Master mitotic kinases regulate viral genome delivery during papillomavirus cell entry

Abstract

Mitosis induces cellular rearrangements like spindle formation, Golgi fragmentation, and nuclear envelope breakdown. Similar to certain retroviruses, nuclear delivery during entry of human papillomavirus (HPV) genomes is facilitated by mitosis, during which minor capsid protein L2 tethers viral DNA to mitotic chromosomes. However, the mechanism of viral genome delivery and tethering to condensed chromosomes is barely understood. It is unclear, which cellular proteins facilitate this process or how this process is regulated. This work identifies crucial phosphorylations on HPV minor capsid protein L2 occurring at mitosis onset. L2's chromosome binding region (CBR) is sequentially phosphorylated by the master mitotic kinases CDK1 and PLK1. L2 phosphorylation, thus, regulates timely delivery of HPV vDNA to mitotic chromatin during mitosis. In summary, our work demonstrates a crucial role of mitotic kinases for nuclear delivery of viral DNA and provides important insights into the molecular mechanism of pathogen import into the nucleus during mitosis.

Fig. 1. A highly conserved SSTP motif on HPV16-L2 is essential for the chromosomal association during mitosis.

A Alignment of minor capsid protein L2 sequences for the indicated human and animal PVs (UniProtKB P03107, P06793, P17389, P06918, P03109, and Q84358) was generated using PRALINE (Bawono and Heringa, 2014). B Chromosomal association assay of ectopically expressed wild type and mutant L2-EGFPs in HeLa H2B-mCherry cells during mitosis; images display representative single medial planes of spinning disk confocal microscopy with L2-EGFP in green and H2B-mCherry in red as indicated. Scale bar: 10 µm. C Quantification of B, displaying the chromosomal association index (CAI) of individual cells (circles), i.e., the intensity of H2B-mCherry-overlapping EGFP signal over total intensity. Values were normalized to EGFP alone (0) and L2-GFP (1). 50 cells from three independent experiments were analyzed. D HeLa cells were infected with L2-WT and mutant HPV16 PsVs as indicated. Infectivity was assessed 48 h post infection (p.i.) by scoring GFP-positive cells by flow cytometry. Virus amounts correspond to 1× = 25 ng L1, 10× = 250 ng L1. Displayed is the average of three independent experiments ± standard deviation (SD). E, G HeLa cells were infected with WT and SSTP212AAAA L2 mutant HPV16-EdU PsV for 20 h. Displayed are representative medial confocal slices of the subcellular localization of vDNA (EdU, green) and nuclei (Hoechst–blue) in interphase (E) and mitotic G cells. Scale bars: 5 µm. F Quantification of E, and H quantification of G for three independent experiments with eight cells/experiment. The overlap of vDNA/chromatin was quantified using intensity-based colocalization analysis (IMARIS Coloc function). Displayed is the average of three independent experiments ± SD. For all bar graphs, colored dots represent data points of individual experiments. For all quantifications, statistical significance was assessed by two-tailed Student’s t test to wildtype (WT). Source data are provided as a Source Data file.

Fig. 2. L2 is phosphorylated by CDK1 and PLK1 during mitosis.

A HeLa cells were transfected with WT and SSTP212AAAA L2-EGFP expression constructs, and subsequently arrested in G₁/S- or M-phase using aphidicolin (3 µM) or nocodazole (330 nM), respectively. Depicted is a representative example of five independent experiments from Western blot analysis against L2 (Santa Cruz #sc-65709) of SDS-PA and Phos-Tag gel electrophoresis of cell lysates. Red numbers annotate different L2 phosphorylation bands. B As in A, but with L2-EGFP only. HeLa cells were arrested in S-phase by double thymidine block. Cell lysates from indicated time points after block release were subjected to analysis as in A. Depicted is a representative example of five independent experiments from Western blot analysis of SDS-PA and Phos-Tag gel electrophoresis. Red numbers annotate different L2 phosphorylation bands. C HEK293 cells were transfected with a WT L2-3xHA expression construct. Subsequently cells were arrested in G₁/S-phase (left), in mitosis after double thymidine arrest in S-phase and release into nocodazole (M, middle), or in the subsequent G₁/S-phase (right). L2 was immunoprecipitated from cell lysates (input) using an HA antibody and analyzed by Western blotting against indicated targets. Depicted is a representative example of three independent experiments. D HEK293 cells were transfected with WT and SSTP212AAAA L2-3xHA constructs, arrested in mitosis or G₁/S by nocodazole (330 nM) and aphidicolin (3 µM), and subjected to immunoprecipitation (IP) using an anti-HA.11 antibody (BioLegend #901516). IPs were subsequently prepared for mass spectrometry analysis. Depicted is a representative LC-MS/MS spectrum of mitotic L2-3xHA peptides of three independent experiments containing the phosphorylated L2 SSTP motif; numbers between parenthesis represent phosphorylation probabilities of residues. E In vitro kinase assay of WT and SSTP212AAAA purified 6xHis-L2 in presence of 6xHis-PLK1, or of 6xHis-CDK1-CyclinB:CKS1 (CCC) complex, or of both kinases. Depicted are representative examples from Western blot analysis against L2 (Santa Cruz sc-65709), PLK1 (Abcam ab17057) and CDK1 (Santa Cruz #sc-54) of SDS-PA and Phos-Tag gel electrophoresis of five independent in vitro kinase assays. Red numbers indicate differential phosphorylation states of L2. Source data are provided as a Source Data file.

