The Role of m6A RNA methylation in chikungunya virus Replication

Chikungunya virus (CHIKV) is an emerging human pathogen transmitted through the mosquitoes Aedes aegypti and Aedes albopictus. CHIKV has a widening distribution due to the impact of globalization, including changing land use and international travel and trade. Infection with CHIKV in humans can present as severe fever and chronic arthritis, with the virus showing a tropism for the joints. Viruses manipulate hosts cells in a variety of ways including through gene expression at the level of RNA. N6-methyl-adenosine RNA modification (m6A) is a common and dynamic RNA modification, which involves the reversible addition of a methyl-group to an adenosine. m6A has been implicated as an important regulatory signal for replication of other RNA viruses, including Zika virus (ZIKV). This work sought to build a more comprehensive understanding of CHIKV biology and its lifecycle, including viral genome transcription in both human and mosquito cell line systems. Huh7 cells, a human liver cell line, and C636 cells, a mosquito cell line, were primarily used in this study. This work sought to investigate the role of m6A methylation on CHIKV replication. A small molecule inhibitor, c3Ado, previously used in other studies of m6A was used to treat human and mosquito cell lines. c3Ado reduces the amount of the methyl donor in the m6A reaction, SAM, leading to lower levels of methylation in the cell. Treatment with the small molecule inhibitor resulted in decreased viral replication and viral genome transcription in both Huh7 and C636 cells. Knockdown of proteins in the m6A pathway, Fat mass and obesity-associated protein (FTO) and Wilms’ Tumor 1 Associated Protein (WTAP), and site-directed mutagenesis of potential m6A sites in the CHIKV genome were also performed. These results showed that knockdown of WTAP in Huh7 cells resulted in a decrease in viral replication. This work also suggested the importance of an adenosine (A) at nucleotide position 96 in the CHIKV genome to overall viral replication in mammalian and insect cells. While the importance of 96A to viral genome replication was shown in a human cell line this could not be conclusively shown in a mosquito cell line. This work adds weight to the hypothesis that 96A and potentially m6A methylation of that position has a pro-viral effect, through an increase in viral genomic transcription in human cells.