A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection

Abstract

Latent Epstein-Barr virus (EBV) infection contributes to both B-cell and epithelial-cell malignancies. However, whether lytic EBV infection also contributes to tumors is unclear, although the association between malaria infection and Burkitt lymphomas (BLs) may involve excessive lytic EBV replication. A particular variant of the viral promoter (Zp) that controls lytic EBV reactivation is over-represented, relative to its frequency in non-malignant tissue, in EBV-positive nasopharyngeal carcinomas and AIDS-related lymphomas. To date, no functional differences between the prototype Zp (Zp-P) and the cancer-associated variant (Zp-V3) have been identified. Here we show that a single nucleotide difference between the Zp-V3 and Zp-P promoters creates a binding site for the cellular transcription factor, NFATc1, in the Zp-V3 (but not Zp-P) variant, and greatly enhances Zp activity and lytic viral reactivation in response to NFATc1-inducing stimuli such as B-cell receptor activation and ionomycin. Furthermore, we demonstrate that restoring this NFATc1-motif to the Zp-P variant in the context of the intact EBV B95.8 strain genome greatly enhances lytic viral reactivation in response to the NFATc1-activating agent, ionomycin, and this effect is blocked by the NFAT inhibitory agent, cyclosporine, as well as NFATc1 siRNA. We also show that the Zp-V3 variant is over-represented in EBV-positive BLs and gastric cancers, and in EBV-transformed B-cell lines derived from EBV-infected breast milk of Kenyan mothers that had malaria during pregnancy. These results demonstrate that the Zp-V3 enhances EBV lytic reactivation to physiologically-relevant stimuli, and suggest that increased lytic infection may contribute to the increased prevalence of this variant in EBV-associated malignancies.

More than just oncogenes: mechanisms of tumorigenesis by human viruses

“…Moreover, a large-scale prospective study in Taiwan showed that patients that had antibodies to at least one of two lytic proteins were at least four times more likely to develop nasopharyngeal carcinoma than seronegative patients [93]. Direct evidence of the importance of lytic reactivation in EBV tumors was recently provided by a study examining a naturally occurring polymorphism in the promoter of the EBV BZLF1 protein, which controls the latent-to-lytic switch of the virus [94]. This polymorphism is over-represented in the EBV sequences isolated from tumor tissues of patients with EBV-associated nasopharyngeal carcinoma, AIDS-associated lymphomas, Burkitt’s lymphomas and gastric carcinomas [94].…”

“…Direct evidence of the importance of lytic reactivation in EBV tumors was recently provided by a study examining a naturally occurring polymorphism in the promoter of the EBV BZLF1 protein, which controls the latent-to-lytic switch of the virus [94]. This polymorphism is over-represented in the EBV sequences isolated from tumor tissues of patients with EBV-associated nasopharyngeal carcinoma, AIDS-associated lymphomas, Burkitt’s lymphomas and gastric carcinomas [94]. Interestingly, this cancer-associated polymorphism leads to increased BZLF1 expression in response to reactivation stimuli such as B cell receptor activation and, consequently, increased lytic reactivation [94].…”

“…This polymorphism is over-represented in the EBV sequences isolated from tumor tissues of patients with EBV-associated nasopharyngeal carcinoma, AIDS-associated lymphomas, Burkitt’s lymphomas and gastric carcinomas [94]. Interestingly, this cancer-associated polymorphism leads to increased BZLF1 expression in response to reactivation stimuli such as B cell receptor activation and, consequently, increased lytic reactivation [94]. The over-representation of this polymorphism in tumors suggests that increased lytic reactivation promotes tumor development.…”

Epstein‐Barr virus strain variation and cancer

“…Research interest has focused on a viral BZLF1 gene, a master regulator of viral lytic replication . BZLF1 is highly polymorphic both for its coding sequence and promoter region …”

A novel Epstein‐Barr virus subtype associated with nasopharyngeal carcinoma found in South China

“…One hypothesis is that NPC‐associated EBV strains or subtypes are functionally different from lymphoma‐derived strains. Recent studies have found sequence differences in the EBV genome in NPC in the promoter region driving expression of the BamHIZ leftward frame 1 (BZLF1) [10], the immediate early protein responsible for the switch from latent to lytic infection, and in the Epstein‐Barr virus nuclear antigen 1 (EBNA1) protein [11], both of which appear to promote EBV replication. These observations are consistent with those from a study on an NPC‐derived EBV strain (M81), which was found to be more lytic and to have enhanced tropism for epithelial cells [12].…”