Arsenicals, the Integrated Stress Response, and Epstein-Barr Virus Lytic Gene Expression.
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ABSTRACT:
SUBMITTER: Lee J
PROVIDER: S-EPMC8147158 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Episomal reporter plasmids containing the Epstein-Barr virus (EBV) oriP sequence stably transfected into Akata Burkitt's lymphoma cells were used to analyze EBV lytic cycle gene regulation. First, we found that the Zp promoter of EBV, but not the Rp promoter, can be activated in the absence of protein synthesis in these oriP plasmids, casting doubt on the immediate early status of Rp. An additional level of regulation of Zp was implied by analysis of a mutation of the ZV element. Second, our analysis of late lytic cycle promoters revealed that the correct relative timing, dependence on ori lyt in cis, and sensitivity to inhibitors of DNA replication were reconstituted on the oriP plasmids. Late promoter luciferase activity from oriP plasmids also incorporating replication-competent ori lyt was phosphonoacetic acid sensitive, a hallmark of EBV late genes. A minimal ori lyt, which only replicates weakly, was sufficient to confer late timing of expression specifically on late promoters. Finally, deletion analysis of EBV late promoter sequences upstream of the transcription start site confirmed that sequences between -49 and +30 are sufficient for late gene expression, which is dependent on ori lyt in cis. However, the TATT version of the TATA box found in many late genes was not essential for late expression.
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Project description:This SuperSeries is composed of the SubSeries listed below.
2020-04-20 | GSE140653 | GEO
Project description:Epstein-Barr virus (EBV) is associated with multiple human malignancies. To evade immune detection, EBV switches between latent and lytic programs. How viral latency is maintained in tumors or in memory B cells, the reservoir for lifelong EBV infection, remains incompletely understood. To gain insights, we performed a human genome-wide CRISPR/Cas9 screen in Burkitt lymphoma B cells. Our analyses identified a network of host factors that repress lytic reactivation, centered on the transcription factor MYC, including cohesins, FACT, STAGA, and Mediator. Depletion of MYC or factors important for MYC expression reactivated the lytic cycle, including in Burkitt xenografts. MYC bound the EBV genome origin of lytic replication and suppressed its looping to the lytic cycle initiator BZLF1 promoter. Notably, MYC abundance decreases with plasma cell differentiation, a key lytic reactivation trigger. Our results suggest that EBV senses MYC abundance as a readout of B cell state and highlights Burkitt latency reversal therapeutic targets.
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Project description:The cellular microRNA miR-155 has been shown to be involved in lymphocyte activation and is expressed in Epstein-Barr virus (EBV)-infected cells displaying type III latency gene expression but not type I latency gene expression. We show here that the elevated levels of miR-155 in type III latency cells is due to EBV gene expression and not epigenetic differences in cell lines tested, and we show that expression in EBV-infected cells requires a conserved AP-1 element in the miR-155 promoter. Gene expression analysis was carried out in a type I latency cell line transduced with an miR-155-expressing retrovirus. This analysis identified both miR-155-suppressed and -induced cellular mRNAs and suggested that in addition to direct targeting of 3' untranslated regions (UTRs), miR-155 alters gene expression in part through the alteration of signal transduction pathways. 3' UTR reporter analysis of predicted miR-155 target genes identified the transcriptional regulatory genes encoding BACH1, ZIC3, HIVEP2, CEBPB, ZNF652, ARID2, and SMAD5 as miR-155 targets. Western blot analysis of the most highly suppressed of these, BACH1, showed lower expression in cells transduced with a miR-155 retrovirus. Inspection of the promoters from genes regulated in EBV-infected cells and in cells infected with an miR-155 retrovirus identified potential binding sequences for BACH1 and ZIC3. Together, these experiments suggest that the induction of miR-155 by EBV contributes to EBV-mediated signaling in part through the modulation of transcriptional regulatory factors.
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Project description:Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.
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Project description:Epstein-Barr virus (EBV) is a ubiquitous human lymphotropic herpesvirus that is causally associated with several malignancies. In addition to latent factors, lytic replication contributes to cancer development. In this study, we examined whether the lytic gene BNRF1, which is conserved among gamma-herpesviruses, has an important role in lymphomagenesis. We found that lymphoblastoid cell lines (LCLs) established by BNRF1-knockout EBV exhibited remarkably lower pathogenicity in a mice xenograft model than LCLs produced by wild-type EBV (LCLs-WT). RNA-seq analyses revealed that BNRF1 elicited the expression of interferon-inducible protein 27 (IFI27), which promotes cell proliferation. IFI27 knockdown in LCLs-WT resulted in excessive production of reactive oxygen species, leading to cell death and significantly decreased their pathogenicity in vivo. We also confirmed that IFI27 was upregulated during primary infection in B-cells. Our findings revealed that BNRF1 promoted robust proliferation of the B-cells that were transformed by EBV latent infection via IFI27 upregulation both in vitro and in vivo.
