Bisulfite Sequencing to Map 5-Methylcytosine Sites in Human Respiratory Syncytial Virus RNA.

Viral epigenetic modification is an emerging field. As an obligate intracellular parasite, viral genetic material is exposed to host RNA-modifying enzymes. Currently, more than 180 types of RNA modifications have been identified. Like all non-segmented negative-sense RNA viruses, human respiratory syncytial virus (RSV) infection produces three types of RNA: genome and antigenome that are encapsidated by viral nucleocapsid and 10 mRNAs that are capped, methylated, and polyadenylated. Recently, the RSV genome, antigenome, and mRNAs have been shown to be modified by N6-methyladenosine (mA), which plays critical roles in replication, gene expression, and innate immune responses. However, whether RSV RNAs contain other types of modification remains unclear. Here, we describe an optimized bisulfite sequencing method and rigorous data analysis procedure to quantitatively map 5-methylcytosine (mC), one of the most prevalent RNA modifications, in RNA isolated from A549 cells infected by RSV and vesicular stomatitis virus (VSV) at single-base resolution. This method identified 20 mC sites in RSV genome but no mC sites were found in RSV antigenome, RSV mRNAs, or VSV RNAs. This protocol can be easily adapted for mC mapping of the RNAs of other viruses.