EB virus-encoded RNAs are recognized by RIG-I and activate signaling to induce type I IFN

Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) are nonpolyadenylated, untranslated RNAs, exist most abundantly in latently EBV-infected cells, and are expected to show secondary structures with many short stem-loops. Retinoic acid-inducible gene I (RIG-I) is a cytosolic protein that detects viral double-stranded RNA (dsRNA) inside the cell and initiates signaling pathways leading to the induction of protective cellular genes, including type I interferons (IFNs). We investigated whether EBERs were recognized by RIG-I as dsRNA. Transfection of RIG-I plasmid induced IFNs and IFN-stimulated genes (ISGs) in EBV-positive Burkitt's lymphoma (BL) cells, but not in their EBV-negative counterparts or EBER-knockout EBV-infected BL cells. Transfection of EBER plasmid or in vitro-synthesized EBERs induced expression of type I IFNs and ISGs in RIG-I-expressing, EBV-negative BL cells, but not in RIG-I-minus counterparts. EBERs activated RIG-I's substrates, NF-kappaB and IFN regulatory factor 3, which were necessary for type I IFN activation. It was also shown that EBERs co-precipitated with RIG-I. These results indicate that EBERs are recognized by RIG-I and activate signaling to induce type I IFN in EBV-infected cells.     

Expression of interferon and interferon-induced genes in salmonids in response to virus infection, interferon-inducing compounds and vaccination

Interferons (IFNs) involved in innate immunity against viruses have recently been cloned from Atlantic salmon and rainbow trout. Moreover, several IFN-stimulated genes (ISGs) have been cloned from salmonids although only Mx has been shown to possess antiviral properties. Much less in known about how viruses induce IFNs in salmonids, but synthetic ligands for some of the main mammalian viral sensors also induce IFNs and ISGs in salmonids. Analysis of the promoters of the salmon IFN-alpha1 and IFN-alpha2 genes shows that activation is dependent on both NFkappaB and IRFs similar to human IFN-beta. Furthermore, several IFN-stimulated genes (ISGs) have been cloned from salmonids although only Mx has been shown to possess antiviral properties. The synthetic compounds poly I:C, imidazoquinolines and CpG oligonucleotides induce IFNs and ISGs in salmonids, probably through the same pathways as in mammals. Salmonid viruses show potent ability to stimulate expression of IFN and ISGs in vivo. Differences between viruses in the ability to stimulate host gene expression are often more evident in cell culture, but more work is needed to pinpoint how salmonid viruses antagonize the IFN system of their host. Finally, existing data suggest that IFNs play a role in the early non-specific protection observed after vaccination of salmonids with rhabdoviral DNA vaccines and conventional polyvalent vaccines.     

Epstein-Barr virus RNA confers resistance to interferon-alpha-induced apoptosis in Burkitt's lymphoma

We investigated whether Epstein--Barr virus (EBV) infection could counteract the antitumor effect of interferon (IFN)-alpha. EBV-negative subclones isolated from EBV-positive Burkitt's lymphoma (BL) cell lines Akata, Daudi and Mutu were found to fall into apoptosis after IFN-alpha treatment. On the other hand, EBV-positive counterparts exhibited striking resistance against IFN-alpha-induced apoptosis. Transfection of an individual EBV latent gene into EBV-negative BL cells revealed that EBV-encoded poly(A)(-) RNAs (EBERs) were responsible for IFN resistance. EBERs bound double-stranded (ds) RNA-activated protein kinase (PKR), a key mediator of the antiviral effect of IFN-alpha, and inhibited its phosphorylation. Transfection of dominant-negative PKR, which was catalytically inactive and could block phosphorylation of endogenous PKR, made EBV-negative BL cells resistant to IFN-alpha-induced apoptosis. Furthermore, EBERs did not bind mutant PKR, which was catalytically active but lacked dsRNA-binding activity, nor did they inhibit its phosphorylation. These results indicate that EBERs confer resistance to IFN-alpha-induced apoptosis via binding to PKR and inhibition of its phosphorylation. This is the first report that the virus counteracts IFN-induced apoptosis in virus-associated tumors.     

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.     