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Project description:EBV has two lytic origins (oriLyt) of DNA replication lying at divergent sites on the viral genome within a duplicated sequence (DS). The latter contains potential hairpin loops, 'hinge' elements and the promoters for transcripts from viral genes BHLF1 and LF3. These genes themselves consist largely of 125 and 102 bp repetitive sequences, respectively, and encode basic proteins. We have examined these genomic regions in detail in attempts to understand why lytic replication--necessary for virus survival--is so inefficient, and to identify controlling elements. Our studies uncovered a diverse family of promoters (P) for BHLF1 and LF3, only one pair of which (P1) proved sensitive to chemical inducing agents. The others (P2-P3/4), abutting the replication 'core' origin elements in DS and extending into 5'-unique sequences, may play roles in the maintenance of viral latency. We further identified a family of overlapping small complementary-strand RNAs that transverse the replication 'core' origin elements in a manner suggesting a role for them as 'antisense' species and/or DNA replication primers. Our data are discussed in terms of alternative lytic replication models. We suggest our findings might prove useful in seeking better control over viral lytic replication and devising strategies for therapy.
| S-EPMC1904260 | biostudies-literature
Project description:The key viral gene responsible for initiating the replicative cycle of Epstein-Barr virus (EBV), termed BZLF1, encodes the multifunctional protein Zta (ZEBRA or Z). It interacts with DNA as both a transcription and a replication factor, modulates both intracellular signal transduction and the DNA-damage response and manipulates cell cycle progression. Muller and colleagues have resolved the structure of Zta bound to DNA, which confirms some structural predictions but reveals an unexpected twist and a complex dimerization interface. Because EBV is associated with human disease, Zta presents a prime target for drug design.
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Project description:The effect of Epstein-Barr virus (EBV) SM protein on EBV gene expression was examined using a recombinant EBV strain with the SM gene deleted and DNA microarrays representing all known EBV coding regions. Induction of lytic EBV replication in the absence of SM led to expression of approximately 40% of EBV genes, but a block in expression of over 50% of EBV genes. Contrary to previous findings, several early genes were SM dependent, and lytic EBV DNA replication did not occur in the absence of SM. Notably, two genes essential for lytic EBV DNA replication, BSLF1 and BALF5, encoding EBV DNA primase and polymerase, respectively, were SM dependent. Lytic DNA replication was partially rescued by ectopic expression of EBV primase and polymerase, but virion production was not. Rescue of DNA replication only enhanced expression of a subset of late genes, consistent with a direct requirement for SM for late gene expression in addition to its contribution to DNA replication. Therefore, while SM is essential for most late gene expression, the proximate block to virion production by the EBV SM deletion strain is an inability to replicate linear DNA. The block to DNA replication combined with the direct effect of SM on late gene expression leads to a global deficiency of late gene expression. SM also inhibited BHRF1 expression during productive replication in comparison to that of cells induced into lytic replication in the absence of SM. Thus, SM plays a role in multiple steps of lytic cycle EBV gene expression and that it is transcript-specific in both activation and repression functions.
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Project description:Cellular topoisomerases and helicases are thought to play an essential role in herpesvirus replication and gene expression and are considered to be potential targets for antiviral therapies. Topoisomerase I (Topo I) and Topo II inhibitors can selectively inhibit Epstein-Barr virus (EBV) lytic cycle DNA replication. We found that the Topo I inhibitor camptothecin and, to a lesser extent, the Topo II inhibitor etoposide are potent inhibitors of the transcription and replication function of the EBV-encoded immediate-early protein Zta (also referred to as ZEBRA, EB1, and BZLF1). Camptothecin inhibited the Zta transcription activation of endogenous and reporter-linked viral promoters. Small interfering RNA depletion of Topo I also inhibited the Zta-dependent activation of lytic cycle DNA replication. Topo I could be coimmunoprecipitated with Zta, but this interaction was restricted to EBV-positive cells, suggesting that other viral proteins stabilize the interaction between Zta and Topo I. We also found that the RecQL1 helicase, which is known to associate with Kaposi's sarcoma-associated herpesvirus (KSHV) OriLyt, interacts with EBV OriLyt. Treatment with camptothecin reduced both Zta and RecQL1 binding to OriLyt in vivo, suggesting that Topo I promotes replication protein assembly at OriLyt.
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