Extended Data Fig. 4: PRV pUL36 WD motifs are required for kinesin-1 binding.

(A) Schematic representation of two potential bipartite kinesin-binding motifs (WD1/WD2 and WD3/WD4) in region 5 of pUL36. Corresponding regions from ten different alphaherpesvirinae are aligned. Prospective WD motifs in PRV are highlighted in red. Conserved tryptophans and/or surrounding E, D, N and Q at positions ±1 and +2 in pUL36 orthologs are in blue. The number of amino acids between the WD3 and WD4 tryptophans are indicated only when no other tryptophan was present in between (W-W); otherwise, n.a. is indicated. Table on the right summarizes mutants used for transient expression (RKB, reduced kinesin binding mutant that was subsequently introduced into PRV – see Extended Data Fig. 5 & 6). (B–D) Co-immunoprecipitations of pUL36 proteins and endogenous cellular Kif5 demonstrate that WD3 and WD4 contribute to the pUL36-Kif5 interaction. Interaction with dynactin component, p150/glued (p150) was used as a positive co-immunoprecipitation control (n = 3 independent experiments each). (E) Quantitation of cells displaying pUL36 localization at the cell periphery or juxtanuclear between 24-28 hpt. Representative images of cells and scoring are provided at top. Percentile distributions of pUL36 accumulation are illustrated by Venn diagrams (average of n = 3 independent experiments >250 cells each). Scale bars are 20 µm.