The pUL37 R2 region is dispensable for PRV anterograde axonal transport in culture.

Dissociated DRG neurons were infected in 2 ml of media with 2.5 × 10⁶ PFU/ml of either wild-type PRV (WT) or R2-mutant PRV (R2) encoding a pUL25/mCherry capsid fusion. (A) Egressing capsids were imaged by time-lapse fluorescent microscopy from 10–13 hpi. Capsids were tracked in isolated axons that were unambiguously contiguous with an identifiable neuronal soma, thereby ensuring that all transport included in the analysis was anterograde directed. More than 30 capsids were tracked per virus per experimental replicate. Three independent experimental replicates were completed and capsid velocities and run lengths for each replicate determined. Gaussian (velocity) or decaying exponential (run length) curves were fit by nonlinear regression (r² > 0.97 for all four curve fits). The net displacement represents the total distance away from the soma that individual capsids moved over a 10 sec recording (bottom panel). Values are mean ± s.d. (**, p < 0.01; n.s., not significant based on two-tailed unpaired t test). (B) The number of capsids in axon terminals at 17–18 hpi was quantified across three independent experiments with at least 10 terminals examined per virus per experiment. Inset graph indicates average capsid number per axon terminal. Values are mean ± s.e.m. (n.s., not significant based on two-tailed unpaired t test). Representative images of capsid accumulation at axon terminals are shown at right.