Human Papillomavirus Type 16 E1 ^∧ E4 Contributes to Multiple Facets of the Papillomavirus Life Cycle

Abstract

The life cycle of human papillomaviruses (HPVs) is tightly linked to the differentiation program of the host's stratified epithelia that it infects. E1∧ E4 is a viral protein that has been ascribed multiple biochemical properties of potential biological relevance to the viral life cycle. To identify the role(s) of the viral E1 ∧ E4 protein in the HPV life cycle, we characterized the properties of HPV type 16 (HPV16) genomes harboring mutations in the E4 gene in NIKS cells, a spontaneously immortalized keratinocyte cell line that when grown in organotypic raft cultures supports the HPV life cycle. We learned that E1 ∧ E4 contributes to the replication of the viral plasmid genome as a nuclear plasmid in basal cells, in which we also found E1 ∧ E4 protein to be expressed at low levels. In the suprabasal compartment of organotypic raft cultures harboring E1 ∧ E4 mutant HPV16 genomes there were alterations in the frequency of suprabasal cells supporting DNA synthesis, the levels of viral DNA amplification, and the degree to which the virus perturbs differentiation. Interestingly, the comparison of the phenotypes of various mutations in E4 indicated that the E1 ∧ E4 protein-encoding requirements for these various processes differed. These data support the hypothesis that E1 ∧ E4 is a multifunctional protein and that the different properties of E1 ∧ E4 contribute to different processes in both the early and late stages of the virus life cycle.Human papillomaviruses (HPVs) are small, doublestranded DNA viruses that infect the stratified epithelium lining the skin, anogenital tract, and oral cavity. Viral infection generally causes hyperproliferative lesions such as warts and condyloma. High-risk, mucosotropic (previously termed anogenital) HPVs, most commonly HPV type 16 (HPV16), are also associated with malignant tumors of the anogenital tract and oral cavity and are now accepted as the major causative agent of cervical cancer (64, 69). The life cycle of HPVs is tightly linked to the differentiation program of the host epithelium. HPVs infect basal keratinocytes, presumably at a site of wounding, and they establish their double-stranded, circular DNA genome as an extrachromosomal nuclear plasmid (replicon) at a low copy number. In these proliferating basal cells, early viral genes are selectively expressed, and viral DNA replication occurs along with cellular chromosomal DNA replication to maintain viral DNA copy numbers in both parent and daughter cells. This stage of the viral life cycle within basal cells is called the nonproductive or early stage because no new virus is made. As the infected cells migrate upward and undergo terminal differentiation, the productive or late stage of the viral life cycle begins. In the suprabasal compartment of the epithelium, the viral DNA is amplified, and this is followed by the expression of the late viral genes, including those encoding the structural proteins that form the capsid. Viral DNA is packaged into these capsids to form progeny virions that accumulate in the most superf...

Coordinate regulation of Fanconi anemia gene expression occurs through the Rb/E2F pathway

“…The cells were split 1:4 when they reached 80% confluence and senesced between 8 and 13 passages depending upon the donor. HPV-positive and -negative NIKs were gifts from Paul Lambert, University of Wisconsin-Madison, and were carried as previously described (Nakahara et al, 2005). Irradiated J2-3T3 cells were used as feeder cells.…”

The Indicators of Predicting Disease Outcome in HPV Carcinogenesis

“…By studying this pattern in relation with the presence of the virus, it was obtained an indication on the influence the virus has in individual cells, on the cellular differentiation. For example, E1-E4 contributes to different processes in both the early and late stages of the virus life cycle (Nakahara et al, 2005). The viral E1-E4 protein contributes to the replication of the viral genome as a nuclear plasmid in basal cells.…”

Biology and natural history of human papillomavirus infection

“…31 In the suprabasal layer, the expression of genes E1, E2, E5, E6, and E7 contributes to the maintenance of the viral genome and induces cell proliferation, increasing the number of HPV-infected cells in the epithelium, resulting in a higher number of cells that will eventually produce infectious virions. 31,57 Occurring in the more differentiated cells of this same layer of the epithelium is the activation of the differentiation-dependent promoter and maintenance of the gene expression of E1, E2, E6, and E7. In addition, there is activation of the expression of the E4 gene, whose product will induce amplification of the viral genome replication, greatly increasing the number of virus copies per cell, while at the same time the expression of genes L1 and L2 occurs.…”