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

Abstract

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...

HPV-18 E6 mutants reveal p53 modulation of viral DNA amplification in organotypic cultures

“…For instance, compared with the E1^E4 message ( Table 1, species a), the E5 transcript (Table 1, species b) is relatively minor. This finding is in accordance with our understanding that E5 is a signal transduction regulatory protein (41), whereas E1E4 is an abundant cytoplasmic protein that interacts with cytokeratins (42). Interestingly, the E6 F4L mutant and the WT virus were virtually indistinguishable in abundance of each of the RNA species, whereas the other mutants had significantly reduced RNA levels (Fig.…”

“…The splice site in the E1^E4s RNA is located just downstream of the calpain cleavage site. Thus, E1E4s may have properties similar to the N terminustruncated E4 peptide in inducing E4 amyloid filaments, which are thought to facilitate virus release (42). The E1^E4s transcript is primarily a late RNA, consistent with the proposed functions of E4.…”

“…E1^E4s has an internal deletion of 24 aa entirely within the E4 ORF. An amino-terminus truncated form of the E4 protein has been attributed to calpain cleavage (42). The splice site in the E1^E4s RNA is located just downstream of the calpain cleavage site.…”

Human Papillomavirus Type 1 E1^E4 Protein Is a Potent Inhibitor of the Serine-Arginine (SR) Protein Kinase SRPK1 and Inhibits Phosphorylation of Host SR Proteins and of the Viral Transcription and Replication Regulator E2

“…The number of E4 species expressed in the virus life cycle, however, is expanded by phosphorylation and proteolysis (15,16). Posttranslational modification of E4 likely serves to regulate E4 function during the different late stages of the infectious cycle (9,(17)(18)(19)(20). As well as E4 being a substrate for a range of different protein kinases, the primarily cytoplasmic protein can also interfere with the cellular distribution of some kinases.…”

Bovine papillomavirus productive infection in cell cultures: First evidences

“…Pancreatic keratinocyte HPV-16 E6/E7 promotes cell immortalization [70] Ectocervix keratinocyte HPV-16 E6/E7 promote cell immortalization [71] Normal foreskin keratinocyte E6 increases telomerase activity [72,73] COS7 cells HPV-1 and 8 E6 interacts with E6AP (ubiquitin) and p53 [74] Normal foreskin keratinocyte HPV-16 E6 promotes epithelium stratification reduction [75] Immortalized keratinocyte Evidences of HPV-16 productive infection [76] Murine C127 cells HPV-16 and BPV-1 E6 promotes focal adhesion decrease [77][78][79] Wart and squamous carcinoma HPV E6 inhibits apoptosis in response to UV damage [80] Normal foreskin keratinocyte HPV-16 E6/E7 induce cytogenetic aberration [81,82] Chondrocyte HPV-16 E6/E7 promotes immortalization and collagen type II expression [83] Normal foreskin keratinocyte HPV-16 E6 induces p53 degradation and p53 and hTERT expression, increasing telomerase activity [84] Normal foreskin keratinocyte HPV-16 E6/E7 induce irreversible epitelialmesenchymal transition [85] Cervical keratinocyte HPV-16 E5 interacts with endoplasmic reticulum membrane [86] Normal foreskin keratinocyte HPV-16 E6/E7 promote epigenetic alterations in host cell [87] Normal foreskin keratinocyte HPV-16 E1^E4 induce keratin reorganization [88] Normal foreskin keratinocyte BPV-1 E2 stimulates cell migration [89] Keratinocyte (PM1) HPV-8 E2, E6 e E7 promote the stem-cell phenotype acquisition [90] HEK293 cells Domain PDZ da E6 induces PKAand AKT phosphorylation [91] U2OS cells E2 binds to Brd4 regulating DNA replication [92] freezing medium (70% DMEM, 10% dymethylsulphoxyde and 20% FBS) and stocked at -196ºC. These cell lines are part of biological collection of Genetics Laboratory.…”