N6-methyladenosine (m6A) plays critical roles in RNA metabolism and function. In addition to the internal m6A, N6,2’-O- dimethyladenosine (m6Am) is present at the transcription start nucleotide of capped mRNAs. The methyltransferase that adds this modification, PCIF1, was recently identified but the functional importance of this modification still remained enigmatic. Human cytomegalovirus (HCMV) is an important human pathogen that replicates in the nucleus exploiting cellular machineries. We will investigate if HCMV utilises the m6Am modification by conducting m6A-IP and high throughput sequencing using primary fibroblasts and investigate its functional importance especially to viral titers in the absence of PCIF1.
Our objectives are:
- We will examine m6Am modification landscape during viral infection (on viral and host mRNA) by conducting m6A-IP on HCMV infected primary fibroblasts and analysing the enrichment of the first base compared to input control.
- We will dissect the effect of this modification on viral propagation by generating PCIF1 KO cells.
- We will analyse how m6Am modification affects viral RNA lifecycle using RNA-seq (total RNA), 4sU-seq (RNA production and RNA stability, JMU) and Ribo-seq (RNA translation) applied on infected PCIF1 KO and control cells.