Nancy Raab-Traub

The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation

The linear virion form of Epstein-Barr virus (EBV) DNA has variable numbers of direct tandem 500 bp repeats at each terminus. The terminal restriction endonuclease fragments and the fused terminal fragments in the intracellular episomal form are heterogeneous in size, and vary by increments of 500 bp. The structure of the termini of EBV in carcinomas of the nasopharynx and the parotid gland was compared with the EBV termini in monoclonal and polyclonal tissues or cell lines. A single band representing the EBV joined termini was detected in each of the carcinomas and in the monoclonal lymphoid proliferations. Polyclonal cell lines contained multiple forms of the joined termini. The detection of a homogeneous episomal population suggests that EBV-associated epithelial malignancies are clonal expansions of a single EBV-infected progenitor cell.

Epstein-Barr virus replication in oropharyngeal epithelial cells.

Despite the well-established tropism of the Epstein-Barr virus (EBV) for human B lymphocytes, the cell type within the oropharynx capable of allowing EBV replication has never been conclusively identified. Using in situ cytohybridization, we demonstrated EBV DNA in oropharyngeal epithelial cells from 10 of 12 patients with infectious mononucleosis. In duplicates of specimens found to contain cell-associated EBV DNA, we detected EBV RNA in two of four samples, using a biotin-labeled EBV DNA probe, thereby confirming the intracellular location of the viral genome. In 20 of 28 throat washings analyzed, cytohybridization results and assays for cell-free infectious virus were in agreement. In seven of the eight remaining specimens, cytohybridization identified intracellular EBV DNA in the absence of detectable extracellular virus. We conclude that the oropharyngeal epithelial cell may be the target cell type that is productively infected in infectious mononucleosis.

Epstein–Barr Virus MicroRNAs Are Evolutionarily Conserved and Differentially Expressed

The pathogenic lymphocryptovirus Epstein–Barr virus (EBV) is shown to express at least 17 distinct microRNAs (miRNAs) in latently infected cells. These are arranged in two clusters: 14 miRNAs are located in the introns of the viral BART gene while three are located adjacent to BHRF1. The BART miRNAs are expressed at high levels in latently infected epithelial cells and at lower, albeit detectable, levels in B cells. In contrast to the tissue-specific expression pattern of the BART miRNAs, the BHRF1 miRNAs are found at high levels in B cells undergoing stage III latency but are essentially undetectable in B cells or epithelial cells undergoing stage I or II latency. Induction of lytic EBV replication was found to enhance the expression of many, but not all, of these viral miRNAs. Rhesus lymphocryptovirus, which is separated from EBV by ≥13 million years of evolution, expresses at least 16 distinct miRNAs, seven of which are closely related to EBV miRNAs. Thus, lymphocryptovirus miRNAs are under positive selection and are likely to play important roles in the viral life cycle. Moreover, the differential regulation of EBV miRNA expression implies distinct roles during infection of different human tissues.

Clonal Proliferations of Cells Infected with Epstein–Barr Virus in Preinvasive Lesions Related to Nasopharyngeal Carcinoma

BACKGROUND The Epstein-Barr virus (EBV) is consistently detected in patients with nasopharyngeal carcinoma. To determine whether EBV infection is an early, initiating event in the development of this malignant tumor, we screened nasopharyngeal-biopsy samples, most of which were archival, for preinvasive lesions, including dysplasia and carcinoma in situ. Preinvasive lesions were found in 11 samples, which were tested for the presence of EBV. METHODS EBV infection was detected with in situ hybridization for EBV-encoded RNAs (EBERs) and by immunohistochemical staining for latent membrane protein 1 (LMP-1). The larger samples were also tested for the EBV genome with the use of Southern blotting. The expression of specific EBV RNAs was determined by the amplification of complementary DNA with the polymerase chain reaction. RESULTS Evidence of EBV infection was detected in all 11 tissue samples with dysplasia or carcinoma in situ. EBERs were identified in all eight samples tested, and LMP-1 was detected in all six of the tested samples. Six of the seven samples tested for the EBV termini contained clonal EBV DNA: Transcription of the latent EBV gene products, EBV nuclear antigen 1, LMP-1, LMP-2A, and the BamHI-A fragment, was detected in most of the samples. Viral proteins characteristic of lytic lesions were not detected. CONCLUSIONS Preinvasive lesions of the nasopharynx are infected with EBV. The EBV DNA is clonal, indicating that the lesions represent a focal cellular growth that arose from a single EBV-infected cell and that EBV infection is an early, possibly initiating event in the development of nasopharyngeal carcinoma. Preinvasive lesions contain EBV RNAs that are characteristic of latent infection but not the viral proteins that are characteristic of lytic infection. The detection of the EBV-transforming gene, LMP-1, in all the neoplastic cells suggests that its expression is essential for preinvasive epithelial proliferations associated with nasopharyngeal carcinoma.

