Epstein–Barr Virus (EBV) Rta-Mediated EBV and Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivations in 293 Cells

Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmunoprecipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication. IMPORTANCECatalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme activity executes at the right time and the right place in DNA replication forks, complex formation with other components in the DNA replication machinery provides an important regulation for UDG function. In this study, we provide the mechanism for EBV UDG BKRF3 nuclear targeting and the interacting domains of BKRF3 with viral DNA replication proteins. Through knockout and complementation approaches, we further demonstrate that in addition to UDG activity, the interaction of BKRF3 with viral proteins in the replication compartment is crucial for efficient viral DNA replication.

Section: Discussionmentioning

confidence: 92%

Uracil DNA Glycosylase BKRF3 Contributes to Epstein-Barr Virus DNA Replication through Physical Interactions with Proteins in Viral DNA Replication Complex

Liu

et al. 2014

“…To ensure successful lytic reactivation, EBV modulates various cellular events to facilitate virus production. Some lytic proteins modulate cell cycle progression, such as Rta, which induces G 1 arrest primarily originating at the transcriptional level (6). Some suppress the immune response to facilitate the progression of the viral lytic cycle.…”

mentioning

confidence: 99%

Epstein-Barr Virus BGLF4 Kinase Downregulates NF-κB Transactivation through Phosphorylation of Coactivator UXT

Chang

Wang

Doong

et al. 2012

b Epstein-Barr virus (EBV) BGLF4 is a member of the conserved herpesvirus kinases that regulate multiple cellular and viral substrates and play an important role in the viral lytic cycles. BGLF4 has been found to phosphorylate several cellular and viral transcription factors, modulate their activities, and regulate downstream events. In this study, we identify an NF-B coactivator, UXT, as a substrate of BGLF4. BGLF4 downregulates not only NF-B transactivation in reporter assays in response to tumor necrosis factor alpha (TNF-␣) and poly(I·C) stimulation, but also NF-B-regulated cellular gene expression. Furthermore, BGLF4 attenuates NF-B-mediated repression of the EBV lytic transactivators, Zta and Rta. In EBV-positive NA cells, knockdown of BGLF4 during lytic progression elevates NF-B activity and downregulates the activity of the EBV oriLyt BHLF1 promoter, which is the first promoter activated upon lytic switch. We show that BGLF4 phosphorylates UXT at the Thr3 residue. This modification interferes with the interaction between UXT and NF-B. The data also indicate that BGLF4 reduces the interaction between UXT and NF-B and attenuates NF-B enhanceosome activity. Upon infection with short hairpin RNA (shRNA) lentivirus to knock down UXT, a spontaneous lytic cycle was observed in NA cells, suggesting UXT is required for maintenance of EBV latency. Overexpression of wild-type, but not phosphorylation-deficient, UXT enhances the expression of lytic proteins both in control and UXT knockdown cells. Taking the data together, transcription involving UXT may also be important for EBV lytic protein expression, whereas BGLF4-mediated phosphorylation of UXT at Thr3 plays a critical role in promoting the lytic cycle.

“…This phenomenon is universal in all doxycycline (Dox)-inducible Rta-expressing cell lines, including TW01TetER, 293TetER, and Rta-inducible EBV replication systems in HEK293 cells, dubbed EREV8 ( Fig. 1A and reference 14). Previous studies demonstrated that the consensus Rta binding sites are 16 to 18 nucleotides in length with high GC content, including the two flanking core elements (5=-GNCC-3= and 5=-GGNG-3=) (8)(9)(10).…”

Section: Resultsmentioning

confidence: 83%

“…EBV Rta is known to be a potent transcriptional activator. However, we observed that the expression of Rta efficiently downregulated transcription of a battery of positive cell cycle regulators, including MYC, CCND1, and JUN, effectively leading to cell cycle arrest (14). This phenomenon is universal in all doxycycline (Dox)-inducible Rta-expressing cell lines, including TW01TetER, 293TetER, and Rta-inducible EBV replication systems in HEK293 cells, dubbed EREV8 ( Fig.…”

Section: Resultsmentioning

confidence: 91%

Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes

Chen

Chang

et al. 2017

Self Cite

Rta, an Epstein-Barr virus (EBV) immediate-early protein, reactivates viral lytic replication that is closely associated with tumorigenesis. In previous studies, we demonstrated that in epithelial cells Rta efficiently induced cellular senescence, which is an irreversible G 1 arrest likely to provide a favorable environment for productive replications of EBV and Kaposi's sarcoma-associated herpesvirus (KSHV). To restrict progression of the cell cycle, Rta simultaneously upregulates CDK inhibitors and downregulates MYC, CCND1, and JUN, among others. Rta has long been known as a potent transcriptional activator, thus its role in gene repression is unexpected. In silico analysis revealed that the promoter regions of MYC, CCND1, and JUN are common in (i) the presence of CpG islands, (ii) strong chromatin immunoprecipitation (ChIP) signals of CCCTC-binding factor (CTCF), and (iii) having at least one Rta binding site. By combining ChIP assays and DNA methylation analysis, here we provide evidence showing that Rta binding accumulated CpG methylation and decreased CTCF occupancy in the regulatory regions of MYC, CCND1, and JUN, which were associated with downregulated gene expression. Stable residence of CTCF in the viral latency and reactivation control regions is a hallmark of viral latency. Here, we observed that Rta-mediated decreased binding of CTCF in the viral genome is concurrent with virus reactivation. Via interfering with CTCF binding, in the host genome Rta can function as a transcriptional repressor for gene silencing, while in the viral genome Rta acts as an activator for lytic gene loci by removing a topological constraint established by CTCF.IMPORTANCE CTCF is a multifunctional protein that variously participates in gene expression and higher-order chromatin structure of the cellular and viral genomes. In certain loci of the genome, CTCF occupancy and DNA methylation are mutually exclusive. Here, we demonstrate that the Epstein-Barr virus (EBV) immediate-early protein, Rta, known to be a transcriptional activator, can also function as a transcriptional repressor. Via enriching CpG methylation and decreasing CTCF reloading, Rta binding efficiently shut down the expression of MYC, CCND1, and JUN, thus impeding cell cycle progression. Rta-mediated disruption of CTCF binding was also detected in the latency/reactivation control regions of the EBV genome, and this in turn led to viral lytic cycle progression. As emerging evidence indicates that a methylated EBV genome is a preferable substrate for EBV Zta, the other immediate-early protein, our results suggest a mechanistic link in understanding the molecular processes of viral latent-lytic switch.KEYWORDS Epstein-Barr virus, immediate-early protein Rta, lytic cycle reactivation, CTCF, DNA methylation, cell cycle arrest, genome topology, immediate-early protein Rta