Fig. 3: The tethering partners VAP-B and PTPIP51 become enriched at MENCs to facilitate HCMV production.

A Average peptide abundances (scaled to the mean) of PTPIP51 (circle) and its ER tether VAP-B (hexagon) during HCMV infection, compared to all other ER-mitochondria MCS proteins (light purple). Shaded regions are standard error of the mean, and p-values are by one-way ANOVA to Mock (data is from MCS-PRM quantification as in Fig. 1, N = 6 biological replicates with ≥3 peptides/protein monitored in each replicate, see Supplementary Data 1 for complete list of peptides). B IF of endogenous PTPIP51 (yellow) during HCMV infection, showing regions from two different cells for each timepoint. Line-scans (below) are across ER (cyan) and mitochondria (red) junctions (arrows). Shown are 7×7 µm regions. C Fixed images (7×7 µm) from cells labeled for mitochondria (red), endogenous PTPIP51 (grey), and VAP-B (yellow) late in HCMV infection. Cartoon represents the observed co-localization of PTPIP51 and VAP-B at MENCs. D Images from live cells labeled for VAP-B (yellow), ER (cyan), and mitochondria (red). Top, 7×7 µm stills from two different cells for each timepoint; Lower, 2-second intervals from the region in C (2.5-minute movie, timepoints above). Arrows indicate points of VAP-B accumulation at ER-mitochondria contacts. E, F Scoring PTPIP51 and VAP-B localization from images in B and D (≥20 mitochondria/cell for PTPIP51 N = 59, 35, 43, 35, 21, 43 cells corresponding to three independently collected experiments and VAP-B N = 35, 18, 35, 33, 21, 31 cells corresponding to two independently collected experiments in Mock, 24, 48, 72, 96, and 120 hpi, respectively; error bars are SEM; ***p ≤ 0.0001 by two-way ANOVA to Mock). G Stills (7×7 µm) from live-cell images of cells labeled for mitochondria (red, mito-BFP) and VAP-B (yellow, eGFP-VAP-B) at 96 hours post-HCMV infection. Arrows indicate stable VAP-B fibers along the mitochondrial length. Timepoints are indicated above. H Proximity ligation assay (PLA) of endogenous PTPIP51-VAP-B interactions during HCMV infection. Left, Violin plots of PLA intensity (solid line is mean, dotted lines are quartiles, N ≥ 38 cells/timepoint, ***p ≤ 0.0001 by two-tailed student’s t-test to Mock). Right, Image of the PLA (yellow) analysis at 120 hpi, with regions near the viral AC (top) and ER-mitochondria co-localization (lower). More examples shown in Supplementary Fig. 9. I Virus titers of PTPIP51 KD versus siRNA control, collected at 120 hpi (Log₁₀ scale, N = 4, ***p ≤ 0.0001 by two-tailed student’s t-test to siCtrl).