Mechanisms regulating expression of the HPV 31 L1 and L2 capsid proteins and pseudovirion entry

Human papillomaviruses (HPV) infect stratified epithelia and restrict expression of late capsid genes to highly differentiated cells. In order to begin to understand the processes regulating HPV 31 infection we examined the synthesis of the HPV 31 capsid proteins, L1 and L2, using heterologous expression systems. Similar to studies in HPV 16, expression of wild type HPV 31 L1 and L2 from heterologous promoters resulted in very low levels of synthesis. In contrast, modification of the codons in the capsid genes to ones more commonly used in cellular genes resulted in high-level synthesis. Through the use of chimeric proteins that fused fragments of wild type L1 to Green Fluorescent Protein (GFP) coding sequences, a short region was identified that was sufficient to inhibit high level synthesis and similar elements were detected in L2. One element was localized to the 3' end of the L1 gene while a series of elements were localized at the 3' end of the L2 coding sequences. These observations are most consistent with negative RNA regulatory elements controlling the levels of L1 and L2 synthesis that are distinct from those identified in HPV 16. Expression vectors for the codon modified HPV 31 capsid proteins were then transfected together with GFP reporter plasmids to generate HPV 31 pseudoviruses. Infection of cells with HPV 31 pseudoviruses in the presence of the inhibitors, chlorpromazine, nystatin or methyl-beta-cyclodextrin, demonstrated that HPV 31, like HPV 16, enters human and monkey cells through a clathrin-mediated pathway rather than through caveolae as previously reported. This suggests that high-risk HPV types may enter cells through common mechanisms

Mitotic control of human papillomavirus genome-containing cells is regulated by the function of the PDZ-binding motif of the E6 oncoprotein.

The function of a conserved PDS95/DLG1/ZO1 (PDZ) binding motif (E6 PBM) at the C-termini of E6 oncoproteins of high-risk human papillomavirus (HPV) types contributes to the development of HPV-associated malignancies. Here, using a primary human keratinocyte-based model of the high-risk HPV18 life cycle, we identify a novel link between the E6 PBM and mitotic stability. In cultures containing a mutant genome in which the E6 PBM was deleted there was an increase in the frequency of abnormal mitoses, including multinucleation, compared to cells harboring the wild type HPV18 genome. The loss of the E6 PBM was associated with a significant increase in the frequency of mitotic spindle defects associated with anaphase and telophase. Furthermore, cells carrying this mutant genome had increased chromosome segregation defects and they also exhibited greater levels of genomic instability, as shown by an elevated level of centromere-positive micronuclei. In wild type HPV18 genome-containing organotypic cultures, the majority of mitotic cells reside in the suprabasal layers, in keeping with the hyperplastic morphology of the structures. However, in mutant genome-containing structures a greater proportion of mitotic cells were retained in the basal layer, which were often of undefined polarity, thus correlating with their reduced thickness. We conclude that the ability of E6 to target cellular PDZ proteins plays a critical role in maintaining mitotic stability of HPV infected cells, ensuring stable episome persistence and vegetative amplification.This work was supported by grants from the Wellcome Trust (093450/Z/10/Z and 09345/B/10/Z) and Cancer Research UK (C427/A8049)

Human papillomavirus type 18 E5 oncogene supports cell cycle progression and impairs epithelial differentiation by modulating growth factor receptor signalling during the virus life cycle

Deregulation of proliferation and differentiation-dependent signalling pathways is a hallmark of human papillomavirus (HPV) infection. Although the manipulation of these pathways by E6 and E7 has been extensively studied, controversies surround the role of the E5 oncoprotein during a productive virus life cycle. By integrating primary keratinocytes harbouring wild type or E5 knockout HPV18 genomes with pharmacological and gain/loss of function models, this study aimed to provide molecular information about the role of E5 in epithelial proliferation and differentiation. We show that E5 contributes to cell cycle progression and unscheduled host DNA synthesis in differentiating keratinocytes. E5 function correlates with increased EGFR activation in differentiating cells and blockade of this pathway impairs differentiation-dependent cell cycle progression of HPV18 containing cells. Our findings provide a functional requirement of enhanced EGFR signalling for suprabasal cellular DNA synthesis during the virus life cycle. They also reveal an unrecognised contribution of E5 towards the impaired keratinocyte differentiation observed during a productive HPV infection. E5 suppresses a signalling axis consisting of the keratinocyte growth factor receptor (KGFR) pathway. Inhibition of this pathway compensates for the loss of E5 in knockout cells and re-instates the delay in differentiation. The negative regulation of KGFR involves suppression by the EGFR pathway. Thus our data reveal an unappreciated role for E5-mediated EGFR signalling in orchestrating the balance between proliferation and differentiation in suprabasal cells

