Structural Analysis Reveals an Amyloid Form of the Human Papillomavirus Type 16 E1 ^∧ E4 Protein and Provides a Molecular Basis for Its Accumulation

Abstract

The abundant human papillomavirus (HPV) type 16 E4 protein exists as two distinct structural forms in differentiating epithelial cells. Monomeric full-length 16E1∧ E4 contains a limited tertiary fold constrained by the N and C termini. N-terminal deletions facilitate the assembly of E1 ∧ E4 into amyloid-like fibrils, which bind to thioflavin T. The C-terminal region is highly amyloidogenic, and its deletion abolishes amyloid staining and prevents E1 ∧ E4 accumulation. Amyloid-imaging probes can detect 16E1 ∧ E4 in biopsy material, as well as 18E1 ∧ E4 and 33E1 ∧ E4 in monolayer cells, indicating structural conservation. Our results suggest a role for fibril formation in facilitating the accumulation of E1 ∧ E4 during HPV infection.Cervical cancer and its precursors usually arise from areas of low-grade disease caused by high-risk human papillomavirus (HPV) types such as HPV-16 (5, 32). The HPV E4 protein (also known as E1 ∧ E4) is expressed from an E1 ∧ E4 spliced mRNA prior to the assembly of infectious virions and accumulates to very high levels in cells supporting productive infection (10, 11). Abundant E1 ∧ E4 is seen in lesions caused by many different HPV types (10,21,26), suggesting that E1 ∧ E4 accumulation plays an important role in the virus life cycle.Although E1 ∧ E4 function is not yet fully understood, its association with keratin filaments is thought to compromise the mechanical integrity of the cell during the late stages of infection (8,33). Keratin binding is not, however, essential for E1 ∧ E4 accumulation, as keratin-binding mutants still accumulate to wild-type levels (24). Rather, it appears that E1 ∧ E4 accumulation depends on sequences at the C terminus of the protein. To understand 16E1∧ E4 function at the molecular level, and in particular to explain its ability to accumulate within the infected cell, we have carried out a structural analysis of the protein. We have found that monomeric 16E1 ∧ E4 does not have a stable tertiary fold but does contain elements of secondary structure that are key to its assembly into fibrils following N-terminal truncation. This work not only enhances our understanding of the HPV-16 E1 ∧ E4 protein but also contributes a viral protein to the growing family of proteins capable of using the amyloid fold (4, 13).Two distinct forms of 16E1 ∧ E4 are expressed during productive infection. Although the E1 ∧ E4 protein of HPV-1 is known to be N terminally cleaved during natural infection (2, 9), it is not yet clear whether HPV-16 E1∧ E4 is similarly modified. To identify the E1 ∧ E4 species present in productively HPV-16-infected squamous epithelium, NIKS cells harboring the full HPV-16 genome were grown in raft culture (24), harvested at day 11 postlifting, and lysed in phosphate-buffered saline containing 6% sodium dodecyl sulfate (SDS) and 1.5 M urea. The E1 ∧ E4 species were characterized using the 16E1 ∧ E4 antibodies TVG402, which binds an epitope between amino acids 29 and 40 (33), and anti-N-term (10), which binds only to species which retain an intact N-t...

E1^E4-mediated keratin phosphorylation and ubiquitylation: a mechanism for keratin depletion in HPV16-infected epithelium

“…3B,C; supplementary material Movie 2). 16E1^E4-mediated keratin reorganisation has been shown to be dependent on C-terminal amino acids (Wang et al, 2004); these residues facilitate multimerisation into amyloid-like fibrils when not restrained by the N-terminal residues (McIntosh et al, 2008). The formation of 16E1^E4 fibrils on keratin might facilitate keratin IF crosslinking, accounting for the static nature of the network.…”

Role of Calpain in the Formation of Human Papillomavirus Type 16 E1^E4 Amyloid Fibers and Reorganization of the Keratin Network

“…The lysate was centrifuged at 10,000 ϫ g for 5 min at 4°C to yield the supernatant. C-terminally His-tagged 16E1 E4 (16E1 E4-His) was prepared as previously described (23), and 400 ng of protein was incubated at 37°C for 30 min with either Cos7 cell lysate supernatant or -calpain (Calbiochem) in lysis buffer containing 1 mM CaCl 2 in a total volume of 200 l. When required, the following inhibitors were included in the reactions: AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride], (1 mM), 6-aminohexanoic acid (5 mg ml Ϫ1 ), antipain (100 M), aprotinin (800 nM), benzamidine HCl (4 mM), bestatin (40 M), chymostatin (100 M), E64 (10 M), EDTA (10 mM), N-ethylmaleimide (1 mM), leupeptin (100 M), pepstatin (1 g ml Ϫ1 ), and phosphoramidon (10 M) from Sigma and ALLN (100 M), calpain inhibitor III (100 M), calpeptin (100 M), EST (100 M), and PD 150606 (100 M) from Calbiochem. Reactions were stopped by addition of 2ϫ Red Loading Buffer containing 100 mM DTT (New England Biolabs).…”

“…The C terminus of 16E1 E4 binds to a cellular DEAD-box RNA helicase, but the role of this interaction is unknown (11). Multimerization of the C terminus of 16E1 E4 is thought to be involved in producing amyloid-like fibers, which are hypothesized to crosslink and disrupt the cytokeratin network (23,28,37).It is becoming increasingly apparent, both from work on 16E1 E4 and from studies of related proteins from other PV types, that the E1 E4 protein is subject to various posttranslational modifications. As well as the aforementioned multimerization, 16E1 E4 can also be phosphorylated by extracellular signal-regulated kinase (ERK), cyclin-dependent kinase 1 (Cdk1), Cdk2, protein kinase A (PKA), and PKC (38).…”

“…In multiple HPV types, E1 E4 species smaller than full-length protein have been observed (type 1 E1 E4) (10). In particular, HPV16-containing differentiating epithelium contains at least two different species of 16E1 E4 that can be distinguished by their antibody-binding capabilities (15,23). Failure of one of these forms to bind to an antibody to the N terminus suggests that at least some in vivo species lacks this end of the protein.…”

“…The C terminus of 16E1 E4 binds to a cellular DEAD-box RNA helicase, but the role of this interaction is unknown (11). Multimerization of the C terminus of 16E1 E4 is thought to be involved in producing amyloid-like fibers, which are hypothesized to crosslink and disrupt the cytokeratin network (23,28,37).…”