Role of the E1 ^∧ E4 Protein in the Differentiation-Dependent Life Cycle of Human Papillomavirus Type 31

Abstract

The most highly expressed protein in the productive life cycle of human papillomaviruses (HPVs) is E1 ∧ E4, but its function is not well understood. To investigate the role of E1 ∧ E4, we undertook a genetic analysis in the context of the complete HPV type 31 (HPV31) genome. A mutant HPV31 genome (E4M9) was constructed that contained a stop codon in the E4 open reading frame at amino acid 9 and was silent in the overlapping E2 coding sequence. Wild-type and mutant genomes were transfected into normal human foreskin keratinocytes (HFKs) and selected for drug resistance, and pooled cultures were examined for effects of E1 ∧ E4 on viral functions. Southern blot analyses of transfected HFKs demonstrated that cells carrying the E4M9 mutant genomes were maintained as episomes at copy numbers similar to those in keratinocytes transfected with wild-type HPV31. Both sets of cells grew at similar rates, exhibited comparable extensions of life spans, and had equivalent levels of early transcripts. Following suspension of the cells in a semisolid medium, differentiation-dependent genome amplification and late gene expression were significantly decreased in cells maintaining the E4M9 mutant genome compared to those with wild-type HPV31. One explanation for these effects could be a reduction in the number of cells harboring mutant genomes that enter S phase upon differentiation. An analysis of cells containing E4M9 mutant genomes in organotypic raft cultures indicated a reduction in bromodeoxyuridine incorporation in differentiated suprabasal cells compared to that seen in wild-type rafts. Our results indicate that the HPV31 E1∧ E4 protein plays a significant role in promoting HPV genome amplification and S phase maintenance during differentiation.Human papillomaviruses (HPVs) are small double-stranded DNA viruses that infect cutaneous and mucosal epithelial tissues (22). Infection by HPVs induces a range of responses, from benign papillomas to invasive carcinoma. The HPV types that infect the genital epithelia are among the best characterized and are categorized as either high-or low-risk viruses based on their association with cervical cancer. The high-risk HPV types include types 16, 18, 31, 33, and 45, and these viral sequences are found in 99% of cervical carcinomas (33, 53). Low-risk HPVs are not associated with the development of malignancy but rather with benign genital warts and include HPV viral types 6 and 11 (5,52,53).Human papillomaviruses infect cells in the basal layer of stratified epithelium. Infection occurs through microlesions of the skin that expose basal cells to viral entry. Following entry, viral genomes are maintained as nuclear episomes at approximately 50 to 100 copies/cell (22). As infected basal cells divide, HPV episomes are coordinately replicated with cellular DNA and distributed to daughter cells. Following cell division, one daughter cell detaches from the basement membrane and migrates towards the suprabasal layers of the epithelium. This process occurs coordinately with cellular differentia...

Biology and natural history of human papillomavirus infection

“…As a result, the virus copy number amplifies from 50-200 copies to several thousands of copies per cell. 63 This replication strategy adopted by HPV reduces expression of proteins in undifferentiated cells and only intensifies the expression of their proteins, particularly those that comprise the viral capsid; in the more differentiated cells, the replication strategy functions as an escape mechanism of the immune response.…”

Molecular Bases of Human Papillomavirus Pathogenesis in the Development of Cervical Cancer

“…The E4 protein sequesters the CDK1/Cyc B1 complex (CDK, Cyclin dependent kinase and Cyc, Cyclin) onto the cytokeratin network, preventing their nuclear accumulation and therefore inducing inhibition of the G2/M transition of the cell cycle (Davy et al, 2002;Nakahara et al, 2002) and allowing viral and genomic DNA replication. The HPV16 E4 coding region possesses a splicing enhancer element required for the early viral mRNA splicing (Rush et al, 2005), especially for the expression of late viral transcripts of E1^E2, E1, E4 and E5, thus regulating the viral DNA amplification (Wilson et al, 2005).…”

“…It has been observed that the viral integration occurs mainly between E1-E2 ORFs, event that produces the loss of E2 and E4 genes expression. Thus, the loss of E2 and E4 genes generates a down-replication of the viral genome, G2 arrest, and E6 and E7 over-expression (Jeon & Lambert, 1995;Wilson et al, 2005). Thus, the integration of the HPV DNA in the host genome represents an important event in cervical carcinogenesis (Pett & Coleman, 2007), as this may cause cellular immortalization (Band et al, 1990;Jeon & Lambert, 1995;Münger et al, 1992;zur Hausen, 2000), reduction of cellular differentiation, cellular dysplasia and TNF-α non-responder cells (TNF, Tumor Necrosis Factor) (Syrjänen & Syrjänen, 1999;zur Hausen, 2000).…”

Identification of an Arginine-Rich Motif in Human Papillomavirus Type 1 E1^E4 Protein Necessary for E4-Mediated Inhibition of Cellular DNA Synthesis In Vitro and in Cells

“…It is expressed as an E1^E4 fusion protein from spliced transcripts formed between the N terminus of the E1 open reading frame and almost the complete open reading frame of E4 (21). The precise function of E4 has not been defined, but loss of expression of the full-length E1^E4 polypeptide has a severe adverse effect on viral genome amplification of HPV type 16 (HPV16), HPV18, and HPV31 following introduction of mutant genomes unable to support E1^E4 expression into keratinocytes and subsequent induction of cellular differentiation (20,38,39). Failure to complete the vegetative stage of the virus life cycle is also the outcome of loss of E1^E4 expression in rabbit papillomas induced by a mutant cottontail rabbit papillomavirus genome (22).…”