Epstein-Barr virus nuclear antigen 2 transactivates the long terminal repeat of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1)-infected subjects show a high incidence of Epstein-Barr virus (EBV) infection. This suggests that EBV may function as a cofactor that affects HIV-1 activation and may play a major role in the progression of AIDS. To test this hypothesis, we generated two EBV-negative human B-cell lines that stably express the EBNA2 gene of EBV. These EBNA2-positive cell lines were transiently transfected with plasmids that carry either the wild type or deletion mutants of the HIV-1 long terminal repeat (LTR) fused to the chloramphenicol acetyltransferase (CAT) gene. There was a consistently higher HIV-1 LTR activation in EBNA2-expressing cells than in control cells, which suggested that EBNA2 proteins could activate the HIV-1 promoter, possibly by inducing nuclear factors binding to HIV-1 cis-regulatory sequences. To test this possibility, we used CAT-based plasmids carrying deletions of the NF-kappa B (pNFA-CAT), Sp1 (pSpA-CAT), or TAR (pTAR-CAT) region of the HIV-1 LTR and retardation assays in which nuclear proteins from EBNA2-expressing cells were challenged with oligonucleotides encompassing the NF-kappa B or Sp1 region of the HIV-1 LTR. We found that both the NF-kappa B and the Sp1 sites of the HIV-1 LTR are necessary for EBNA2 transactivation and that increased expression resulted from the induction of NF-kappa B-like factors. Moreover, experiments with the TAR-deleted pTAR-CAT and with the tat-expressing pAR-TAT plasmids indicated that endogenous Tat-like proteins could participate in EBNA2-mediated activation of the HIV-1 LTR and that EBNA2 proteins can synergize with the viral tat transactivator. Transfection experiments with plasmids expressing the EBNA1, EBNA3, and EBNALP genes did not cause a significant HIV-1 LTR activation. Thus, it appears that among the latent EBV genes tested, EBNA2 was the only EBV gene active on the HIV-1 LTR. The transactivation function of EBNA2 was also observed in the HeLa epithelial cell line, which suggests that EBV and HIV-1 infection of non-B cells may result in HIV-1 promoter activation. Therefore, a specific gene product of EBV, EBNA2, can transactivate HIV-1 and possibly contribute to the clinical progression of AIDS.     

Expressed gene clusters associated with cellular sensitivity and resistance towards anti-viral and anti-proliferative actions of interferon

Interferons (IFN) are multi-functional proteins that induce a large number of genes which mediate many biological processes including host defense, cell growth control, signaling, and metabolism. Bioinformatics analysis of the 3'-untranslated regions of IFN-stimulated genes (ISGs) showed that the AU-rich elements (ARE) exist in approximately 10% of the mRNA induced by IFN. The human epithelial cell lines, WISH and 293, and the human B cell lines, Daudi and RPMI 1788, were assessed for their response to type-I IFN. Due to their differential response to the anti-viral and anti-proliferative action of IFN-alpha, they were used as cellular models for genome wide ARE-gene expression. The anti-viral and anti-proliferative actions of IFN-alpha were substantially more potent against WISH and Daudi cells than 293 and RPMI 1788 cells, respectively. These results correlated with the Stat1-driven gene expression as assessed by monitoring the expression of Stat1-mediated IFN-inducible 6-16 mRNA. Interferons were able to induce a significant proportion of common and distinct ARE-genes, but the patterns of expression were different and dependent on the type of the cell, type of IFN, and status of the cellular sensitivity to IFN. Clustering algorithms generated two informative expressed gene clusters that were selectively associated with cellular sensitivity and resistance to the anti-viral and anti-proliferative action of IFN. Use of rationally designed microarray experiments in IFN biology yielded informative clusters that may provide candidate genes for diagnostic or for evaluation of therapeutic possibilities.     

Epstein-barr virus-induced resistance to drugs that activate the mitotic spindle assembly checkpoint in Burkitt's lymphoma cells

Epstein-Barr virus (EBV) is associated with a number of human cancers, and latent EBV gene expression has been reported to interfere with cell cycle checkpoints and cell death pathways. Here we show that latent EBV can compromise the mitotic spindle assembly checkpoint and rescue Burkitt's lymphoma (BL)-derived cells from caspase-dependent cell death initiated in aberrant mitosis. This leads to unscheduled mitotic progression, resulting in polyploidy and multi- and/or micronucleation. The EBV latent genes responsible for this phenotype are expressed from the P3HR1 strain of virus and several viruses with similar genomic deletions that remove the EBNA2 gene. Although EBNA2 and the latent membrane proteins are not expressed, the EBNA3 proteins are present in these BL cells. Survival of the EBV-positive cells is not consistently associated with EBV lytic gene expression or with the genes that are expressed in EBV latency I BL cells (i.e., EBNA1, EBERs, and BARTs) but correlates with reduced expression of the cellular proapoptotic BH3-only protein Bim. These data suggest that a subset of latent EBV gene products may increase the likelihood of damaged DNA being inherited because of the impaired checkpoint and enhanced survival capacity. This could lead to greater genetic diversity in progeny cells and contribute to tumorigenesis. Furthermore, since it appears that this restricted latent EBV expression interferes with the responses of Burkitt's lymphoma-derived cells to cytotoxic drugs, the results of this study may have important therapeutic implications in the treatment of some BL.