Forms Of Epstein-Barr Virus Latency Research Articles

Characterization of Epstein-Barr virus-encoded small RNA gene variations in virus associated lymphomas in Northern China.

Epstein-Barr virus (EBV)-encoded small RNAs (EBER1 and EBER2) are highly expressed in all forms of EBV latency in EBV-associated malignancies. EBER gene variations and their association with EBV-associated disease still remain poorly characterized. To investigate the patterns of EBER gene variations and their roles in tumorigenesis, EBER gene sequences were analyzed by nested-PCR and DNA sequencing in 101 lymphomas from Northern China, a non-nasopharyngeal carcinoma (NPC) endemic area. In addition, EBV type 1 and type 2 classifications were made by using nested-PCR assays across type-specific regions in the EBNA2 gene. EB-6m was the dominant subtype (95.0%, 96/101) in lymphoma. The distribution of the EBER subtypes in the four lymphoma groups was not significantly different (p>0.05), neither was that of the EBNA2 type (p>0.05). Compared with previous data in the same area, the distribution of EBER subtypes in lymphoma was similar to that in EBV-associated gastric carcinoma (EBVaGC) and throat washing (TW) from healthy donors (p>0.05), but was significantly different from that of NPC. The EBNA2 type distribution between lymphoma and the other three groups was significantly different (p<0.05). The proportion of type 1 and type 2 dual infections was higher in lymphoma than that in GC, NPC and TW. The mutation 7123nt A→T was identified in 11 of 101 (10.9%, 11/101) lymphomas, significantly more than that in EBV-associated gastric carcinomas (EBVaGC) (0%, 0/50) and throat washings (TWs) from healthy donors (3.3%, 3/92) (p<0.05). These findings indicate that EBER subtypes may not be associated with pathogenesis of lymphoma, but that a point mutation at position 7123nt (A→T) provides a new area for further exploration. Furthermore it is necessary to investigate the role of EBNA2-subtype mixed infections in the establishment of lymphoma.

Epstein–Barr virus microRNAs reduce immune surveillance by virus-specific CD8+ T cells

Infection with Epstein-Barr virus (EBV) affects most humans worldwide and persists life-long in the presence of robust virus-specific T-cell responses. In both immunocompromised and some immunocompetent people, EBV causes several cancers and lymphoproliferative diseases. EBV transforms B cells in vitro and encodes at least 44 microRNAs (miRNAs), most of which are expressed in EBV-transformed B cells, but their functions are largely unknown. Recently, we showed that EBV miRNAs inhibit CD4+ T-cell responses to infected B cells by targeting IL-12, MHC class II, and lysosomal proteases. Here we investigated whether EBV miRNAs also counteract surveillance by CD8+ T cells. We have found that EBV miRNAs strongly inhibit recognition and killing of infected B cells by EBV-specific CD8+ T cells through multiple mechanisms. EBV miRNAs directly target the peptide transporter subunit TAP2 and reduce levels of the TAP1 subunit, MHC class I molecules, and EBNA1, a protein expressed in most forms of EBV latency and a target of EBV-specific CD8+ T cells. Moreover, miRNA-mediated down-regulation of the cytokine IL-12 decreases the recognition of infected cells by EBV-specific CD8+ T cells. Thus, EBV miRNAs use multiple, distinct pathways, allowing the virus to evade surveillance not only by CD4+ but also by antiviral CD8+ T cells.

Growth-Promoting Properties of Epstein-Barr Virus EBER-1 RNA Correlate with Ribosomal Protein L22 Binding

The Epstein-Barr virus (EBV)-encoded RNAs, EBER-1 and EBER-2, are highly abundant noncoding nuclear RNAs expressed during all forms of EBV latency. The EBERs have been shown to impart significant tumorigenic potential upon EBV-negative Burkitt lymphoma (BL) cells and to contribute to the growth potential of other B-cell lymphoma-, gastric carcinoma-, and nasopharyngeal carcinoma-derived cell lines. However, the mechanisms underlying this EBER-dependent enhancement of cell growth potential remain to be elucidated. Here we focused on the known interaction between EBER-1 and the cellular ribosomal protein L22 and the consequences of this interaction with respect to the growth-promoting properties of the EBERs. L22, a component of 60S ribosomal subunits, binds three sites on EBER-1, and a substantial fraction of available L22 is relocalized from nucleoli to the nucleoplasm in EBV-infected cells. To investigate the hypothesis that EBER-1-mediated relocalization of L22 in EBV-infected cells is critical for EBER-dependent functions, we investigated whether EBER-1 expression is necessary and sufficient for nucleoplasmic retention of L22. Following demonstration of this, we utilized RNA-protein binding assays and fluorescence localization studies to demonstrate that mutation of the L22 binding sites on EBER-1 prevents L22 binding and inhibits EBER-1-dependent L22 relocalization. Finally, the in vivo consequence of preventing L22 relocalization in EBER-expressing cells was examined in soft agar colony formation assays. We demonstrate that BL cells expressing mutated EBER-1 RNAs rendered incapable of binding L22 have significantly reduced capacity to enhance cell growth potential relative to BL cells expressing wild-type EBERs.

Epstein-Barr virus and Hodgkin's disease: transcriptional analysis of virus latency in the malignant cells.