Fig. 3. L2 SSTP motif is essential for interaction with PLK1 through the Polo-box domain.

A HEK293 cells were transfected with WT and SSTP212AAAA mutant L2-3xHA expression constructs, and subsequently arrested in S-phase, M-Phase or at the G2/M border using aphidicolin (3 µM), nocodazole (330 nM) or RO-3306 (9 µM). Depicted is a representative example from Western blot analysis against HA tag (BioLegend #901516), PLK1 (Abcam ab17057), or GAPDH (Proteintech #10494-1-AP) as loading control of SDS-PA gel electrophoresis of endogenous PLK1 immunoprecipitates (Abcam ab17057). B HEK293 cells were transfected with WT and SSTP212AAAA, S213A, T214A or P215A L2-3xHA expression constructs, and subsequently arrested in M-Phase using nocodazole (330 nM). Depicted is a representative example from Western blot analysis against HA tag (BioLegend #901516), and endogenous PLK1 (Abcam ab17057), of SDS-PA gel electrophoresis of PLK1 immunoprecipitates (Abcam ab17057). C HEK293 cells were co-transfected with WT L2-3xHA and 3xFLAG-PLK1 or 3xFLAG-Ran expression constructs. Depicted is a representative example from Western blot analysis against L2 (Santa Cruz sc-65709), against FLAG tag for 3xFLAG-PLK1 and 3xFLAG-Ran (Sigma-Aldrich #F1804), against GAPDH (Proteintech #10494-1-AP) and of SDS-PA gel electrophoresis of FLAG tag PLK1 and Ran immunoprecipitates (Sigma-Aldrich #F1804). D HEK293 cells were co-transfected with WT L2-3xHA and myc-PBD or myc-Vps26B constructs. Depicted is a representative example from Western blot analysis against L2 (Santa Cruz sc-65709), against myc tag for PBD and Vps26B (ThermoFisher #13-2500), against GAPDH (Proteintech #10494-1-AP) and of SDS-PA gel electrophoresis of myc tag PBD and Vps26B immunoprecipitates (ThermoFisher #13-2500). E HEK293 cells were co-transfected with 3xHA-L2 and 3xFLAG-PLK1 constructs (WT: wild type, KD: kinase dead K82R, hyper: hyper activated T210D, pincer: Polo-box domains dead, H538A K540M). Depicted is a representative example from Western blot analysis against L2 (Santa Cruz sc-65709), against FLAG tag for 3xFLAG-PLK1 (Sigma-Aldrich #F1804), against GAPDH (Proteintech #10494-1-AP) and of SDS-PA gel electrophoresis of L2-3xHA (Santa Cruz sc-65709) and 3xFLAG-PLK1 (Sigma-Aldrich #F1804), immunoprecipitates. Representative blots for three independent experiments are shown. Source data are provided as a Source Data file.

Fig. 4. PLK1 inhibition impairs HPV16-L2 chromosomal association and vDNA delivery to host mitotic chromatin.

