Fig 1: HSV-1 promotes the release of p-tau from infected neurons and the accumulation of p-tau in uninfected neurons adjacent to infection.ReNcell VM cultures were infected with HSV-1 for 24 hours to characterize p-tau’s extracellular release and changes in proximity to viral infection. a−d, 3.5−4-week-old 3D ReNcell VM cultures were infected with serial dilutions of HSV-1 for 24 hours, and cell media and cell lysates were analyzed by MSD Multi-Spot Phospho (Thr 231)/Total Tau Assay for soluble p-tau and total tau. a, Comparison of cell media soluble p-tau at different viral loads (F3, 54 = 7.521 ((uninfected versus 2.55, ***P = 0.0005), (uninfected versus 5.10, ***P = 0.0004)). b, Comparison of cell media soluble total tau at different viral loads (F3, 55 = 10.49, **P = 0.0011). c, Comparison of the ratio between cell media p-tau and total tau at different viral loads (F3, 54 = 19.94, ****P < 0.0001). d, Comparison of the ratio between cell lysate soluble p-tau and soluble total tau at different viral loads (F3, 96 = 202.5, ****P < 0.0001). e, 2D ReNcell VM cultures in microfluidic devices were infected in the left chamber with HSV-1 for 48 hours, immunoprobed with anti-p-tau (PHF1) labeled with a fluorescent secondary antibody and analyzed for neurons (GFP), HSV-1 (RFP) and p-tau (647) fluorescence by confocal microscopy. Fluorescence signals for ReNcell VM, HSV-1 and p-tau were imaged for infected and uninfected conditions. f, GA3 analysis of HSV-1-positive neurons compared intracellular p-tau fluorescence among infected neurons (triangles), uninfected neurons proximal to infected neurons (*) and uninfected neurons not proximal to infected neurons (arrows) (F2, 3,288 = 45.35, ****P < 0.0001). g, Individual neuronal p-tau fluorescence intensity was compared to total proximal HSV-1−RFP fluorescence intensity (R2 = 0.7912). Box plots are representative of ±s.e.m. ((a−c, n = 13), (d, n = 25)) depicting median and interquartile range, with whiskers denoting variability according to Tukey’s method. Statistical mean comparisons were calculated by one-way ANOVA using Dunnett’s multiple comparisons test (a−d), one-way ANOVA using Tukey’s multiple comparisons test (f) and simple linear regression (g).Source data
Fig 2: Tau binding to HSV-1 viral capsid proteins is potentiated by microtubule-binding repeats and phosphorylation.Dilutions of synthetic tau isoforms were incubated with heat-immobilized HSV-1 capsid or whole virion in indirect and competitive ELISAs to measure tau−virus binding affinity. a, 2N4R GSK-3β p-tau binding between HSV-1 isolated capsid or whole virion was compared after normalization of available binding sites assessed using VP21/VP22a antibody (****P < 0.0001). b, Dilutions of 2N4R GSK-3β p-tau (scale of 5 µg ml−1 to 0.3125 µg ml−1) were incubated with immobilized HSV-1 capsids (F4, 55 = 43.18, ****P < 0.0001). c, The binding affinity of 2N4R GSK-3β p-tau to HSV-1 capsids was compared to 2N3R, 50/50 mix of 2N3R/2N4R and 2N4R tau (F3, 44 = 8.996, *P = 0.267 ((2N4R GSK-3β p-tau versus 2N3R tau, ***P = 0.0001), (2N4R GSK-3β p-tau versus 2N3R/2N4R tau, ***P = 0.0007)). d,e, Tau adhesion inhibition was assessed by preincubation of immobilized HSV-1 capsids with an anti-VP21/VP22a antibody (F8, 32 = 7.071, ****P < 0.0001) (d) or an anti-pUL48-VP16 antibody (F8, 32 = 3.978, ***P = 0.004, ****P < 0.0001) (e) before exposure to 2N4R GSK-3β p-tau. f, Tau adhesion inhibition was repeated with the anti-VP21/VP22a antibody using a mannose-incubated 2N4R GSK-3β p-tau. Box plots are representative of ±s.e.m. (n = 12) depicting median and interquartile range, with whiskers denoting variability according to Tukey’s method. Statistical significance was calculated by two-tailed Mann−Whitney test (a) and one-way ANOVA using Tukey’s multiple comparisons test (b−f).Source data
Fig 3: HSV-1 induces intraneuronal aggregation of p-tau and neuritic dystrophy.3D ReNcell VM cultures were infected with HSV-1 to characterize tau’s change in cellular distribution and levels in response to viral infection. As shown in a, 3.5−4-week-old 3D ReNcell VM cultures were infected with a replication-deficient HSV-1 (deletion of UL28 protein) for 24 hours, immunoprobed with anti-p-tau (PHF1) and anti-HSV1 labeled with a fluorescent secondary antibody and analyzed for neurons (GFP), HSV-1 (568) and p-tau (647) fluorescence by confocal microscopy. b,c, Fluorescent image captures from 384 wells over four experiments were compared by GA3 software in Nikon Elements for p-tau fluorescent neurites (triangles) (***P = 0.0002) (b) and cell bodies (arrows) (*P = 0.0022) (c) between uninfected and infected wells. d, Confocal images showing 3D ReNcell VM cultures that were infected with HSV-1 for 24 hours, immunoprobed with anti-p-tau (PHF1) labeled with a fluorescent secondary antibody and immunostained with thioflavin S. e, Pearson’s correlation coefficient (error bars represent 95% confidence interval) was derived from analyzing uninfected and infected wells for co-localization of thioflavin S (green), HSV-1 (RFP) and p-tau (647) fluorescence. f−k, 3.5−4-week-old 3D ReNcell VM cultures were infected with serial dilutions of HSV-1 for 24 hours, and cell lysates were separated into soluble and insoluble tau fractions for analysis by MSD Multi-Spot Phospho (Thr 231)/Total Tau Assay. f, Comparison of cellular soluble p-tau at different viral loads (F3, 96 = 129.6, **P = 0.0027, ****P < 0.0001). g, Comparison of cellular insoluble p-tau at different viral loads. h, Comparison of the ratio between cellular insoluble and soluble p-tau at different viral loads (F3,96 = 26.54, ****P < 0.0001). i, Comparison of cellular soluble total tau at different viral loads (F3, 96 = 9.251, **P = 0.0040, ***P = 0.0001, ****P < 0.0001). j, Comparison of cellular insoluble total tau at different viral loads (F3, 89 = 6.810, **P = 0.0049, ***P = 0.0002). k, Comparison of the ratio between cellular insoluble and soluble total tau at different viral loads (F3, 89 = 7.469, **P = 0.0057, ****P < 0.0001). Box plots are representative of ±s.e.m. ((f−h, n = 25), (i−k, n = 19)) depicting median and interquartile range, with whiskers denoting variability according to Tukey’s method. Statistical mean comparisons were calculated by two-tailed unpaired t-tests (b,c) and one-way ANOVA using Dunnett’s multiple comparisons test (f−k).Source data
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