Epstein-Barr virus (EBV) is associated with a number of different human tumors and appears to play different pathogenetic roles in each case. Thus, immunoblastic B cell lymphomas of the immunosuppressed display the full pattern of EBV latent gene expression (expressing Epstein-Barr nuclear antigen [EBNA]1, 2, 3A, 3B, 3C, and -LP, and latent membrane protein [LMP]1, 2A, and 2B), just as do B lymphoblastoid cell lines transformed by the virus in vitro. In contrast, those EBV-associated tumors with a more complex, multistep pathogenesis show more restricted patterns of viral gene expression, limited in Burkitt's lymphoma to EBNA1 only and in nasopharyngeal carcinoma (NPC) to EBNA1 and LMP1, 2A, and 2B. Recent evidence has implicated EBV in the pathogenesis of another lymphoid tumor, Hodgkin's disease (HD), where the malignant Hodgkin's and Reed-Sternberg (HRS) cells are EBV genome positive in up to 50% of cases. Here we extend preliminary results on viral gene expression in HRS cells by adopting polymerase chain reaction-based and in situ hybridization assays capable of detecting specific EBV latent transcripts diagnostic of the different possible forms of EBV latency. We show that the transcriptional program of the virus in HRS cells is similar to that seen in NPC in several respects: (a) selective expression of EBNA1 mRNA from the BamHI F promoter; (b) downregulation of the BamHI C and W promoters and their associated EBNA mRNAs; (c) expression of LMP1 and, in most cases, LMP2A and 2B transcripts; and (d) expression of the "rightward-running" BamHI A transcripts once thought to be unique to NPC. This form of latency, consistently detected in EBV-positive HD irrespective of histological subtype, implies an active role for the virus in the pathogenesis of HD and also suggests that the tumor may remain sensitive to at least certain facets of the EBV-induced cytotoxic T cell response.

Three transcriptionally distinct forms of epstein-barr virus latency in somatic cell hybrids: Cell phenotype dependence of virus promoter usage

Phenotypically distinct human B cell lines display two transcriptionally distinct forms of Epstein-Barr virus (EBV) latency. Latency I (Lat I) in group I Burkitt's lymphoma (BL) cell lines is characterized by selective expression of the virus-coded nuclear antigen EBNA 1 from a uniquely spliced mRNA driven by the Fp promoter. Latency III (Lat III) in group III BL and EBV-transformed lymphoblastoid cell lines (LCLs) is characterized by expression of EBNAs 1, 2,3a, 3b, 3c, and -LP from mRNAs driven by the Cp or Wp promoter and of the latent membrane proteins (LMPs 1, 2A, and 2B) from mRNAs driven by the LMP promoters. Here we have altered the group I BL and LCL phenotypes by cell hybridization and screened for attendant changes in EBV latency by PCR analysis of viral mRNAs and immunoblotting of viral proteins. Fusion of group I BL cells with LCLs activated the BL virus genome from a Lat I to Lat III pattern of gene expression. Fusion of LCLs with nonlymphoid lines repressed virus gene expression from Lat III either to Lat I or to another form of latency (Lat II) hitherto not seen in vitro and characterized by selective expression of the Fp-driven EBNA 1 mRNA and of the LMP 1, 2A, and 2B transcripts. There are therefore three forms of EBV latency which can be interconverted by altering cellular phenotype and thereby virus promoter usage.

Epstein-Barr virus infection in oral hairy leukoplakia: virus replication in the absence of a detectable latent phase.

Epstein-Barr virus (EBV) infects both B lymphocytes and oropharyngeal epithelium, and it has been argued that the true reservoir of virus persistence in vivo is the self-renewing basal epithelial compartment. The identification of oral hairy leukoplakia (HL) of AIDS patients as a clinically apparent focus of EBV replication in lingual epithelium therefore provides a means of studying the EBV-epithelial cell interaction in situ. Replicative EBV DNA and productive cycle antigens are restricted to the upper, more differentiated epithelial layers in HL, and here we have applied highly sensitive in situ hybridization and immunohistological methods to examine the lower basal/suprabasal layers for evidence of latent EBV infection. We could not detect EBV DNA in these layers using an in situ DNA hybridization protocol which, on reference B cell lines, detected 1 viral genome/cell. Likewise, using sensitive in situ RNA hybridization for both the small non-polyadenylated EBER RNAs (abundant transcripts seen in all known forms of EBV latency) and the latent membrane protein (LMP) mRNA (the most abundant viral mRNA in B lymphoblastoid cell lines), the basal/suprabasal cells in HL were consistently negative; immunohistological staining with specific monoclonal antibodies also gave no evidence of latently infected LMP-positive cells. When the biopsy extracts were analysed by immunoblotting with selected human antisera, in addition to abundant productive cycle antigens, a band of constant size (66K) was observed which also reacted with immunopurified antibodies monospecific for one of the latency-associated nuclear antigens, EBNA 1; the cellular origin of this EBNA 1 could not be ascertained, but it is possible that in HL the protein is expressed during the productive cycle. The absence of demonstrable EBV latency in the basal/suprabasal cells of HL suggests that this is purely a virus replicative lesion which is sustained by continual re-infection of the maturing epithelium, not by the maturation of latently infected cells from the basal compartment.