Challenges to mapping and defining m⁶A function in viral RNA

Viral RNA molecules contain multiple layers of regulatory information. This includes features beyond the primary sequence, such as RNA structures and RNA modifications, including N6-methyladenosine (m⁶A). Many recent studies have identified the presence and location of m⁶A in viral RNA and have found diverse regulatory roles for this modification during viral infection. However, to date, viral m⁶A mapping strategies have limitations that prevent a complete understanding of the function of m⁶A on individual viral RNA molecules. While m⁶A sites have been profiled on bulk RNA from many viruses, the resulting m⁶A maps of viral RNAs described to date present a composite picture of m⁶A across viral RNA molecules in the infected cell. Thus, for most viruses, it is unknown if unique viral m⁶A profiles exist throughout infection, nor if they regulate specific viral life cycle stages. Here, we describe several challenges to defining the function of m⁶A in viral RNA molecules and provide a framework for future studies to help in the understanding of how m⁶A regulates viral infection.