No abstract

Human papillomavirus (HPV) does not induce lysis of infected keratinocytes, and the exact mechanisms of viral escape are not known. As keratinocytes differentiate, the cornified cell envelope (CCE) develops, providing a protective barrier to the host. We previously showed that the normally durable CCE in HPV 11-infected epithelium is fragile compared to CCEs in healthy epithelium. In this study, we examined uninfected and HPV 11-infected human genital epithelium for expression of loricrin, the major CCE protein in healthy epidermis. In HPV 11-infected human genital epithelium, detection of loricrin was reduced in immunoelectron microscopic and immunoblot assays, suggesting that loricrin incorporation into the CCE was reduced or that loricrin synthesis was reduced. Loricrin detection was reduced in immunohistochemical assays in areas of high viral replication. Mathematical modeling by least squares was performed using the amino acid composition of highly purified CCE fragments, confirming that loricrin was markedly reduced and that the small proline-rich proteins and cytokeratins were increased in those derived from HPV 11-infected epithelium compared to healthy genital epithelium. In RNase protection and RT-PCR assays, loricrin transcripts were markedly reduced in HPV 11-infected epithelium compared to uninfected epithelium. Loricrin transcripts were detectable by RNA in situ analysis in isolated cells of HPV 11-infected epithelium, but were absent in large regions of epithelium. We conclude that HPV 11 infection reduces transcription of the loricrin gene and that this leads to a reduction in loricrin incorporation into the CCE. Further studies will examine the effects of specific HPV gene products on transcription of loricrin and other CCE components, as it is likely that viral egress from the infected keratinocyte depends on these effects.

Human papillomavirus (HPV) does not induce lysis of infected keratinocytes, and the exact mechanisms of viral escape are not known. As keratinocytes differentiate, the cornified cell envelope (CCE) develops, providing a protective barrier to the host. Our prior studies have identified abnormalities in CCEs isolated from genital epithelium infected with HPV 11 (a low-risk HPV type) and HPV 59 (a high-risk HPV type). These abnormalities included reduced thickness and increased fragility compared to CCEs in healthy epithelium. Transcription of loricrin is also reduced in HPV 11- and 59-infected epithelium. In this study, uninfected and HPV 11- or 59-infected human genital epithelium were examined for expression of the small proline rich proteins (SPRs), which serve as cross-linking proteins within the CCE. Limiting cycle RT-PCR was performed to detect the various SPR transcripts in HPV 11- and 59-infected, or uninfected epithelium. Immunohistochemical analysis and immunoblot assays were performed to analyze the distribution and quantity of SPR2A, SPR2B, and SPR3. SPR2B transcripts were moderately increased in the HPV 11- and 59-infected tissues and SPR3 transcripts were significantly increased in HPV 11-infected tissues and minimally increased in HPV 59-infected tissues. SPR2B protein quantities were moderately increased while SPR2A was not significantly changed. SPR3 protein, while not present in uninfected epithelium, was detected in abundance in HPV 11-infected tissue. We conclude that low-risk and high-risk HPVs share the ability to alter expression of CCE proteins, although the exact mechanisms may differ. Expression of individual SPRs differed between these types and these alterations may play a role in fragility of CCEs in HPV infection.

Human papillomavirus type 59 (HPV 59) is an oncogenic type related to HPV 18. HPV 59 was recently propagated in the athymic mouse xenograft system. A continuous keratinocyte cell line infected with HPV 59 was created from a foreskin xenograft grown in an athymic mouse. Cells were cultured beyond passage 50. The cells were highly pleomorphic, containing numerous abnormally shaped nuclei and mitotic figures. HPV 59 sequences were detected in the cells by DNA in situ hybridization in a diffuse nuclear distribution. Southern blots were consistent with an episomal state of HPV 59 DNA at approximately 50 copies per cell. Analysis of the cells using a PCR/reverse blot strip assay, which amplifies a portion of the L1 open reading frame, was strongly positive. Differentiation of cells in monolayers was induced by growth in F medium containing 2 mM calcium chloride for 10 days. Cells were harvested as a single tissue-like sheet, and histologic analysis revealed a four-to-six cell-thick layer. Transcripts encoding involucrin, a cornified envelope protein, and the E1/E4 and E1/E4/L1 viral transcripts were detected after several days of growth in F medium containing 2 mM calcium chloride. The E1/E4 and L1 proteins were detected by immunohistochemical analysis, and virus particles were seen in electron micrographs in a subset of differentiated cells. An extract of differentiated cells was prepared by vigorous sonication and was used to infect foreskin fragments. These fragments were implanted into athymic mice. HPV 59 was detected in the foreskin xenografts removed 4 months later by DNA in situ hybridization and PCR/reverse blot assay. Thus, the complete viral growth cycle, including production on infectious virus, was demonstrated in the HPV 59 immortalized cells grown in a simple culture system.

Infection of the genital tract with human papillomaviruses (HPVs) leads to proliferative and dysplastic epithelial lesions. The mechanisms used by the virus to escape the infected keratinocyte are not well understood. Infection of keratinocytes with HPV does not cause lysis, the mechanism used by many viruses to release newly formed virions. For HPV 11, a type associated with a low risk of neoplastic disease, the cornified cell envelope (CCE) of infected keratinocytes is thin and fragile, and transcription of loricrin, the major CCE protein, is reduced. The effects of high-risk HPV infection on components of the CCE have not been previously reported. HPV 59, an oncogenic genital type related to HPV types 18 and 45 was identified in a condylomata acuminata lesion. An extract of this lesion was used to infect human foreskin fragments, which were grown in athymic mice as xenografts. Continued propagation using extracts of xenografts permitted growth of additional HPV 59-infected xenografts. CCEs purified from HPV 59-infected xenografts displayed subtle morphologic abnormalities compared to those derived from uninfected xenografts. HPV 59-infected xenografts revealed dysplastic-appearing cells with mitotic figures. Detection of loricrin, involucrin, and cytokeratin 10 was reduced in HPV 59-infected epithelium, while small proline-rich protein 3 (SPR3) was increased. Reduction in loricrin was most apparent in regions of epithelium containing abundant HPV 59 DNA. Compared to uninfected epithelium, loricrin transcription was decreased in HPV 59-infected epithelium. We conclude that HPV 59 shares with HPV 11 the ability to alter CCE components and to specifically reduce transcription of the loricrin gene. Because loricrin is the major CCE protein, a reduction in this component could alter the physical properties of the CCE, thus facilitating virion release.