(A) Structural comparison of PML mutation sites in JCPyV VP1 (PDB 3NXG) with MuPyV VP1 (PDB 5CPU) residues (Buch et al., 2015; Neu et al., 2010). (B) Kidney LT mRNA levels in mice 4 dpi with A2 or mutant viruses s.c. Data are from two independent experiments, n = 9–10 mice. For A2 vs. A2.V296F p=0.0008, A2 vs. A2.V296Y p=0.0100, A2 vs. A2.N293F p=0.0014, and A2 vs .A2.N293Y p=0.0168. (C) Brain LT mRNA levels in mice 4 dpi with A2 or mutant viruses i.c. Data are from four independent experiments, n = 13–14 mice. For A2 vs. A2.V296F p>0.9999, A2 vs. A2.V296Y p=0.2576, A2 vs. A2.N293F p=0.9974, and A2 vs. A2.N293Y p=0.9928. (D) LT mRNA levels in NMuMG cells 24 hpi with A2.N293F or A2.N293Y preincubated with 8A7H5 or control IgG. Data are from two independent experiments, n = 12. For A2.N293F p<0.0001 and A2.N293Y p<0.0001. (E) Analysis of 8A7H5 avidity for mutant VP1’s using NH4SCN. Left: Relative binding of 8A7H5 to recombinant VP1 with increasing concentrations of NH4SCN. Right: Relative binding of 8A7H5 to recombinant VP1 at 2M NH4SCN. Each point is the average of two technical replicates in an independent repeat, n = 3. For WT vs. V296F p<0.0001, WT vs. N293Y p=0.6440, WT vs. T291D p=0.0003, WT vs. H139R p<0.0001, and WT vs. R77N p<0.0001. (F) Location of novel changes in the VP1 escape mutations indicated by underlines. (G) LT mRNA levels 4 dpi in the kidneys of mice infected s.c. Data are from two independent experiments, n = 8 mice. For A2 vs. A2.N80K p=0.3374, A2 vs. A2.∆294 p=0.0010, A2 vs. A2.∆295 p=0.3724, A2.N80K vs. A2.∆294 p<0.0001, A2.N80K vs. A2.∆295 p=0.9999, and A2.∆294 vs. A2.∆295 p<0.0001. (H) LT mRNA levels 4 dpi in the brains of mice infected i.c. Data are from 2 to 4 independent experiments, n = 8–16 mice. For A2 vs. A2.N80K p<0.0001, A2 vs. A2.∆294 p<0.0001, A2 vs. A2.∆295 p<0.0001, A2.N80K vs. A2.∆294 p=0.8811, A2.N80K vs. A2.∆295 p=0.9979, and A2.∆294 vs. A2.∆295 p=0.6869. (I) Viral shedding in the urine is impaired by V296F, despite antibody escape in the blood. Top: Experimental design for infection of μMT mice and 8A7H5 treatment. Bottom: Frequency of A2.V296F DNA in blood, urine, and brain tissue of mice. Data are from three independent experiments, n = 13–14 mice. For Blood vs. Urine p<0.0001, Blood vs. Brain p=0.0500, and Urine vs. Brain p=0.0028. Data were analyzed by one-way ANOVA (B, C, E Right, G, H, I) or multiple t tests (D). In E, comparisons were only between WT and each mutant. **p<0.01, ***p<0.001, ****p<0.0001.