Epstein–Barr virus in the pathogenesis of NPC

Epstein-Barr virus (EBV) is consistently detected in nasopharyngeal carcinoma (NPC) from regions of high and low incidence. EBV DNA within the tumor is homogeneous with regard to the number of terminal repeats. The detection of a single form of viral DNA suggests that the tumors are clonal proliferations of a single cell that was initially infected with EBV. Specific EBV genes are consistently expressed within the NPC tumors and in early, dysplastic lesions. The viral proteins, latent membrane protein 1 and 2, have profound effects on cellular gene expression and cellular growth, resulting in the highly invasive, malignant growth of NPC tumors. In addition to potential genetic changes, the establishment of a latent, transforming infection in epithelial cells is likely to be a major contributing factor to the development of this tumor.

Epidermal growth factor receptor is a cellular receptor for human cytomegalovirus

Human cytomegalovirus (HCMV) is a widespread opportunistic herpesvirus that causes severe and fatal diseases in immune-compromised individuals, including organ transplant recipients and individuals with AIDS. It is also a leading cause of virus-associated birth defects and is associated with atherosclerosis and coronary restenosis. HCMV initiates infection and intracellular signalling by binding to its cognate cellular receptors and by activating several signalling pathways including those mediated by mitogen-activated protein kinase, phosphatidylinositol-3-OH kinase, interferons, and G proteins. But a cellular receptor responsible for viral entry and HCMV-induced signalling has yet to be identified. Here we show that HCMV infects cells by interacting with epidermal growth factor receptor (EGFR) and inducing signalling. Transfecting EGFR-negative cells with an EGFR complementary DNA renders non-susceptible cells susceptible to HCMV. Ligand displacement and crosslinking analyses show that HCMV interacts with EGFR through gB, its principal envelope glycoprotein. gB preferentially binds EGFR and EGFR–ErbB3 oligomeric molecules in Chinese hamster ovary cells transfected with erbB family cDNAs. Taken together, these data indicate that EGFR is a necessary component for HCMV-triggered signalling and viral entry.

Human tumor virus utilizes exosomes for intercellular communication

The Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) is expressed in multiple human malignancies and has potent effects on cell growth. It has been detected in exosomes and shown to inhibit immune function. Exosomes are small secreted cellular vesicles that contain proteins, mRNAs, and microRNAs (miRNAs). When produced by malignant cells, they can promote angiogenesis, cell proliferation, tumor-cell invasion, and immune evasion. In this study, exosomes released from nasopharyngeal carcinoma (NPC) cells harboring latent EBV were shown to contain LMP1, signal transduction molecules, and virus-encoded miRNAs. Exposure to these NPC exosomes activated the ERK and AKT signaling pathways in the recipient cells. Interestingly, NPC exosomes also contained viral miRNAs, several of which were enriched in comparison with their intracellular levels. LMP1 induces expression of the EGF receptor in an EBV-negative epithelial cell line, and exosomes produced by these cells also contain high levels of EGF receptor in exosomes. These findings suggest that the effects of EBV and LMP1 on cellular expression also modulate exosome content and properties. The exosomes may manipulate the tumor microenvironment to influence the growth of neighboring cells through the intercellular transfer of LMP1, signaling molecules, and viral miRNAs.

