Calpain activity in differentiating epithelium is responsible for cleavage of HPV16 and 18 E1^E4 proteins in the context of the virus life cycle. Organotypic raft cultures of NIKS cells containing HPV16 (A and B) or HPV18 (C and D) genomes were treated with calpain inhibitor III or control DMSO for 15 days from lifting until harvest. Total cell extracts from HPV16 and HPV18 organotypic raft cultures were analyzed by Western blotting with the anti-16E4 antibody TVG402 (A) or the anti-E4 antibody R705 (C), and loading levels are indicated by the Hsp70 blots. Sections of the organotypic raft cultures were immunostained for keratin 10, MCM2, or profilaggrin (red) and 16E1^E4 (B) or 18E1^E4 (D) (green) with a DAPI nuclear stain (blue) or were stained with hematoxylin and eosin (H&E). The images are representative of data obtained from triplicate rafts.

Calpain activity is important for reorganization of the 16E1^E4 and keratin networks. Cos7 cells were transfected with pMV11.16E1^E4 (or left untransfected). Six hours later, 75 μM calpain inhibitor III or an equivalent control volume of DMSO was added to the cells. (A) At 16 or 28 h posttransfection, the cells were Western blotted for 16E1^E4 with TVG402 anti-16E4 antibody (16E1^E4 is not detectable at 8 h posttransfection). (B) Cells immunostained for 16E1^E4 (green) and keratin 18 (red). A DAPI nuclear stain was included (blue). For each treatment, 3 × 10² cells were analyzed for their degree of reorganization of the 16E1^E4 and keratin 18 networks according to the following categories: no apparent reorganization or early-stage, mid-stage, or late-stage reorganization. Late-stage reorganization also included a mitochondrial pattern since 16E1^E4 relocates to these structures once the keratin network has been depleted. Examples are shown of each category (from DMSO-treated cells). The bar charts show the mean number of cells in each category ± the standard deviation for keratin at 0, 8, 16, and 28 h posttransfection and for 16E1^E4 at 16 and 28 h posttransfection.

Calpain cleavage of 16E1^E4 results in the assembly of amyloid fibers. 16E1^E4-His was incubated with calpain or a buffer control for 30 min before addition of the calpain inhibitor calpeptin. (A) Proteins were separated by SDS-PAGE and stained with Coomassie blue. (B) Electron micrographs of individual amyloid-like E4 fibrils formed from 16E1^E4-His treated with calpain (middle panel) or buffer control (top panel). 16E1^E4-His treated with calpain was left for a further 3 weeks at 4°C, whereupon individual fibrils were observed to assemble into twisted bundles (bottom panel). (C) His-16E1^E4 treated or not treated with calpain were mixed with the amyloid stain thioflavin-T, and the fluorescence emission spectrum measured at between 480 and 600 nm. Thioflavin-T mixed with amyloid-β or caspase 14-treated His-16E1^E4 were used as positive and negative controls, respectively. The fluorescence peak observed at 485 nm is typically observed upon association of thioflavin-T with amyloid-like fibrils.

Conservation of E1^E4 cleavage sites and a model for the role of cleavage in the life cycle. (A) Alignment of the N termini of HPV type 16, 18, and 1 E1^E4 amino acid sequences according to the CLUSTAL W method. Vertical dotted line shows the position at which the N-terminally truncated protein begins in types 16 and 1 and the predicted position in type 18. (B) Model showing the proposed role of 16E1^E4 cleavage in reorganization of the keratin networks. (i) Monomeric E1^E4 binds to cytokeratin filaments, which may inhibit E1^E4 cleavage (38). At this stage, the cells are still in cycle, and some of the E1^E4 is phosphorylated by ERK. Keratin binding is facilitated by transient ERK phosphorylation of E1^E4. Keratin binding displaces the C terminus of E1^E4 from its N terminus. (ii) As E1^E4/keratin binding increases, unbound E1^E4 becomes available for cleavage. As the E1^E4-expresing cells exit the cell cycle and begin to differentiate, calpain is activated and cleaves the free E1^E4. Truncated E4 acts as a template to seed the formation of E4 amyloid fibers, which may incorporate the full-length protein in both its free and keratin-bound forms. (iii) These heterogeneous E1^E4 fibers associate with and cross-link the keratin network, preventing normal keratin network dynamics, which may facilitate the release of viral particles.

