Structural Modeling of Protein-DNA Interactions Underlying Genome Copy Number Variation in Nanoviruses

Multipartite nanoviruses possess multiple segmented genomes that accumulate at unequal levels during infection, giving rise to dynamic genome formulas. Although the replication of all segments relies on a common replication-associated protein (Rep), the molecular basis underlying segment-specific differences in genome abundance remains poorly understood. Here, we investigated whether protein-DNA interactions at conserved cis-regulatory elements are associated with this variability by focusing on the intergenic region (IR) of segment S of milk vetch dwarf virus (MDV). We used a structure-based computational approach to analyze interactions between the MDV S-IR and Rep or movement proteins (MPs) from MDV and faba bean necrotic yellows virus under a consistent computational framework. Molecular docking, molecular dynamics simulations, and molecular mechanics/generalized Born surface area-based interaction energy analyses showed that all complexes formed stable interactions with the MDV S-IR. ΔG _bind values were broadly comparable across systems, whereas differences in interaction persistence, structural stability, and residue-level contributions were observed between Rep- and MP-containing complexes. In particular, Rep-IR interactions exhibited more consistent and distributed interaction patterns, while MP-IR interactions showed greater variability and more localized contacts. These interaction features were more consistent with previously reported MDV DNA-S abundance trends than docking geometry alone. Together, these findings provide a structural framework to interpret MDV DNA-S abundance patterns in a comparative and hypothesis-generating context rather than as direct evidence of causal regulation.