Extended Data Fig. 5: The PRV[RKB] mutant is defective for nuclear trafficking and gene expression.

(A) Mutation of WD3 attenuated PRV spread, as measured by plaque size, whereas mutations of WD4 rendered PRV non-viable (n = 200 plaques over 3 independent experiments). (B) Mutation of the WD4 tryptophan alone also rendered PRV non-viable, whereas mutation of the surrounding acidic residues did not impair viral spread. PRV encoding both the ∆WD3 mutation and WD4(DE>AA) attenuated viral spread but could be propagated and further studied and was designated as the reduced-kinesin binding mutant (RKB; also see Figure S4). Plaque diameters were measured 43 hpt. Data are presented as scatter plots, with each dot representing a single plaque (n = 200 plaques over 3 independent experiments). Mean values ± SD are indicated. (P-values were determined by one-way analysis of variance with a post-hoc Tukey test). (C) Incoming capsids of PRV encoding the pUL36 RKB mutant accumulate juxtanuclear in explanted chick dorsal root ganglia (DRG). Representative images of neural soma (transmitted and fluorescent capsid image pairs with nuclei highlighted by dotted lines). Percentile distributions of juxtanuclear capsid accumulation are illustrated at right (n= 3 independent experiments > 500 infected cells each). DRG explants were imaged between 2.5-3 hpi. Scale bars are 20 µm. Mean values ± SD are indicated. (P values based on two-tailed unpaired t test). (D) PRV[RKB] has delayed viral gene expression. PK15 cells were infected at MOI 10 and total RNA was collected at 4 hpi. Relative IE180 mRNA levels were measured by RT-PCR first-strand DNA synthesis and qPCR amplification using primers specific for PRV IE180 and the swine ribosomal S28 rRNA (loading control). Data was normalized to S28 rRNA levels and depicted as a fold change with respect to IE180 mRNA levels during WT infection (n = independent experiments). Mean values ± SD are indicated. (P values based on two-tailed unpaired t test).