Updated project metadata. The mechanisms by which viruses hijack their host’s genetic machinery are of enormous current interest. One mechanism is adenosine diphosphate (ADP) ribosylation, where ADP-ribosyltransferases (ARTs) transfer an ADP-ribose fragment from the ubiquitous co-factor nicotinamide adenine dinucleotide (NAD) to acceptor proteins (Cohen and Chang, 2018). When bacteriophage T4 infects Escherichia coli, three different ARTs reprogram the host’s transcriptional and translational apparatus (Koch et al., 1995; Tiemann et al., 2004). Recently, NAD was identified as a 5’-modification of cellular RNA molecules in bacteria and higher organisms (Cahova et al., 2015; Chen et al., 2009; Jiao et al., 2017). Here, we report that a bacteriophage T4 ART ModB accepts not only NAD but also NAD-RNA as substrate, thereby covalently linking entire RNA chains to acceptor proteins in an “RNAylation” reaction. This enzyme specifically RNAylates its host protein targets, ribosomal proteins rS1 and rL2, at arginine residues and prefers NAD-RNA over NAD. RNAylation of specific ribosomal proteins decreases ribosome activity. We identify specific E. coli and T4 phage RNAs, which are RNAylated to rS1 in vivo.T4 phages expressing an inactive mutant of ModB show a decreased burst size and a decelerated lysis of E. coli during T4 infection. Our findings not only challenge the established views of the phage replication cycle but also reveal a distinct biological role of NAD-RNA, namely activation of the RNA for enzymatic transfer. Our work exemplifies the first direct connection between RNA modification and post-translational protein modification. As ARTs play important roles far beyond viral infections (Fehr et al., 2020), RNAylation may have far-reaching implications.