with the tumor-associated antigen LMP2 were present in the infused lines, expanded in peripheral blood after infusion, and also entered tumor. Viral load decreased, demonstrating the biologic activity of the infused CTLs. Clinically, EBV CTLs were well tolerated, could control type B symptoms (fever, night sweats, and weight loss), and had antitumor activity. After CTL infusion, five patients were in complete remission at up to 40 mo, two of whom had clearly measurable tumor at the time of treatment. One additional patient had a partial response, and five had stable disease. The performance and fate of these human tumor antigen-specific T cells in vivo suggests that they might be of value for the treatment of EBV+ Hodgkin lymphoma.

Epstein-Barr virus-encoded LMP2A regulates viral and cellular gene expression by modulation of the NF-kappaB transcription factor pathway.

Epstein-Barr virus (EBV)-associated malignancies display distinct patterns of virus latent gene expression that reflect the complex interplay between the virus and its host cell. In the EBV-associated epithelial tumor nasopharyngeal carcinoma (NPC), the virus-encoded latent membrane protein LMP2A is consistently expressed whereas the oncogenic LMP1 protein appears to be restricted to only a proportion of tumors. In an attempt to understand the contribution of LMP2A to the pathogenesis of NPC, we established carcinoma cell lines stably infected in vitro with either a wild-type recombinant EBV (rEBV) or a mutant rEBV in which LMP2A is deleted (rEBV-2A). An NPC-like pattern of EBV gene expression including LMP2A but not LMP1 was consistently observed in carcinoma cells infected with rEBV. However, carcinoma cells infected with rEBV-2A expressed high levels of LMP1 from the signal transducer and activator of transcription (STAT)-regulated L1-TR promoter. Consistent with this effect, basal STAT activity was reduced in rEBV-infected carcinoma cells, and this repression was relieved in the absence of LMP2A. This modulation of STAT activity correlated with the ability of LMP2A to inhibit the autocrine secretion of IL-6 from carcinoma cell lines. Exogenous IL-6 was able to induce expression of LMP1 by means of STAT3 activation both in rEBV-infected carcinoma cell lines and in the EBV-positive C666-1 NPC cell line. The LMP2A-mediated suppression of IL-6 was a consequence of NF-kappaB inhibition. These data reveal that LMP2A modulates two key transcription factor pathways in carcinoma cells and suggest that this finding may be important in the pathogenesis of EBV-associated tumors.

Peripheral blood lymphocytes express recombination-activating genes 1 and 2 during Epstein-Barr virus-induced infectious mononucleosis.

Implicit in the persistence of Epstein-Barr virus (EBV) in B lymphocytes is the successful circumvention of ongoing cell selection for competence of B cell receptors (BCRs). Because the EBV infection of B cells in vitro induces enzymatic machinery that is responsible for secondary immunoglobulin gene rearrangement, we examined the expression of the recombination-activating genes (RAGs) in peripheral blood mononuclear cells (PBMCs) from 26 patients with infectious mononucleosis (IM). RAG1 and/or RAG2 RNA was detected in PBMCs from 42% of patients with IM but not from healthy control subjects. EBV may usurp the cellular mechanism that diversifies the BCR, to guarantee a level of survival signaling sufficient for its own persistence.

Length of Epstein-Barr virus termini as a determinant of epithelial cell clonal emergence.

Reiterated terminal sequences of Epstein-Barr virus (EBV) DNA are numerically heterogeneous among infectious virions, providing a viral measure of clonality in infected cells. After in vitro infection, carcinoma cells bearing EBV episomes with fewer terminal repeats (TRs) proliferated faster. In single-cell clones, TR number varied inversely to the quantity of latent membrane protein 2A (LMP2A) transcripts whose unspliced precursors cross joined TRs. Thus, EBV clonality may reflect selection for a TR number that optimizes LMP2A-enhanced tumor progression, with infection occurring after epithelial cell transformation.

A defective, rearranged Epstein-Barr virus genome in EBER-negative and EBER-positive Hodgkin's disease.

A ubiquitous herpesvirus that establishes life-long infection, the Epstein-Barr virus (EBV) has yielded little insight into how a single agent in general accord with its host can produce diverse pathologies ranging from oral hairy leukoplakia to nasopharyngeal carcinoma, from infectious mononucleosis to Hodgkin's disease (HD) and Burkitt's lymphoma. Its pathogenesis is further confounded by the less than total association of virus with histologically similar tumors. In other viral systems, defective (interfering) viral genomes are known to modulate outcome of infection, with either ameliorating or intensifying effects on disease processes initiated by prototype strains. To ascertain whether defective EBV genomes are present in HD, we examined paraffin-embedded tissue from 56 HD cases whose EBV status was first determined by cytohybridization for nonpolyadenylated EBV RNAs (EBERs). Using both standard polymerase chain reaction (PCR) and PCR in situ hybridization, we successfully amplified sequences that span abnormally juxtaposed BamHI W and Z fragments characteristic of defective heterogeneous (het) EBV DNA from 10 of 32 (31%) EBER-positive tumors. Of 24 EBER-negative HD, 8 yielded PCR products indicating presence of het EBV DNA. Two of these contained defective EBV in the apparent absence of the prototype virus. Of the 42 tumors analyzed for defective EBV by both PCR techniques, there was concordance of results in 38 (90%). Detection of defective EBV genomes with the potential to disrupt viral gene regulation suggests one mechanism for pathogenic diversity that may also account for loss of prototypic EBV from individual tumor cells.