Natural Variations in BRLF1 Promoter Contribute to the Elevated Reactivation Level of Epstein-Barr Virus in Endemic Areas of Nasopharyngeal Carcinoma

Abstract

Background: Epstein-Barr virus (EBV) infection is a crucial risk factor for nasopharyngeal carcinoma (NPC), but the mechanism for its elevated activation level in NPC endemic areas remains unclear. This study aims to identify the EBV natural variations contributed to the different reactivation potential between NPC endemic and non-endemic areas. Methods: 1030 subjects were recruited in China, including 303 healthy individuals from two NPC non-endemic areas, 483 healthy people from three endemic areas and 244 NPC patients. Among which, saliva DNA samples from 244 participants were sequenced for the EBV immediate early (IE) genes of BRLF1 and BZLF1, their promoters were included; the rest 786 subjects were used for the validation of significant variations among three different populations. Haplotype and population structure analysis were conducted. Dual-luciferase assay was used to detect the promoter activity. Results: A total of 246 distinct variations were detected, 29 showed significant difference in the frequencies between healthy people from NPC endemic area and non-endemic area. Population structure analysis clustered EBV strains into 9 subgroups mostly in accordance with the geographical origin of samples. Interestingly, two EBV genotypes, Rp-V1 and Rp-V2, were identified according to the linkage relationship of the variations in BRLF1 promoter (Rp). Rp-V1 has higher frequency in NPC endemic areas than in non-endemic areas (52.38% vs 18.15%, P=2.07×10-14), and was associated with higher oral EBV DNA levels (adjusted OR=1.64, 95% CI=1.21-2.24, P=0.002), suggesting a more powerful activation ability of Rp-V1 than that of the wild type Rp-WT of the EBV strain; On the contrary, Rp-V2 has higher frequency in NPC non-endemic areas than in endemic areas (18.48% vs 0.38%, P=1.17×10-7), might represent a reduced activation potential of EBV. Further dual-luciferase assay showed Rp-V1 has higher promoter activity while compared with Rp-V2 (P<0.0001). Notably, Rp-V1 impaired the transcription repression effect of YY1 while Rp-V2 strengthened the transcription repression effect of EBF1 on Rp. In addition, significant differences of Rta 393-407 CTL epitope which may influence the recognition of Rta by CD8 T cells were detected between healthy people from NPC endemic area and non-endemic area. Conclusions: This study identified natural variations in cis-acting elements (YY1 and EBF1) of EBV Rp altering Rp transcription activities, which may contribute to the elevated EBV activation level in NPC endemic areas than non-endemic areas. Funding Statement: This work was supported by the National Key Research and Development Program of China [grant number: 2016YFC1302704], the Sino-Sweden joint research program [grant number: 81861138006], the Science and Technology Planning Project of Guangzhou City, China [grant number: 201804020094] and the National Natural Science Foundation of China [grant number: 81502056, 81325018 and 81220108022]. The funder had no role in the study design, participant recruitment, data collection, data analysis, data interpretation, or writing of the report. Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The study was reviewed and approved by the Human Ethics Approval Committee at Sun Yat-sen University Cancer Center.