Abstract
The 2022 global mpox outbreak was characterized by an unusual accumulation of more than fifty APOBEC-signature substitutions in MPXV genomes. A previous study suggested that these substitutions preferentially occur within and near hairpins or cruciforms - stem-loop structures formed by inverted repeats. Here, we show that this conclusion resulted from an inappropriate statistical analysis and, using two substantially larger datasets, demonstrate that APOBEC-signature mutations are specifically enriched at TC motifs located at the 3′ ends of hairpin loops, but not in other regions of hairpins/cruciforms or in their vicinity. We further identified a preference for stem-loop structures with longer stems and shorter loops, consistent with patterns previously reported for the APOBEC mutagenesis in cancer genomes. Our analysis also revealed a positive association between APOBEC-signature mutation propensity and hairpin stability in single-stranded DNA. Nevertheless, stem-loop structures with long stems and short loops are relatively rare in the MPXV genome, limiting their contribution to the genome-wide distribution of APOBEC-signature mutations to a predominantly local effect. In contrast, extended nucleotide context downstream of the TC motif showed strong genome-wide predictive value, with a marked preference for guanine-rich trinucleotide immediately downstream. Analysis of the upstream nucleotide context was consistent with the mutational specificity of APOBEC3A. Together, these results identify distinct structural and sequence determinants of the APOBEC-associated mutagenesis in MPXV and show that local hairpin/cruciform architecture and extended nucleotide context contribute at different genomic scales.