Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization

Abstract

The switch from Epstein-Barr virus (EBV) latent infection to lytic replication is governed by two viral transactivators, Zta and Rta. We previously reported that the EBV protein LF2 binds Rta, inhibits Rta promoter activation, and blocks EBV replication in cells. In addition, LF2 induces SUMO2/3 modification of Rta. We now show that this modification occurs at four lysines within the Rta activation domain (426, 446, 517, and 530) and that sumoylation of Rta is not essential for its repression. Coexpression studies demonstrated that Rta is sequestered to the extranuclear cytoskeleton in the presence of LF2. We mapped the LF2 binding site to Rta amino acids (aa) 476 to 519 and showed that LF2 binding is critical for Rta relocalization and repression. The core of this binding site, Rta aa 500 to 526, confers LF2-mediated relocalization and repression onto the artificial transcription factor GAL4-VP16. Mutational analysis of LF2 provided further evidence that Rta redistribution is essential for repression. Rta localization changes during replication of the LF2-positive P3HR1 genome, but not during replication of the LF2-negative B95-8 genome. BLRF2 protein expression was decreased and delayed in P3HR1 cells compared with B95-8 cells, consistent with reduced Rta activity. By contrast, BMRF1 expression, regulated primarily by Zta, did not differ significantly between the two cell lines. Our results support a model in which LF2 regulates EBV replication by binding to Rta and redistributing it out of the nucleus. Epstein-Barr virus (EBV), the prototypical gammaherpesvirus, causes infectious mononucleosis in healthy individuals, B-cell lymphoproliferative disease in immunosuppressed individuals, and rarely, B-cell lymphomas, Hodgkin lymphoma, and nasopharyngeal carcinoma in otherwise-healthy persons (45, 65). Gammaherpesviruses, including EBV and Kaposi's sarcoma-associated herpesvirus (KSHV), differ from other viruses because their associated diseases are not the consequence of virus replication. Instead, EBV-associated malignancies are a by-product of the growth and survival signals triggered by limited viral gene expression that allows EBV to persist in a latent state in infected cells, and hence the human population (44). Because viral replication is not occurring in most EBV-infected cells, inhibitors of replication are not efficacious in treating infectious mononucleosis or EBV-associated malignancies. On the contrary, activation of EBV replication has been suggested as therapy, because virus replication can directly kill EBV-infected tumor cells, sensitize them to nucleoside analogues, and stimulate immunemediated killing via increased virus antigen expression in tumor cells (21,22,55).Entry into replication is regulated by the EBV genes BZLF1 and BRLF1, encoding the transcriptional activators Z (Zta) and R (Rta), respectively (44, 53). Zta and Rta must act in concert for EBV replication to occur: deletion of either BZLF1 or BRLF1 renders the virus incompetent for DNA replication and virion production (20). Some E...

Genome-Wide Analysis of Epstein-Barr Virus Rta DNA Binding

“…That synthesis of new viral DNA is not required for Rta interactions with late promoters narrows down the possibilities for cis or trans signals that direct Rta to late promoters (2,69). Chromatin changes in the parental episome, viral or cellular gene expression independent of viral DNA replication, Rta modifications, such as sumoylation (11,33), or translocation of the episome to nuclear sites, such as PML bodies (3), might play a role in the recruitment of Rta to late gene promoters. Recent studies in murine gammaherpesvirus 68 (MHV68) have demonstrated that late gene expression depends not only on DNA replication but also on a subset of genes present in beta-and gammaherpesviruses but absent in alphaherpesviruses.…”

“…pcDNA3-Rta, pGL3-BALF2p, pGL3-BMLF1p, pGL3-BMRF1p, pGL3-Zp, and pGL3-Rp have been described previously (8,33). The BLRF2 (Ϫ349/ϩ28; B95-8 nucleotides 88548 to 88924), BFRF3 (Ϫ727/ϩ128; B95-8 nucleotides 60585 to 61439), and BALF5 (Ϫ376/ ϩ16; B95-8 nucleotides 156859 to 157250) promoters were amplified from B95-8 genomic DNA and cloned into the pGL3 promoter (Promega, Madison, WI).…”

“…Rta activates its own promoter via Sp1 sites, potentially via interactions with the Sp1 binding protein MCAF1 (10,64). However, it is not clear whether nuclear Rta is required for Rp activation, and thus the Sp1 sites may instead be downstream of cytoplasmic signaling cascades triggered by Rta expression (10,33,56,64). Rta activation of Zp requires the Zp promoter element ZII and is thought to be mediated by Rta-dependent activation of cellular signaling pathways in the cytoplasm (1,16,71).…”

“…Multiple mechanisms have been proposed for Rta promoter activation. Rta consists of an amino-terminal dimerization (amino acids 1 to 231) and DNA binding (amino acids 1 to 280) domain (60) fused to a carboxy-terminal activation domain (amino acids 416 to 605) that interacts with basal cellular transcription factors (32,59,60) and the EBV Rta regulator LF2 (8,9,33). Rta activates several promoters through direct binding to cognate DNA at sites called Rta response elements (RREs).…”

RanBPM regulates Zta-mediated transcriptional activity in Epstein–Barr virus

“…In addition, sumoylation by SUMO-1 has been shown to increase Rta transactivation activity (Chang et al, 2004). It is also known that LF2 enhances Rta conjugation to SUMO-2 and SUMO-3 (Calderwood et al, 2008), but this is unrelated to LF2-mediated repression of Rta functions (Heilmann et al, 2010). However, we have found that SUMO-2-Rta can be ubiquitinated via RNF4, leading to decreased Rta stability during lytic progression .…”