Epstein-Barr virus LMP2A transforms epithelial cells, inhibits cell differentiation, and activates Akt

ABSTRACT The Epstein-Barr virus LMP2A protein was expressed in a human keratinocyte cell line, HaCaT, and effects on epithelial cell growth were detected in organotypic raft cultures and in vivo in nude mice. Raft cultures derived from LMP2A-expressing cells were hyperproliferative, and epithelial differentiation was inhibited. The LMP2A-expressing HaCaT cells were able to grow anchorage independently and formed colonies in soft agar. HaCaT cells expressing LMP2A were highly tumorigenic and formed aggressive tumors in nude mice. The LMP2A tumors were poorly differentiated and highly proliferative, in contrast to occasional tumors that arose from parental HaCaT cells and vector control cells, which grew slowly and remained highly differentiated. Animals injected with LMP2A-expressing cells developed frequent metastases, which predominantly involved lymphoid organs. Involucrin, a marker of epithelial differentiation, and E-cadherin, involved in the maintenance of intercellular contact, were downregulated in LMP2A tumors. Whereas activation of the mitogen-activated protein kinase pathway was not observed, phosphatidylinositol-3-kinase (PI3-kinase)-dependent activation of the serine-threonine kinase Akt was detected in LMP2A-expressing cells and LMP2A tumors. Inhibition of this pathway blocked growth in soft agar. These data indicate that LMP2A greatly affects cell growth and differentiation pathways in epithelial cells, in part through activation of the PI3-kinase–Akt pathway.

Microvesicles and Viral Infection

ABSTRACT Cells secrete various membrane-enclosed microvesicles from their cell surface (shedding microvesicles) and from internal, endosome-derived membranes (exosomes). Intriguingly, these vesicles have many characteristics in common with enveloped viruses, including biophysical properties, biogenesis, and uptake by cells. Recent discoveries describing the microvesicle-mediated intercellular transfer of functional cellular proteins, RNAs, and mRNAs have revealed additional similarities between viruses and cellular microvesicles. Apparent differences include the complexity of viral entry, temporally regulated viral expression, and self-replication proceeding to infection of new cells. Interestingly, many virally infected cells secrete microvesicles that differ in content from their virion counterparts but may contain various viral proteins and RNAs. For the most part, these particles have not been analyzed for their content or functions during viral infection. However, early studies of microvesicles (L-particles) secreted from herpes simplex virus-infected cells provided the first evidence of microvesicle-mediated intercellular communication. In the case of Epstein-Barr virus, recent evidence suggests that this tumorigenic herpesvirus also utilizes exosomes as a mechanism of cell-to-cell communication through the transfer of signaling competent proteins and functional microRNAs to uninfected cells. This review focuses on aspects of the biology of microvesicles with an emphasis on their potential contributions to viral infection and pathogenesis.

EBV-Encoded Latent Membrane Protein 1 Cooperates with BAFF/BLyS and APRIL to Induce T Cell-Independent Ig Heavy Chain Class Switching

By substituting the H chain C region of IgM with that of IgG, IgA, or IgE, class switching enables Abs to acquire new effector functions that are crucial for the neutralization of invading pathogens. Class switching occurs through class switch DNA recombination (CSR) and usually requires engagement of CD40 on B cells by CD40 ligand on Ag-activated CD4+ T cells. CSR must be tightly regulated because abnormal IgG and IgA production favors the onset of autoimmunity, whereas increased switching to IgE leads to atopy. These inflammatory disorders can be triggered or exacerbated by EBV infection. In this study, we show that EBV induces CD40-independent CSR from Cμ to multiple downstream Cγ, Cα, and Cε genes through latent membrane protein 1 (LMP1), a CD40-like viral protein that signals in a ligand-independent fashion. LMP1-induced CSR is associated with transcriptional activation of germline Cγ, Cα, and Cε genes and triggers the up-regulation of activation-induced cytidine deaminase, a crucial component of the CSR machinery. In addition, LMP1 induces B cells to express B cell-activating factor of the TNF family and a proliferation-inducing ligand, two molecules that mediate B cell survival and T cell-independent Ab production. B cell-activating factor of the TNF family and a proliferation-inducing ligand cooperate with LMP1 to induce Ig class switching because their neutralization by appropriate soluble decoy receptors attenuates CSR in LMP1-expressing B cells. By showing that LMP1 triggers T cell-independent CSR, our findings suggest that EBV could play an important role in the pathogenesis of disorders with aberrant IgG, IgA, and/or IgE production.