Fig 2: The NPC subunit NUP188 binds to Nesprin-2 and promotes SV40 nuclear entry.(A) CV-1 cells were infected with SV40 (MOI ~1) and treated with either a dimethyl sulfoxide (DMSO) control or importazole. At 24 hpi, cells were fixed and stained for large TAg. Data were normalized to the DMSO control. (B) Unique peptides corresponding to SV40-interacting host proteins identified by IP mass spectrometry [32]. (C) CV-1 cells were transfected with scrambled control siRNA, 50 nM siRNA against NUP35, NUP88, NUP93, NUP98, and NUP133, or 10 nM siRNA against NUP188 for 48 h. Protein levels were assessed by immunoblotting. Hsp90 was used as a loading control. (D) CV-1 cells were transfected as in (C) and infected with SV40 (MOI ~1) for 24 h. Cells were fixed and stained for large TAg. Data were normalized to the scrambled control. (E) CV-1 cells were infected with SV40 (MOI ~25) for 20 h. Endogenous NUP188 was immunoprecipitated (IP) from whole-cell extracts and the eluted samples subjected to SDS-PAGE followed by immunoblotting for VP1. (F) CV-1 cells were infected with SV40 (MOI ~25) for 20 h. Endogenous Nesprin-2 was immunoprecipitated (IP) from whole-cell extracts and eluted samples subjected to SDS-PAGE followed by immunoblotting for NUP188 and BICD2. (G) CV-1 cells were transfected with either the scrambled control siRNA or siRNA against Nesprin-2 for 48 h. Cells were then infected with SV40 (MOI ~25) for 20 h and endogenous NUP188 immunoprecipitated from whole-cell extracts. The samples were subjected to SDS-PAGE followed by immunoblotting for VP1. (H) As in (G), except samples were immunoblotted for NUP93. (I) Confocal analysis of CV-1 cells that were transfected with either scramble control siRNA or siRNA against NUP188 for 48 h and then infected with SV40 (MOI ~25) for 20 h in the presence of 1 µM actinomycin D (ActD). Cells were stained with anti-VP2/3 (green) and anti-MAB414 that stains the NPC (red). Cells were counterstained with DAPI (blue). White arrow = VP2/3 at nuclear membrane, orange arrow = VP3 inside the nucleus. (J) Quantification of the percentage of cells in (I) with VP2/3 at the nuclear membrane normalized to the scrambled control. Actual values are shown in parenthesis. (K) As in J, except the percentage of cells with VP3 inside of the nucleus is quantified. Actual values are shown in parenthesis. Values are averages of the means (n = 3) ± SD. A standard Student’s t test was used to determine statistical significance. **, P = 0.005; ***, P = 0.0005; ****, P = 0.0001. Scale bars, 10 µm.

Fig 3: ATR interactors.a Schematic summary of ATR interactors and their relative roles at the nuclear envelope and perinuclear areas b Validation of Nesprin-2 and ATR interaction using GFP-ATR immunoprecipitation and western blot analysis (full blots of Nesprin-2 with molecular weights included in Supplementary Fig. 4h and in Source data file). c Example of ATR–Nesprin-2 interaction foci generated by proximity ligation assay (PLA) in interphase and prophase cells (scale bar = 10 µm). d Quantification of total number of foci present in interphase and prophase cells (n = 65 for interphase and 10 for mitosis, pooled from 2 independent experiments). Graph presented as mean ± SEM. p-value calculated using two-tailed Student’s t-test.

Fig 4: Model.Following virus escape from the endoplasmic reticulum (ER) at the viral-induced ER-foci and BICD2-dependent disassembly in the cytosol, SV40 is transported towards the nucleus where it is captured at the nuclear membrane through direct binding to Neprin-2 (step 1). Nesprin-2 is physically positioned proximal to the nuclear pore complex (NPC), in part, through its association with nucleoporin NUP188. This enables the successful transfer of SV40 from Nesprin-2 into the nucleoplasm through the NPC (step 2). During nuclear entry, the viral particle undergoes a final disassembly step such that the VP1 and VP3, but not VP2, capsid proteins accompany the viral genome into the nucleus to cause infection. The fate of VP2 is unknown. Inset: SV40 structural organization. The circular, double-stranded DNA viral genome is encased within a capsid shell composed of 72 pentamers of the major capsid protein VP1 (teal). Inside, each pentamer is associated with one copy of either the VP2 (beige) or VP3 (blue) minor capsid proteins.

Fig 5: ATR preserves nuclear mechanics.a, b Elastic modulus measurements using AFM. a Cellular stiffness (n = 171, 161, and 179 measurements for shCtrl, shATR1, and ShATR2, respectively) and b stiffness of isolated nuclei from control and ATR-depleted cells (n = 144, 110, and 98 measurements; N = 2 independent experiments). c Membrane phospholipid composition analysis; total cell and nuclear membrane PC/PE ratio, nuclear PC/PE ratio of individual species (from three biological and two technical replicates; two-way ANOVA test; Bonferroni’s multiple comparisons test; N = 2 independent experiments). d FLIM-FRET analysis of cells expressing H2B-GFP and H2B-mCherry; sample images of Fluorescent lifetime (FLIM-FRET index) and overall Lifetime in Hela cells infected with shATR (n = 13) or control (n = 7), or treated with DMSO (n = 10) or ATR inhibitor for 4 h (n = 8). e Examples of FRET signals at the NE as measured by Nesprin-2 FRET sensor in control, shATR, and control cells in the presence of ATR inhibitor (3 h), and quantification of FRET signals (n = 112, 88, and 96 measurements for shCtrl, shATR1, and ShATR2 respectively; data pooled from two or three independent experiments). f Immunofluorescent images of YAP cellular distribution in control and shATR cells, (below) quantifications of nuclear to cytoplasmic YAP signal ratio (n = 69, 66, and 58 shCtrl, shATR1, and ShATR2, respectively). g Western blotting of ser127-phosphorylated YAP (N = 2 independent experiments. Uncropped images available in Source Data file). Scale bar is 20 µm in all images. Bar graphs presented as mean ± SEM and box plot whiskers and outliers plotted using Tukey’s method in prism7 software. p-values calculated using one-way ANOVA test with Tukey’s multiple comparisons test (for d) or Dunnett’s multiple comparisons test (for a, b, e, f). (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; n.s., not significant).