Interaction of the Mouse Polyomavirus Capsid Proteins with Importins Is Required for Efficient Import of Viral DNA into the Cell Nucleus

Figures

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  Figure 1. In vivo interactions between importin β1 and viral particles at early times post infection. Western blots of 3T6 cell lysates (labeled as Input) or complexes precipitated through antibody against importin β1 (labeled as IP) were performed with antibody against importin β1 (A), VP1 (B) or VP2/VP3 (C,D). As a negative control (NC), samples precipitated through nonspecific anti-mouse IgG were used. The viral DNA was isolated from complexes and amplified by PCR at indicated times post infection (E).

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  Figure 2. Proximity ligation assay of VP1 and importin β1 at 6 and 8 hpi. (A) PLA assay was performed on 3T6 cells infected with MPyV (200 virus particles per cell), using primary mouse and rabbit antibodies against VP1 or mouse antibody against VP1 and rabbit antibody against importin β1. Next, the oligoprobes tagged anti-mouse and anti-rabbit antibodies were used. Red spots represent the products of amplification after oligonucleotide ligation. DNA was stained by DAPI. As controls, the infected cells were stained with two antibodies (mouse and rabbit) against VP1 and non-infected cells were stained with anti-VP1 and anti-importin β1 antibodies. At the top of each image, the average numbers of the PLA spots quantified in three independent experiments are presented (for each experiment 50 cells were analyzed). The pictures were taken 20× magnification; (B) The graph represents the mean values of three independent experiments ± SD. Samples were compared by the Student’s t-test. p values are given and asterisks represent statistically significant differences (** p ≤ 0.01).

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  Figure 3. Mutagenesis design. Wild type MPyV overlapping coding sequences for VP1 and VP2/3 genes are displayed with the corresponding aa at the top of the nucleotide triplet for VP1 or at the bottom of the nucleotide sequence for VP2/3 (brackets show the codons). Positions of aa residues are given for VP1 (top) and VP2 (bottom) proteins, respectively. For better differentiation, codons for VP1 are alternate in bold and not bold letters. Mutants 1, 2, and 3 are also presented. For better display, the basic aa (K and R) are shown in red and the mutated nucleotides and corresponding aa are shown in green. NucPred score values for NLS of VP1 and VP2/3 capsid proteins of the wt or mutated variants are presented on the right.

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  Figure 4. Characterization of the mutated viruses. The wt virus or mutant viruses 1, 2, and 3, isolated from transfected cells were contrasted by phosphotungstic acid and visualized by electron microscopy (Bars = 100 nm) (A); Presence of the capsid proteins was detected by Western blot using specific antibody against VP1 and VP2/3 (B).

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  Figure 5. Effects of mutations in the NLS of VP1 and VP2/3 on viral infection and on binding of viruses to importin β1. (A) 3T6 cells were infected with wt or mutant viruses 1, 2, or 3 (using virus equivalent representing 1.5 × 104 genomes per cell). Cells were fixed at 24 hpi and LT was stained by specific antibody. The presented data correspond to the mean of three independent experiments +/− standard deviation (s.d.). At least 300 cells were counted per experiment. Samples were compared by the t-test. p values are given and asterisks represent statistically significant differences (* p ≤ 0.1, ** p ≤0.01 *** p ≤ 0.001); (B) PLA assay was performed in 3T6 cells at 6 hpi with wt MPyV or the mutant 2 virus (200 virus particles per cell). For this experiment, we used primary mouse and rabbit antibodies against VP1 or mouse antibody against VP1 and rabbit antibody against importin β1. Next, the oligoprobes tagged anti-mouse and anti-rabbit antibodies were used. Red spots represent the products of amplification after oligonucleotide ligation. DNA was stained by DAPI. As controls, the infected cells were stained with two antibodies (mouse and rabbit), both against VP1 and non-infected cells…

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  Figure 6. Effect of mutations in NLS of MPyV structural proteins VP1, VP2, and VP3 on their cellular localization during infection. Confocal sections of 3T6 cells infected with wt (m–p) or mutated viruses 1 (a–d), 2 (e–h), or 3 (i–l). Cells were fixed at 24 hpi and stained by antibodies against VP1 (red), VP2/3 (green), and DNA by DAPI (blue). Bars 5 µm.

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  Figure 7. Subcellular distribution of wild type or mutated VP1 capsid proteins. Confocal sections of 3T3 cells expressing wt or mutated VP1 proteins. Cells were fixed at 24 hpt and stained with anti-VP1 antibody (green) and DNA by DAPI (blue) (A); For analysis of subcellular distribution of wt (i–ii) or mutated proteins (iii–v), 50 cells were examined for each variant. Bars 5 µm. Scores are given as percentage (B).

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  Figure 8. Subcellular localization of wild type or mutated VP2 proteins. Confocal sections of 3T3 cells expressing wt or mutated VP2 proteins. Cells were fixed at 5 hpt and stained by antibodies against VP2 (green) and DNA by DAPI (blue). Bars 5 µm (A); Scores are given as percentage (B).

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  Figure 9. Nuclear entry of wt and mutant variants of the VP1 and VP2 proteins. Confocal sections of 3T3 cells co-expressing combinations of wt VP1 and wt VP2 (i) or wt VP1 and mutated variants of VP2 (ii–iv) or wt VP2 with the mutated variant of the VP1 (v). Cells were fixed at 24 hpt and stained with antibodies against VP1 (red) and VP2 (green). DNA was stained by DAPI (blue). Bars 5 µm.