Impact of the Potential m⁶A Modification Sites at the 3′UTR of Alfalfa Mosaic Virus RNA3 in the Viral Infection

We have previously reported the presence of m⁶A in the AMV (Alfamovirus, Bromoviridae) genome. Interestingly, two of these putative m⁶A-sites are in hairpin (hp) structures in the 3’UTR of the viral RNA3. One site (₂₀₁₂AAACU₂₀₁₆) is in the loop of hpB, within the coat protein binding site 1 (CPB1), while the other (₁₉₀₀UGACC₁₉₀₄) 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 m⁶A-sites in the AMV cycle, by introducing compensatory point mutations to interfere with or abolish the m⁶A-tag of these sites. Our results suggest that the loop of hpB could be involved in viral replication/accumulation. Meanwhile, in the ₁₉₀₀UGACC₁₉₀₄ 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.