The Picornavirales include viruses that infect vertebrates, insects, and plants. It was believed that they pack only their genomic mRNA in the particles; thus, we envisaged these viruses as excellent model systems for studies of mRNA modifications. We used LC-MS to analyze digested RNA isolated from particles of the sacbrood and deformed wing iflaviruses as well as of the echovirus 18 and rhinovirus 2 picornaviruses. Whereas in the picornavirus RNAs we detected only N6 -methyladenosine and 2'-O-methylated nucleosides, the iflavirus RNAs contained a wide range of methylated nucleosides, such as 1-methyladenosine (m1 A) and 5-methylcytidine (m5 C). Mapping of m1 A and m5 C through RNA sequencing of the SBV and DWV RNAs revealed the presence of tRNA molecules. Both modifications were detected only in tRNA. Further analysis revealed that tRNAs are present in form of 3' and 5' fragments and they are packed selectively. Moreover, these tRNAs are typically packed by other viruses.

1st Faculty of Medicine Charles University Katerinská 32 12108 Prague 2 Czech Republic

Department of Molecular Biology and Genetics Faculty of Science University of South Bohemia Branisovska 1760 37005 České Budějovice Czech Republic

Institute of Molecular Genetics of the Czech Academy of Sciences 14220 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo nam 2 16610 Prague 6 Czech Republic

Structural Virology Central European Institute of Technology Masaryk University Kamenice 753 5 62500 Brno Czech Republic

University of Chemistry and Technology 16628 Prague Czech Republic

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Exploring RNA modifications in infectious non-coding circular RNAs