Optogenetic and temperature-dependent modulation of vesicular stomatitis virus polymerase activity by an intramolecular dimerizer

Optogenetic dimerization modules are widely used to regulate protein function, yet their behaviour within folded enzymes remains incompletely understood. Here, we engineered vesicular stomatitis virus by inserting an intramolecularly tethered light-inducible dimerizer into the viral RNA-dependent RNA polymerase [large (L) protein], enabling assessment of its effects on early viral transcription and viral replication. Blue light illumination enhanced viral replication, but reduced temperature produced a similar increase in viral replication even in the absence of light. In addition, a thermally stabilized dimerizer variant supported robust replication under dark conditions. These observations suggest that early viral transcription and viral replication are influenced by the structural and biophysical properties of the inserted dimerizer and their temperature dependence, rather than by light alone. Structural modelling suggested that permissive insertion sites are located within or adjacent to regions with low predicted Local Distance Difference Test (pLDDT) scores, whereas non-permissive insertions showed greater predicted local structural deviations, particularly in the capping domain. Although these analyses are based on predicted structures, the observed structural differences are consistent with the functional differences among insertion sites. Together, our results show that intramolecular optogenetic modules enable light- and temperature-dependent modulation of early viral transcription and replication. While the structural analyses are based on predicted models, they provide a useful framework for identifying permissive insertion sites and generate testable hypotheses regarding how the structural and biophysical properties of the inserted dimerizer may influence polymerase function.