Employing viruses to unravel the functional significance of the m5C epitranscriptome

5-methylcytosine (m5C) is an important RNA modification studied mostly for its role in tRNA biology. However, its roles in other aspects of RNA biology remain understudied. Our preliminary results, using bisulfite treatment of RNA followed by high-throughput sequencing, show that the genomes of many RNA viruses are m5C methylated in a site-specific manner, including Sindbis virus (SINV), chikungunya virus (CHIKV) and Coxsackievirus B3 (CVB3). The presence of m5C in diverse viruses, whose RNAs undergo many processes including translation, replication, transcription, and virion packaging, provides an attractive starting point for understanding the broader significance of this modification in regulating RNA function. A single dominant m5C site in SINV allowed us to generate an m5C-null mutant that exhibited cell-type dependent effects on virus replication. The host tRNA methyltransferase (MTase), NSUN2, which is important for host neuronal development and stem cell differentiation, appears to be the “writer” required for m5C modification of SINV. NSUN5, an MTase of ribosomal RNA is required for CVB3 methylation. We hypothesize that m5C plays a role in regulating viral RNA functions impacting virus-host interactions and viral life cycles. In three aims, using virologic, molecular, biochemical, high-throughput sequencing, and small animal model approaches: i) SINV will be exploited to learn how m5C is deposited and how it regulates RNA functions and viral infection and pathogenesis; studies of the related alphavirus CHIKV will allow conserved and virus-specific features to be uncovered, ii) how m5C regulates CVB3 RNA and what effects the modification has on virus replication and CVB3-associated myocarditis will be determined, and iii) SINV and CVB3 will be leveraged to characterize unknown functions of the NSUN2 and NSUN5 MTases, respectively, and as probes to discover novel m5C binding proteins that can exert their effect by direct binding (“readers”) or by removal of the m5C mark (“erasers”). This work will contribute to our understanding of human biology by revealing fundamental principles and functions of this widespread mark in the epitranscriptome with implications for its roles in maintaining cellular homeostasis. Since m5C methylation is a cellular process exploited by numerous viruses, this study could yield new targets for antiviral intervention.