SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

Figures

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  Fig 1. SUN1 is required for SV40 infection. (A) CV-1 cells transfected with a scrambled (Scr) control siRNA or siRNA against SUN1 or SUN2 were harvested and the resulting whole cell extracts subjected to SDS-PAGE and immunoblotting with the indicated antibodies. β actin was used as a loading control. (B) CV-1 cells transfected with the indicated siRNA were infected with SV40, fixed, and stained for large T antigen (T-Ag). Data were normalized to the Scr control. (C) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE and immunoblotting. (D) The T-Ag band intensity in C was quantified by the FIJI software. Data were normalized to the Scr control. (E) Schematic of full-length (FL) SUN1 containing an N-terminal GFP tag (GFP-SUN1 FL), and a truncated SUN1 lacking the coiled-coil and SUN domain with also an N-terminal GFP tag (GFP-SUN1 ΔLU). (F) CV-1 cells transfected with the indicated constructs were fixed and stained for GFP (green) and counterstained with DAPI (blue). Scale bar: 10 µm. (G) CV-1 cells transfected with the Scr control siRNA or siRNA against SUN1 were also transfected with either the…

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  Fig 2. SUN1 promotes nuclear targeting of SV40. (A) CV-1 cells transfected with either a scrambled (Scr) control siRNA or siRNA against SUN1 or SUN2 were infected with SV40 in the presence of actinomycin D (ActD). Cells were stained with anti-VP2/3 (green), mAb414 (red), and counterstained with DAPI (blue). Scale bar: 10 µm. (B) The percent of cells with a discrete VP2/3 + signal on or proximal to (i.e., within 0.3 µm) the nuclear membrane were quantified and normalized to the Scr control. (C) As in A, except stained with BAP31 (red). Scale bar: 10 µm. (D) CV-1 cells transfected with the indicated siRNA were infected with SV40 and processed using the ER-to-cytosol transport assay as described in ([20]). The resulting cytosol and membrane fractions were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. (E) The cytosol fraction from D was layered on a discontinuous sucrose gradient and centrifuged to generate individual fractions (see Materials and Methods). Fractions were subjected to SDS-PAGE followed by immunoblotting for VP1. The VP1 signal from each fraction was quantified for Scr and SUN1 KD cells relative to respective total signal across all…

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  Fig 3. SUN1 and Nesprin-2 act coordinately to recruit SV40. (A) Endogenous SUN1 was immunoprecipitated (IPed) from whole cell extracts derived from SV40-infected CV-1 cells. Bound DNA was eluted from the precipitated material and subjected to PCR to detect the SV40 genomic DNA. (B) CV-1 cells expressing the indicated FLAG-tagged constructs were infected with SV40. FLAG-tagged proteins were IPed from the resulting whole cell extracts, the precipitated samples eluted and subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. * indicates degraded SUN proteins. (C) As in B, except the precipitated samples were subjected to PCR to detect the SV40 genome. (D) Nesprin-2 was IPed from whole cell extracts derived from CV-1 cells expressing the indicated constructs. The precipitated samples were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. * indicates degraded SUN proteins (E) As in B, except SUN1 ΔLU-3xFLAG was also used. (F) CV-1 cells transfected with the indicated siRNA and construct were infected with SV40. FLAG-tagged proteins were IPed from the resulting whole cell extracts and processed as in B. * indicates degraded SUN1 protein. (G)…

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  Fig 4. The SUN domain of SUN1 is crucial to promote SV40 binding and infection. (A) Schematic of full-length N-terminal FLAG-tagged SUN1 and SUN2, and of chimeric SUN proteins with swapped SUN domains. (B) CV-1 cells transfected with the indicated construct were fixed, stained for FLAG (green), and counterstained with DAPI (blue). Scale bar: 10 µm. (C) CV-1 cells transfected with the indicated construct and siRNA were infected with SV40, fixed, and stained for T-antigen, FLAG, and counterstained with DAPI. The percentage of transfected cells expressing T-antigen was quantified and normalized to the GFP + Scr control condition. (D) CV-1 cells transfected with the indicated construct were infected with SV40. FLAG-tagged proteins were IPed from the resulting whole cell extracts. The precipitated materials were subjected to PCR to detect SV40 genomic DNA or subjected to SDS-PAGE and immunoblotting. The dotted line indicates intervening lanes were removed with adjacent lanes spliced from the same immunoblot. ** p ≤ 0.01.

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  Fig 5. KPNA4 is important for SV40 infection. (A) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE followed by immunoblotting. The extent of depletion by the KPNA1, KPNA2, and KPNA4 siRNA is shown in the Western blots below. Data were normalized to the Scr control. (B) The T-Ag band intensity in A was quantified by the FIJI software. Data were normalized to the Scr control. (C) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE and immunoblotting. (D) The T-Ag band intensity in C was quantified by the FIJI software. Data were normalized to the Scr control. (E) CV-1 cells were fixed and stained for Nesprin-2 (red) and either one of the KPNA1, KPNA2, or KPNA4 protein (green) and counterstained with DAPI (blue). Scale bar: 10 µm. (F) Pearson’s coefficient was used to quantify colocalization between KPNA1, KPNA2, or KPNA4 with Nesprin-2. Each data point represents one field of view with at least 10 cells. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

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  Fig 6. KPNA4 binds to SV40 after Nesprin-2-SUN1-dependent targeting to promote virus nuclear entry. (A) Endogenous KPNA4 was IPed from whole cell extracts derived from SV40-infected CV-1 cells. The precipitated samples were subjected to SDS-PAGE and immunoblotting. (B) As in A, except CV-1 cells were subjected to SUN1 KD. (C) CV-1 cells transfected with the indicated siRNA and SUN1-3xFLAG were infected with SV40. SUN1-3xFLAG was IPed from the resulting whole cell extracts, and the precipitated samples subjected to SDS-PAGE and immunoblotting. (D) The VP1 band intensity in C was quantified by the FIJI software. Data were normalized to the Scr condition. (E) As in Fig 2A, except cells were depleted of KPNA4 and stained for FLAG (red), VP2/3 (green), and counterstained with DAPI (blue). Scale bar: 10 µm. (F) Manders’ coefficient was used to quantify overlap of VP2/3 + signal on SUN1-3xFLAG. Each data point represents one cell. (G) CV-1 cells transfected with the indicated siRNA were infected with SV40. Cells were fixed and stained for mAb414 (red), VP2/3 (green), and counterstained with DAPI (blue). Scale bar: 10 µm. (H) The percent of cells with a discrete VP2/3 + signal in the nucleus in…