A central region in the minor capsid protein of papillomaviruses facilitates viral genome tethering and membrane penetration for mitotic nuclear entry

Abstract

Incoming papillomaviruses (PVs) depend on mitotic nuclear envelope breakdown to gain initial access to the nucleus for viral transcription and replication. In our previous work, we hypothesized that the minor capsid protein L2 of PVs tethers the incoming vDNA to mitotic chromosomes to direct them into the nascent nuclei. To re-evaluate how dynamic L2 recruitment to cellular chromosomes occurs specifically during prometaphase, we developed a quantitative, microscopy-based assay for measuring the degree of chromosome recruitment of L2-EGFP. Analyzing various HPV16 L2 truncation-mutants revealed a central chromosome-binding region (CBR) of 147 amino acids that confers binding to mitotic chromosomes. Specific mutations of conserved motifs (IVAL286AAAA, RR302/5AA, and RTR313EEE) within the CBR interfered with chromosomal binding. Moreover, assembly-competent HPV16 containing the chromosome-binding deficient L2(RTR313EEE) or L2(IVAL286AAAA) were inhibited for infection despite their ability to be transported to intracellular compartments. Since vDNA and L2 were not associated with mitotic chromosomes either, the infectivity was likely impaired by a defect in tethering of the vDNA to mitotic chromosomes. However, L2 mutations that abrogated chromatin association also compromised translocation of L2 across membranes of intracellular organelles. Thus, chromatin recruitment of L2 may in itself be a requirement for successful penetration of the limiting membrane thereby linking both processes mechanistically. Furthermore, we demonstrate that the association of L2 with mitotic chromosomes is conserved among the alpha, beta, gamma, and iota genera of Papillomaviridae. However, different binding patterns point to a certain variance amongst the different genera. Overall, our data suggest a common strategy among various PVs, in which a central region of L2 mediates tethering of vDNA to mitotic chromosomes during cell division thereby coordinating membrane translocation and delivery to daughter nuclei.

Fig 1. HPV16 vDNA and L2-EGFP associate with mitotic chromosomes.

(A) HeLa cells and HaCaT cells were infected with EdU-HPV16, fixed at 20 h p.i., and stained for host DNA and vDNA using Hoechst and EdU-click chemistry, respectively. Cells were analyzed by CLSM. Shown is a representative confocal section of a metaphase cell with the host DNA (left, red), vDNA (center, green), and merge (right). (B) HeLa cells were infected with HPV16 PsV, fixed at 20 h p.i., and stained for incoming L2 protein using K1L2 and host DNA with Hoechst. Shown is a representative confocal section of a metaphase cell as above. (C) HeLa H2B-mCherry cells were transiently transfected with a HPV16 L2-EGFP expression plasmid, and synchronized by a single thymidine block. High magnification images of fixed cells were acquired using a spinning disc microscope. Depicted are representative images of the subcellular localization of L2-EGFP during the different cell cycle phases as determined by the histone marker.

Fig 2. Chromosomal association of N-terminal or C-terminal deletion mutants of HPV16 L2.

(A) Schematic illustration of L2 that can be divided into an N-terminal (N), middle (M), and C-terminal (C) part according to the clusters of described functional domains for PV L2. Numbers refer to the amino acid positions in HPV16 L2. (B) Overview of the N-terminally truncated HPV16 L2-EGFP mutants that were analyzed for chromosomal association during mitosis (numbers indicate the amino acid residues). The chromosomal association assay was performed upon transfection with L2-EGFP expression plasmids of the indicated PVs. Images were acquired using a spinning disc microscope with 40x magnification, and analyzed computationally. (C) Shown are single confocal slices of full-length and selected truncated HPV16 L2-EGFP (upper row, green), H2B-mCherry (center row, red), and merges (lower row) for representative cells (the remaining constructs are shown in S3 Fig). (D) Chromosomal association of L2 was quantified by a semi-automated image analysis algorithm of at least 50 cells. The chromosomal association was assessed by the chromosomal association index (CAI), i.e. the ratio of mean EGFP fluorescence intensity (intensity per area) of chromosomally associated L2-EGFP over cytoplasmic L2-EGFP normalized by subtracting the median ratio for EGFP. The CAI is depicted as dot plot with solid lines indicating the median, and grey areas marking the middle 50% range of data points for HPV16 L2-EGFP (light grey) and EGFP (dark grey). Statistical significances (two-tailed Student’s t-test) relative to full-length HPV16 L2-EGFP were assessed (***P < 0.001). (E) Overview of the C-terminal deletion mutants of HPV16 L2-EGFP that were examined in the chromosomal association assay. (F) Single confocal slices of full-length and selected C-terminally shortened HPV16 L2-EGFP (upper row, green), H2B-mCherry (center row, red) and merges (lower row) are shown for representative cells (the remaining constructs are shown in S3 Fig). (G) The chromosomal association indices for C-terminal deletion mutants are displayed as in (D).

