Purification and biophysical characterization of 16E1^∧E4. (A) The sedimentation velocity [c(s)] profile illustrates that 16E1^∧E4 is primarily monomeric with a small proportion in a dimeric state (left). Storage of 16E1^∧E4 results in the formation of aggregates here observed by negative staining under EM (right) (inset magnified ×2). OD, optical density. (B) The far-UV CD spectrum of 16E1^∧E4 reveals elements of both alpha helix and beta strand (left); however, the near-UV CD spectrum indicates a limited tertiary structure (right). Δɛ^mrw, CD extinction coefficient calculated using mean residue weight. (C) The predicted secondary structure of 16E1^∧E4 (blue indicates beta strand, and red indicates helical regions) with confidence levels (0, low; 9, high) assigned by PSIPRED indicates that the beta-stranded region of 16E1^∧E4 is located at the C terminus and the helical region is N terminal, as indicated in the proposed model.

Detection of the E1^∧E4 protein with the amyloid binding probes BTA-1 and NIAD-4 is consistent with the presence of E1^∧E4 amyloid-like fibrils in vivo and indicates that fibril formation is key to the accumulation of E1^∧E4. (A) Immunofluorescence images illustrating that 16E1^∧E4 expressed in COS-7 cells is readily detected with BTA-1 48 h after transfection. Specificity of NIAD-4 for 16E1^∧E4 amyloid is shown in vitro, where NIAD-4 exhibits a fluorescence maximum at 600 nm, indicative of its association with β-strand-based fibrils, with both 16E4Δ2-5 (blue) and amyloid-β(1-42) (turquoise) but not with 16E1^∧E4 (black) or BSA (yellow), NIAD-4 alone is also shown (green). Specificity of NIAD-4 for 16E1^∧E4 amyloid is shown in vitro, where NIAD-4 exhibits a fluorescence maximum at 600 nm, indicative of its association with β-strand-based fibrils, with both 16E4Δ2-5 (blue) and amyloid-β(1-42) (turquoise) but not with 16E1^∧E4 (back) or BSA (yellow); NIAD-4 alone is also shown (green). The immunofluorescence colocalization of NIAD-4 and 16E1^∧E4 in 16E1^∧E4-transfected COS-7 cells illustrates that NIAD-4 is specific for E1^∧E4 in tissue culture. Immunofluorescence images (magnification, ×10) of an HPV-16 CIN lesion reveals NIAD-4 staining within the 16E1^∧E4-positive regions; a dashed white line has been superimposed on each panel to indicate the limits of the upper cornified layer of the lesion. A higher-power image of a single 16E1^∧E4-positive cell is shown in the bottom panel. au, arbitrary units. (B) Alignment of the E1^∧E4 protein sequences of a number of high-risk alpha group viruses illustrates considerable sequence homology in the C-terminal region (shown here); amino acids are shaded according to percent conservation. In the context of the above amino acid alignment, the average and standard deviation values for the beta-aggregation propensity (TANGO) of each amino acid position calculated for the E1^∧E4 proteins of HPV-16, -18, -33, and -45 illustrate the high amyloidogenic potential of this conserved C-terminal region. COS-7 cells infected with HPV-18 and HPV-33 E1^∧E4 were double stained for E1^∧E4 with a polyclonal (HPV-18 or -33) antibody and for amyloid with BTA-1; the colocalization observed indicates that both 18E1^∧E4 and 33E1^∧E4 form amyloid-like structures. (C) The intracellular aggregates observed on expression of the keratin binding mutant, E4Δ12-16, are readily detected in COS-7 cells with BTA-1, confirming its assembly into amyloid-like fibrils. In contrast, the C-terminal mutant (16Ε4Δ86-92) is unable to form amyloid-like structures and is not detected by BTA-1. (D) Western blot showing the relative distribution of mutant and wild-type E1^∧E4 proteins between the soluble and insoluble fractions. 16E4Δ86-92 exists as an exclusively soluble protein and does not, like the wild-type and 16E4Δ2-5 mutant proteins, accumulate in the insoluble fraction. (E) Structural model of the intracellular assembly of 16E1^∧E4 into amyloid-like fibers following N-terminal truncation during epithelial differentiation. Previous data suggested the existence of hexameric 16E1^∧E4 structures, which may be assembly intermediates. 16E4Δ2-5 and 16E4Δ12-16, which have lost N-terminal sequences and are predicted to behave like N-terminally truncated 16E4, accumulate in the cell and stain with the amyloid imaging probe BTA-1. The C-terminal E4 mutant protein 16E4Δ86-92 retains weak keratin binding ability but does not accumulate in the cell and does not stain with amyloid imaging probes.

An N-terminally truncated form of 16E1^∧E4 is observed in tissue culture and in HPV-16-induced cervical intraepithelial lesions. (A) Western blot analysis of the 16E1^∧E4 species observed in extracts of COS-7 cells at 24, 48, and 72 h after transfection with MV11 16E1^∧E4 and in extracts of an HPV-16 raft. Full-length 16E1^∧E4 (band 1) is detected by both TVG402 and anti-N-term, whereas the lower-molecular-mass species (band 2) is detected by only TVG402, indicating that it is N terminally truncated. (B) Immunofluorescence images of E1^∧E4 (top panel) in an HPV-16-induced CIN tissue section (magnification, ×100, confocal) and of E1^∧E4 (bottom panel) in an HPV-16 raft (magnification, ×20). A 4′,6′-diamidino-2-phenylindole (DAPI) (blue) nuclear stain is included in both images.

Monomeric 16E1^∧E4 has a limited tertiary structure which, in the absence of N-terminal residues, forms amyloid-like fibrils; fibril formation is dependent on C-terminal amino acids. (A) Stern-Voler analyses of the acrylamide quenching of 16E1^∧E4 reveals that not all of the tryptophan residues are fully solvent exposed (left). Fluorescence excitation spectra were recorded to assess the contributions of W20 (green), W34 (blue), and W67 (red), observed at 280 nm, to the fluorescence spectrum of the MIANS label on C61. Background MIANS fluorescence is shown in black (middle). Excitation polarization spectra were recorded for the W34F (green) and W20F (red) mutant proteins to monitor tryptophan homotransfer for the W20/W64 pair and the W34/W64 pair, respectively (right). F and FO are the fluorescence in the presence and absence of acrylamide, respectively; au, arbitrary units. (B) Proposed arrangement of the 16E1^∧E4 polypeptide based on the tryptophan fluorescence data in which the polypeptide is folded so as to bring W20, W34, W67, and C61 into close proximity. The C-terminal region could occupy a number of orientations, as indicated by the letters A, B, and C; however, a less solvent-exposed orientation such as arrangement A may favor the highly hydrophobic C terminus. (C) Electron micrograph (inset magnified ×2) of fibers assembled from N-terminally truncated E1^∧E4 (16Ε4Δ2-5) (left). Thioflavin T fluorescence at 485 nm, indicative of amyloid-like fibril structures, is observed in the presence of these fibers and fibers formed from 16E4 amino acids 66 to 92 (16E4 66-92) but not in the presence of 16E1^∧E4. Positive (amyloid-β) and negative (BSA) control samples are also included. (D) Far-UV CD spectrum of the C-terminal 26-amino-acid peptide of 16E1^∧E4 (16E4 amino acids 66 to 92) indicating a beta-stranded structure (left). The electron micrograph (right) shows that this peptide also assembles into fibrillar structures, as indicated by the thioflavin T binding data (inset magnified ×2). Δɛ^mrw, CD extinction coefficient calculated using mean residue weight.