Proteolytic processing of full-length 16E1^E4 by a cysteine protease generates an N-terminally truncated species. (A) Total cell extracts from one negative and four positive HPV16 raft cultures were separated on Tricine gels and analyzed by Western blotting with anti-16E4 antibodies: TVG402 (top panel, the gel has split such that lane 4 appears in two sections), anti-N-term (second panel), and anti-PETPAT (low exposure, third panel; high exposure, bottom panel). An arrow indicates a truncated species that is only apparent with an antibody that binds to an epitope of 16E1^E4 within amino acids 52 to 61. (B) Total cell extracts from HPV16-positive and HPV16-negative raft cultures were compared to an extract from Cos7 cells transfected with a plasmid that expresses 16E1^E4, using glycine gels and Western blotting with the anti-16E4 antibody TVG402. (C) 16E1^E4-His expressed and purified from E. coli (lane 1) was incubated with Cos7 cell lysate (lane 3) or, as a control, the lysis buffer used to break open the Cos7 cells (lane 2). The reactions were analyzed by Western blotting with the anti-16E4 antibodies TVG402 (upper panel) and anti-N-term (lower panel). (D) Cos7 cell lysate was incubated with or without 16E1^E4-His and analyzed by Western blotting with the anti-16E4 antibody TVG402. (E) 16E1^E4-His expressed and purified from E. coli was incubated with Cos7 cell lysate containing a range of protease inhibitors. The reactions were analyzed by Western blotting with the anti-16E4 antibodies TVG402 (upper panels) and anti-N-term (lower panels).

Cleavage of 16E1^E4 by calpain results in a predominant fragment consisting of amino acids 18 to 92. (A) 16E1^E4-His expressed and purified from E. coli (lane 1) was incubated with Cos7 cell lysate (lane 2) or with lysis buffer only (lane 3) or with 10, 1, and 0.1 U of human μ-calpain (lanes 4 to 6 and lanes 7 to 9). The reactions were analyzed by Western blotting with the anti-16E4 antibodies TVG402 (upper left panel) or anti-N-term (lower left panel) or with anti-His-tag (right panel). (B) 16E1^E4-His expressed and purified from E. coli (lane 1) was incubated with calpain (lane 2) or calpain treated with an inhibitor of calpain (lanes 5 to 9) or, as controls, calpain treated with the solvents used to dissolve the inhibitors (lanes 3 and 4). The reactions were analyzed by Western blotting with the anti-16E4 antibody TVG402. (C) 16E1^E4-His expressed and purified from E. coli was incubated with Cos7 cell lysate (lane 1) or Cos7 cell lysate treated with calpain inhibitors (lanes 2 to 6) or, as controls, Cos7 cell lysate treated with the solvents used to dissolve the inhibitors (lanes 7 and 8) or with only lysis buffer (lane 9). The reactions were analyzed by Western blotting with the anti-16E4 antibodies TVG402 (upper panel) and anti-N-term (lower panel). (D) Calpain inhibitors (lanes 3 to 7) or, as controls, the solvents used to dissolve the inhibitors (lanes 1 and 2) were added to Cos7 cells 6 h after the cells were transfected with pMV11.16E1^E4. At 24 h posttransfection, the cells were harvested and analyzed by Western blotting with the anti-16E4 antibodies TVG402 (upper panel) and anti-N-term (middle panel) and with anti-GAPDH (lower panel). (E) 16E1^E4-His expressed and purified from E. coli was incubated with calpain for 30 or 60 min before being separated by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was stained with Coomassie blue, and the lower 16E1^E4 bands were excised and subjected to N-terminal sequencing analysis. Peptides beginning at serine 18 or leucine 15 were identified with the serine 18 peptides predominating with the longer exposure to calpain. SI-MS of calpain-digested 16E1^E4-His identified two predominant peaks corresponding to N-terminal peptides ending either in lysine 14 or glycine 17. (F) Analysis of the amino acids close to the cleavage sites (dotted line) in 16E1^E4 shows some (in boxes) to be residues conserved in a number of calpain substrates.