Fig 3. Chromosomal association of bilateral truncations of HPV16 L2.

(A) Overview of the bilaterally truncated HPV16 L2-EGFP that were assayed for chromosomal association (numbers indicate the amino acid residues). (B) Images of full-length and selected fragments of HPV16 L2-EGFP (left, green), H2B-mCherry (center, red), and merges (right, merge) are depicted for representative cells (the remaining constructs are shown in S3 Fig). (C) The chromosomal association indices of the N- and C-terminally truncated versions of L2-EGFP are plotted as in Fig 2D. Statistical significances (two-tailed Student’s t-test) relative to full-length HPV16 L2-EGFP were determined (n.s. = not significant, **P < 0.01, ***P < 0.001).

Fig 4. Association of L2 with mitotic chromosomes is conserved among PVs.

(A) The phylogram illustrates the evolutionary relationships between the mucosal high-risk alpha-PVs HPV16 and HPV18, the cutaneous beta-PV HPV5, and the animal delta- and iota-PV BPV1 and MnPV, respectively, based on their L2 amino acid sequences. The branch length represents the amount of genetic changes (scale bar: 0.6 substitutions/site). (B) Images of L2-EGFP (left, green), H2B-mCherry (center, red), and merges (right) are shown for representative cells. (C) The chromosomal association indices for N-terminal deletion mutants are depicted as in Fig 2D. As an exception, only 33 cells were analyzed for BPV1 L2.

Fig 5. Chromosomal association of point mutants of HPV16 L2.

(A) Full-length L2 is depicted with N-terminal, middle and C-terminal functional domains. The minimal chromosomal binding region (CBR) is highlighted in orange. Within the CBR (insert) the nuclear retention signal and the SUMO interaction motif are indicated at their relative location with the CBR. Conserved regions within the CBR are depicted in red, whereas regions with lower conservation are white. Single conserved residues are highlighted. The sites of the point mutations in the CBR are color-coded according to their impact on chromosomal association (black = interfering; grey = silent). (B) The alignment of the amino acid (aa) sequences of the NRS and upstream residues of HPV16, 18, 5, BPV1 and MnPV L2 are written in the single-letter code. The aa residue numbers, and mutated residues (bold font) are denoted for HPV16 L2. The conservation between the indicated L2 aa sequences was scored by PRALINE [78], and obtained scores were grouped into five categories: no (white), low (blue), intermediate-low (green), intermediate-high (orange) and high (red) conservation. (C), (D) The aa substitutions IVAL286AAAA, RR297EE, RR302/5AA and RTR313EEE in HPV16 L2-EGFP were analyzed for chromosomal association during mitosis as in Fig 2C and 2D. (C) Images of wild-type and mutant HPV16 L2-EGFP (upper row, green), H2B-mCherry (center row, red), and merges (lower row) are shown for representative cells. (D) The chromosomal association indices are depicted in a dot plot as in Fig 2D. Statistical significances (two-tailed Student’s t-test) relative to wild-type HPV16 L2-EGFP were assessed (**P < 0.01, ***P < 0.001).

Fig 6. Mutant HPV16 PsVs are impaired in nuclear delivery of vDNA.