A HeLa cells stably expressing L2-EGFP /H2B-mCherry were incubated in presence of the indicated PLK1 inhibitors (100 nM) for 16 h, arrested in mitosis using nocodazole (330 nM), and processed for chromosomal association assay as in Fig. 1B. Depicted are representative single medial planes of spinning disk confocal microscopy with L2-EGFP in green and H2B-mCherry in red as indicated. Scale Bar: 10 µm. B Quantification of A, displaying the chromosomal association index (CAI) of individual cells (circles), i.e. the intensity of H2B-mCherry-overlapping EGFP signal over total intensity. Values were normalized to EGFP alone (0) and L2-GFP (1). 50 cells from three independent experiments were analyzed. Displayed is the average of three independent experiments ± SD. C HeLa cells were infected with WT L2 HPV16-EdU PsV for 20 h, then released into mitosis in presence of nocodazole (330 nM – control), nocodazole and BI2536 (100 nM), or nocodazole and BI6727 (100 nM). Displayed are representative medial confocal slices of the subcellular localization of vDNA (EdU, green) and nuclei (Hoechst - blue) in mitotic cells. Scale bars: 5 µm. D Quantification of C for three independent experiments with eight cells/experiment. The overlap of vDNA/chromatin was quantified using intensity-based colocalization analysis (IMARIS Coloc function). Displayed is the average of three independent experiments ± SD. Colored dots represent data points of individual experiments. Statistical significance was assessed by two-tailed Student’s t test to control. Source data are provided as a Source Data file.

Fig. 5. L2 T265 is a putative PLK1 phosphosite required for proper chromosomal association and vDNA tethering to host chromatin.

A Chromosomal association assay of ectopically expressed wild type and T209A, T265A and S319A L2-EGFP in HeLa H2B-mCherry cells during mitosis. Images display representative single medial planes of spinning disk confocal microscopy with L2-EGFP in green and H2B-mCherry in red as indicated. Scale bar: 10 µm. B Quantification of A, displaying the chromosomal association index (CAI) of individual cells (circles), i.e., the intensity of H2B-mCherry-overlapping EGFP signal over total intensity. 50 cells from three independent experiments were analyzed. Displayed is the average of three independent experiments ± SD. C, E HeLa cells were infected with WT and T265A L2 mutant HPV16-EdU PsV for 24 h. Images displayed are representative medial confocal slices of the subcellular localization of vDNA (EdU, green), Golgi (Giantin – red), and nuclei (Hoechst - blue) in interphase C and mitotic E cells. Scale bars: 5 µm. D Quantification of C, and F quantification of E for three independent experiments with eight cells/experiment. The overlap of vDNA/chromatin and vDNA/Golgi were quantified using intensity-based colocalization analysis (IMARIS Coloc function). Displayed is the average of three independent experiments ± SD. Colored dots represent data points of individual experiments. Statistical significance was assessed by two-tailed Student’s t test to wildtype (WT). Source data are provided as a Source Data file.

Fig. 6. CDK1 phosphorylation is primarily required to recruit PLK1 to L2.

A Schematic depiction of L2-GFP-PLK1 WT and SSTP212AAAA mutant chimeras generated to study chromosomal association. B Chromosomal association assay of ectopically expressed wild type and SSTP 212 L2-EGFP-PLK1 chimera in HeLa H2B-mCherry cells arrested with nocodazole (330 nM) in mitosis; images display representative single medial planes of spinning disk confocal microscopy with L2-EGFP-PLK1 in green and H2B-mCherry in red as indicated. The chromosomal association index (CAI) of individual cells (circles), i.e. the intensity of H2B-mCherry-overlapping EGFP signal over total intensity. 50 cells from three independent experiments were analyzed. Displayed is the average of three independent experiments ± SD. Statistical significance was assessed by two-tailed Student’s t test. C Quantitative colony formation assay was used to test the ability of HPV18 genomes to persist in keratinocytes. Infected cells were treated with G418 for the duration of the experiment. Colonies were visualized using MMT. Representative images are shown. D Quantification of data shown in C. Three separate donors were used for the primary human keratinocytes. E Proposed model for sequential phosphorylation of L2 by master mitotic kinases CDK1 and PLK1, which regulate L2 delivery to mitotic chromosomes: L2 spanning across the Golgi limiting membrane allows for CDK1 phosphorylation at the SSTP motif (1), thus priming the viral protein for interaction with PLK1 PBD (2), resulting in further L2 phosphorylations, including the T265 residue (3). Phosphorylated L2, spanning from a bona fide Golgi-derived vesicle travels via MTs (4) and is tethered to mitotic chromosomes (5), promoted by the phosphorylation state, which allows interaction. Source data are provided as a Source Data file.