Impact of the Potential m6 A Modification Sites at the 3′UTR of Alfalfa Mosaic Virus RNA3 in the Viral Infection

We have previously reported the presence of m(6)A in the AMV (Alfamovirus, Bromoviridae) genome. Interestingly, two of these putative m(6)A-sites are in hairpin (hp) structures in the 3'UTR of the viral RNA3. One site ((2012)AAACU(2016)) is in the loop of hpB, within the coat protein binding site 1 (CPB1), while the other ((1900)UGACC(1904)) is in the lower stem of hpE, a loop previously associated with AMV negative-strand RNA synthesis. In this work, we have performed in vivo experiments to assess the role of these two regions, containing the putative m(6)A-sites in the AMV cycle, by introducing compensatory point mutations to interfere with or abolish the m(6)A-tag of these sites. Our results suggest that the loop of hpB could be involved in viral replication/accumulation. Meanwhile, in the (1900)UGACC(1904) motif of the hpE, the maintenance of the adenosine residue and the lower stem hpE structure are necessary for in vivo plus-strand accumulation. These results extend our understanding of the requirements for hpE in the AMV infection cycle, indicating that both the residue identity and the base-pairing capacity in this structure are essential for viral accumulation.