(A) HeLa cells and HaCaT cells were infected with low and high amounts of HPV16, HPV16-L2(RTR313EEE) and HPV16-L2(IVAL286AAAA) PsVs carrying a GFP reporter plasmid, fixed at 48 h p.i., and analyzed by flow cytometry. Depicted is the fraction of GFP-positive (i.e. infected) cells in percent ± SD. (B) Subcellular localization of incoming vDNA upon infection with HPV16, HPV16-L2(RTR313EEE) and HPV16-L2(IVAL286AAAA) PsVs carrying EdU-labeled vDNA in the absence or presence of 15 μM aphidicolin was analyzed in HeLa cells at 20 h p.i. by CLSM. Depicted are maximum intensity projections of three medial confocal slices of vDNA detected by EdU-click chemistry (green), host nucleus stained with Hoechst (blue), and the trans-Golgi network immunofluorescently stained for p230 (TGN, red). Shown are merge images of vDNA and nucleus (upper row), and vDNA and TGN in the absence and presence of aphidicolin (middle an lower row, respectively). (C) Quantification of intensity-based colocalization (in percent ± SD) of vDNA from EdU-HPV16, EdU-HPV16-L2(RTR313EEE) and EdU-HPV16-L2(IVAL286AAAA) with the host nucleus. (D) Quantification of intensity-based colocalization (in percent ± SD) of vDNA from EdU-HPV16, and EdU-HPV16-L2(RTR313EEE) with the TGN upon infection in the absence or presence of aphidicolin. (E) Subcellular localization of incoming vDNA upon infection with HPV16, HPV16-L2(RTR313EEE) and HPV16-L2(IVAL286AAAA) PsVs carrying EdU-labeled vDNA was analyzed in HaCaT cells at 20 h p.i. by CLSM. Depicted are maximum intensity projections of three medial confocal slices of vDNA detected by EdU-click chemistry (green) and host nucleus stained with Hoechst (blue). Shown are merge images of vDNA and nucleus (top panel), and vDNA with the nuclear outline (lower panel). (F) Quantification of intensity-based colocalization was performed as in (C).

Fig 7. Mutant HPV16 PsVs are impaired in nuclear delivery of L2(RTR313EEE).

(A) HeLa or HaCaT cells were infected with HPV16 wild-type or HPV16-L2(RTR313EEE) PsVs. At 20 h p.i. cells were fix with 4% PFA and stained for the localization of incoming L2 protein with K1L2 antibody and with Hoechst for the host DNA. Depicted are merge images of L2 and nucleus (left panel) and L2 signal with nuclear outline (right panel) for HPV16 wild-type (upper row) and HPV16-L2(RTR313EEE) (lower row). (B) Quantification of intensity-based colocalization (in percent ± SD) of L2 protein from HPV16 wild-type and HPV16-L2(RTR313EEE) with the host nucleus in HeLa and HaCaT cells. (C) HeLa H2B-mCherry were infected with HPV16 wild-type or HPV16-L2(RTR313EEE) PsVs. At 20 h p.i. cells were fixed, stained and analyzed as in (A). (D) Quantification of intensity-based colocalization (in percent ± SD) of L2 protein from HPV16 wild-type and HPV16-L2(RTR313EEE) with the mitotic host chomosomes in HeLa H2B-mCherry cells.

Fig 8. Mutant PsVs are defective in mitotic chromosome association of vDNA and membrane penetration of L2 C-terminus.

(A), (B) Subcellular localization of incoming vDNA upon infection with EdU-HPV16, EdU-HPV16-L2(RTR313EEE) and EdU-HPV16-L2(IVAL286AAAA) in mitotic HeLa H2B-mCherry and HaCaT cells. Cellular chromatin in HaCaT cells was visualized by Hoechst staining. (A) Depicted are maximum intensity projections of confocal slices of host chromatin (left panel, red) and vDNA (center panel, green), and merge images (right panel). (B) Quantification of intensity-based colocalization (in percent ± SD) of vDNA from EdU-HPV16 and EdU-HPV16-L2(RTR313EEE) with the host chromatin. (C) Infection and translocation in HaCaT GFP-BAP cells infected with HPV16 L2, L2(RTR313EEE), L2(RR302/05AA), or L2(IVAL286AAAA)-BirA PsV at equal multiplicities of infection. GFP-biotin, total GFP, and intracellular L2-BirA were visualized by Western blot and immunostaining with neutravidin, anti-GFP, and anti-L2 (K4) respectively, as described in [47]. (D) Infection values were determined by luciferase measurements and represent mean relative infection values compared to wt L2-BirA infected cells and normalized to GAPDH levels.