Conformational Dynamics and Phase Separation Biophysical Drivers of Virus Transmission: AlphaFOLD2-processed Flexible Folding Behavior, Biomolecular condensates and Deep Learning-driven Protein Interface Epitopes Redefine the Evolutionary and Functional Modulators of Plant Virus Movement Proteins

Cell-to-cell trafficking during most viral infection in plants relies on virus-encoded movement proteins (MPs). MPs essentially contribute to virus intercellular progression, systemic by assisting the transport of plant virus RNA through plasmodesmata (intercellular junctions in plants). Yet many annotated MPs remain structurally unclassified and ill-characterized. Though unique proteins, MPs are well represented over evolutionarily diverse taxonomic lineages of plant viruses, but their biophysical and functional features remain unknown. Understanding these aspects is particularly important to better characterize pathogenicity and transmission of viruses, especially on economically important crops and improve disease resistance. In this study, a comprehensive and manually curated sequence dataset of MPs (n=425) was first retrieved, and their 3D structures predicted using the state-of-the-art machine learning-based structure prediction method, AlphaFOLD2 (AF2). Corresponding MP Structures were then compared through diverse databases using the FoldSeek software to probe structural homologs, robustly uncover evolutionarily related folds and their potentially exaptive shared origins with different proteins. In an attempt to predict MPs biological functions, we used MetaVIRIA, our new semi-automated fast Nextflow-based framework, to link per-residue AF2 structural confidence metrics (pLDDTs) with highly accurate sequence-based predictors of biophysical behavior such as conformational sequence-ensembles relationships of intrinsically disordered regions (IDRs), biomolecular condensates and protein-interface epitopes, to demonstrate correlation between these biophysical propensities and the biological functions of diverse MPs. First, to the best of our knowledge, as nothing is known about the biophysical properties and functional role the unique N- and C-terminal IDRs of unknown origins, we demonstrate here for the first time, important biophysical features associated to the terminal IDRs of MPs and how these driving forces may correlate with biological context. Second, while our results confirm the hypothesized evolutionary origin of MPs 30K superfamily from CPs through horizontal acquisition followed by neofunctionalization, based on our large multiscale biophysical and structural prediction, new uncharted evolutionary trajectories have emerged on MPs assigned to different proteins classes and taxonomical lineages. Third, we found that more than the highly conserved core structural domain, IDRs of most plant virus MPs investigated here are highly diverse in terms of biophysical propensities. Some of those IDRs could be related to viral transmission. We also summarize novel insights on the potential tuning roles of electrostatics, entropy fluctuation in low-complexity regions (LCRs), context-sensitive fold-upon-binding dynamics and sequence segregation patterns, towards capturing MPs-host and vector specificity. Coarse-grained examination of non-random sequence states highlights IDRs ensembles and phase separation as unexplored modulating drivers of nematodes and arthropods vector-borne transmission specificity, with widespread multimodal binding affinities.

Across the sequence-to-conformation biophysical landscape of MPs, statistically well-supported correlations analysis between their folded core domain conformational dynamics and their natively unfolded flexible regions uncovered diverse generalizable avenues to streamline the evolutionary adaptive role of these IDRs with drivers of phase separation as novel opportunities in plant virology to capture their significant and complex involvement in plant virus pathogenicity and viral transmission in general.