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...

The E4 (also called E1--E4) and E2 proteins of human papillomavirus type 16 are thought to be expressed within the same cells of a lesion, and their open reading frames overlap, suggesting that they may have a functional relationship. We have examined the effect of co-expression of these two proteins and found that each enhances the level of the other. We also identified the N-terminus of E2 as the first example of a viral protein that directly binds the HPV16 E1--E4 protein. This appears to result in the E2 becoming less soluble and promotes its relocation from the nucleus to the cytoplasm. In addition, the turnover of the E2 protein is decreased in the presence of E1--E4. All this raises the possibility that E1--E4 acts to influence E2 activity by varying the amount of available E2 in the cell.

The human papillomavirus (HPV) type 16 E1 E4 (16E1 E4) protein is expressed in the middle to upper layers of infected epithelium and has several roles within the virus life cycle. It is apparent that within the epithelium there are multiple species of 16E1 E4 that differ in length and/or degree of phosphorylation and that some or all of these can associate with the cellular keratin networks, leading to network disruption. We show here that the cellular cysteine protease calpain cleaves the 16E1 E4 protein after amino acid 17 to generate species that lack the N terminus. These C-terminal fragments are able to multimerize and form amyloid-like fibers. This can lead to accumulation of 16E1 E4 and disruption of the normal dynamics of the keratin networks. The cleavage of E1 E4 proteins by calpain may be a common strategy used by ␣-group viruses, since we show that cleavage of type 18 E1 E4 in raft culture is also dependent on calpain. Interestingly, the cleavage of 16E1 E4 by calpain appears to be highly regulated as differentiation of HPV genome-containing cells by methylcellulose is insufficient to induce cleavage. We hypothesize that this is important since it ensures that the formation of the amyloid fibers is not prematurely triggered in the lower layers and is restricted to the upper layers, where calpain is active and where disruption of the keratin networks may aid virus release.Papillomaviruses are small DNA viruses that infect epithelial tissue and can give rise to hyperproliferative lesions (16). The majority of these lesions are benign, but a small number of papillomaviruses, so called high-risk types, cause lesions that may become malignant. Human papillomavirus type 16 (HPV16) is such a virus and is responsible for the majority of cases of cervical cancer (35). As with all PV, the life cycle of HPV16 is tightly linked to the differentiation of the host epithelium. Initial infection occurs in the basal cells, and as these cells divide and move toward the surface of the epithelium, differentiating as they ascend, the expression of different viral proteins is triggered (reviewed in reference 9). In the upper layers of the epithelium, the viral DNA is vastly amplified for packaging into new virions. Coincident with this is the highlevel expression of the viral E1 E4 protein, the product of a spliced transcript of the E1 and E4 open reading frames (ORFs) (15). However, while viral DNA replication occurs in the nucleus, 16E1 E4 appears to be cytoplasmic, and its exact function in the virus life cycle is not fully understood.The HPV16 E1 E4 protein appears to have multiple activities that may contribute to different aspects of the virus life cycle. HPV16 mutants with disrupted E4 ORFs show altered abilities to replicate viral DNA (24). In particular, consistent with other PV types, these mutants have less viral DNA amplification in the late stages of the virus life cycle. Possibly related to this, it has been shown that 16E1 E4 has the ability to bind to Cdk/cyclin complexes and arrest the cell cycle prior to...

Human papillomavirus (HPV) genomes are replicated and maintained as extrachromosomal plasmids during persistent infection. The viral E2 proteins are thought to promote stable maintenance replication by tethering the viral DNA to host chromatin. However, this has been very difficult to prove genetically, as the E2 protein is involved in transcriptional regulation and initiation of replication, as well as its assumed role in genome maintenance. This makes mutational analysis of viral trans factors and cis elements in the background of the viral genome problematic and difficult to interpret. To circumvent this problem, we have developed a complementation assay in which the complete wild-type HPV18 genome is transfected into primary human keratinocytes along with subgenomic or mutated replicons that contain the minimal replication origin. The wild-type genome provides the E1 and E2 proteins in trans, allowing us to determine additional cis elements that are required for long-term replication and partitioning of the replicon. We found that, in addition to the core replication origin (and the three E2 binding sites located therein), additional sequences from the transcriptional enhancer portion of the URR (upstream regulatory region) are required in cis for long-term genome replication.