Russell Barkley, Cornell University

Jack Crowley, Cornell University Andrew Brodrick, University of Maryland Warren Zipfel, Cornell University John Parker, Cornell University

Virus Replication: Entry, Exit and Everything in Between

Viruses concentrate their components into compartments for replication and assembly, and phase separation often underlies the formation of these viral compartments. Phase-separated inclusions have liquid-like properties that are often characterized with fluorescent protein (FP) tags despite the unknown and understudied effects of FP tags on phase separation. We expressed FP-tagged µNS constructs (FP::µNS) in cells to study the major component of mammalian orthoreovirus viral factories and we quantified the dynamics of fluorescent µNS inclusions. We found that FP::µNS inclusions coalesced with internal molecular rearrangement and that µNS had a defined saturation concentration for droplet formation, consistent with our hypothesis that reovirus µNS forms phase-separated compartments. With an interest in the intrinsic factors influencing FP::µNS nucleation, we monitored the emergence of nascent diffraction-limited puncta and measured the droplet nucleation rates of full-length µNS and a minimal(er) region variant and found that the first 471 residues of µNS enhanced droplet formation. By quantifying the droplet density of condensates formed by untagged µNS to that of FP::µNS constructs, we found that FP tags enhanced the formation of µNS condensates in an FP-dependent manner. We then used a panel of µNS mutants that do not phase separate to show that FP tags variably rescued µNS phase separation. We screened nine FP tags and found one that minimally perturbed µNS condensation. This work shows that reovirus µNS forms phase-separated inclusions and we uncovered an important and previously unrecognized problem with FP tags affecting the condensation of biomolecular condensates.