N6-methyladenosine modification of HCMV IE1 transcript promotes the repressive state of viral genome to achieve latent infection

Human cytomegalovirus (HCMV) is a prevalent pathogen that chronically infects the majority of human population. Among the many features that allow such widespread HCMV infection, one is its ability to maintain a transcriptionally dormant immune-evasive state called latency by suppressing its own major immediate early promoter (MIEP) via epigenetic alterations. In this study, we show a novel mechanism of MIEP regulation in which the major immediate early (MIE) gene product, IE1, downregulates its own promoter activity in an m6A modification-dependent manner. We found that the loss of the m6A writer, METTL3, in host cell results in the failure of HCMV to establish latency in these cells. Through transcriptome-wide m6A profiling of latently infected monocytes, we identified that the major immediate early gene product IE1 is m6A-modified during latent infection, with an m6A profile that is distinct from that found during lytic infection. Using an IE1-specific m6A-abolished mutant, we found that latent infection-specific m6A modification of the IE1 transcript was necessary for the efficient repression of MIEP, and this mutant virus failed to establish latency and progressed toward lytic-like infection in THP-1 cells. Our findings demonstrate that HCMV exploits the host m6A machinery to suppress its own lytic program to establish latency and uncover an unexpected role of immediate early gene mRNA in regulating its own expression. Overall design: MeRIP-seq data were generated from HCMV TB40/E-infected THP-1 cells at two time points (1 dpi and 7 dpi) under different treatment conditions (7 dpi + DMSO and 7 dpi + PMA), using two biological replicates. At 5 dpi, a subset of samples was treated with DMSO or PMA and harvested at 7 dpi for MeRIP-seq analysis.