Abstract
The switch between latent and lytic Epstein-Barr virus (EBV) infection is mediated by the viral immediate-early (IE) protein, BZLF1 (Z). Z, a homologue of c-jun that binds to AP1-like motifs (ZREs), induces expression of the BRLF1 (R) and BRRF1 (Na) viral proteins, which cooperatively activate transcription of the Z promoter and thereby establish a positive autoregulatory loop. A unique feature of Z is its ability to preferentially bind to, and activate, the methylated form of the BRLF1 promoter (Rp). To date, however, Rp is the only EBV promoter known to be regulated in this unusual manner. We now demonstrate that the promoter driving transcription of the early BRRF1 gene (Nap) has two CpG-containing ZREs (ACGCTCA and TCGCCCG) that are only bound by Z in the methylated state. Both Nap ZREs are highly methylated in cells with latent EBV infection. Z efficiently activates the methylated, but not unmethylated, form of Nap in reporter gene assays, and both ZREs are required. Z serine residue 186, which was previously shown to be required for Z binding to methylated ZREs in Rp, but not for Z binding to the AP1 site, is required for Z binding to methylated Nap ZREs. The Z(S186A) mutant cannot activate methylated Nap in reporter gene assays and does not induce Na expression in cells with latent EBV infection. Molecular modeling studies of Z bound to the methylated Nap ZREs help to explain why methylation is required for Z binding, and the role of the Z Ser186 residue. Methylation-dependent Z binding to critical viral promoters may enhance lytic reactivation in latently infected cells, where the viral genome is heavily methylated. Conversely, since the incoming viral genome is initially unmethylated, methylation-dependent Z activation may also help the virus to establish latency following infection.
Highlights
Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with B-cell lymphomas, nasopharyngeal carcinoma (NPC) and gastric cancer [1,2]
We show that another EBV promoter (Nap, driving transcription of the BRRF1 gene) likewise has two methylation-dependent Z binding sites, and that Z only activates the Na promoter (Nap) efficiently in the methylated form
Since the BRLF1 and BRRF1 genes encode essential viral transcription factors that work cooperatively with Z to induce the lytic form of viral infection, our results indicate that methylation of the EBV genome enhances Z-mediated disruption of viral latency
Summary
Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with B-cell lymphomas, nasopharyngeal carcinoma (NPC) and gastric cancer [1,2]. Lytic replication is mediated by the virally encoded DNA polymerase using the oriLyt replication origin, and results in the release of infectious viral particles [8]. During latent viral infection (which normally occurs in memory B cells), only a subset of viral genes is expressed, the genome is replicated once per cell cycle using the cellular DNA polymerase and the oriP replication origin, and progeny virus is not released. Latent EBV infection allows the virus to persist for the life of the host and avoid detection by the immune system [1,4]. Both the latent and lytic forms of EBV infection are essential for viral pathogenesis
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