Expression of AP1 during cellular differentiation determines human papillomavirus E6/E7 expression in stratified epithelial cells

E6 and E7 oncoproteins of human papillomavirus (HPV) play significant roles in the pathogenesis of cervical cancer. However, the pattern of E6/E7 expression during the productive virus life cycle in differentiating epithelia of the uterine cervix remains unclear. In addition, little is known about the cellular factors regulating E6/E7 expression in differentiating epithelia. In the present study, using transient expression assays and DNA binding assays, we demonstrated that E6/E7 transcription is critically regulated by the cellular factor AP1, a Jun/Fos heterodimer complex. Immunohistochemical analyses of various uterine cervical lesions showed AP1 expression in lower cell layers of normal cervix and low-grade cervical intraepithelial neoplasia (CIN), while it was detected throughout all layers in high-grade CIN and invasive cancer. In situ RNA-RNA hybridization analyses of organotypic raft culture specimens of an HPV-31-containing cell line revealed that E6/E7 transcripts were expressed in most cell layers, with reduced expression in differentiated cells. This pattern of HPV expression correlated with the pattern of AP1 expression detected by immunohistochemicaI analyses. These findings suggest that E6/E7 expression in differentiating epithelia is dependent on AP1, which appears to be associated with proliferative activity of the cells. Since E6/E7 expression induces cell proliferation, co-expression of AP1 and E6/E7 in undifferentiated cell layers might create a positive regulatory loop, probably contributing to maintenance of initial HPV infection and subsequent activation in basal and suprabasal cell layers

Human Papillomaviruses Recruit Cellular DNA Repair and Homologous Recombination Factors to Viral Replication Centers

Human papillomaviruses (HPV) activate the ataxia telangiectasia mutated (ATM)-dependent DNA damage response to induce viral genome amplification upon epithelial differentiation. Our studies show that along with members of the ATM pathway, HPV proteins also localize factors involved in homologous DNA recombination to distinct nuclear foci that contain HPV genomes and cellular replication factors. These studies indicate that HPV activates the ATM pathway to recruit repair factors to viral genomes and allow for efficient replication

Hypoxia-specific stabilization of HIF-1alpha by human papillomaviruses

金沢大学附属病院産科婦人科Human papillomaviruses (HPV) are the causative agents of cervical cancer and have been shown to increase expression of pro-angiogenic factors from infected cells. Many angiogenic factors are regulated by hypoxia inducible factor 1α (HIF-1α). We investigated whether HPV31 affects the levels of HIF-1α under normal and hypoxic conditions. Our studies indicate that cells containing complete HPV31 genomes showed enhanced levels of HIF-1α upon treatment with the hypoxia mimic DFO, which resulted from protein stabilization and led to increased expression of some but not all HIF-1α target genes. Both HPV E6 and E7 were able independently to enhance induction of HIF-1α upon DFO treatment. Enhancement of HIF-1α stability was not restricted to high-risk HPV types, as HPV11, a low risk HPV type, mediated a similar effect. These findings shed light on mechanisms by which HPV contributes to angiogenesis both in benign cervical lesions and in cervical cancers. © 2009 Elsevier Inc. All rights reserved

Altered Toll-like receptor expression and function in HPV-associated oropharyngeal carcinoma

Toll-like receptors (TLRs) have been widely investigated due to their importance in the inflammatory response and possible links to tumor promotion/regression and prognosis. In cancers with an infective etiology, such as human papillomavirus (HPV)-associated Oropharyngeal Squamous Cell Carcinoma (OPSCC), TLR responses may be activated and play a role in tumorigenesis. Our aim was to assess the expression of all TLRs in OPSCC cell lines (both HPV+and HPV-) by qPCR, Western Blot and flow cytometry and assess their response to TLR ligands lipopolysaccharide (LPS), LPS ultra-pure (LPS-UP) and peptidoglycan (PGN) by analyzing IL-8 and IL-6 production. We also immunostained 61 OPSCC tissue samples with anti-TLR4. Results showed lower TLR1 and TLR6 mRNA expression and higher TLR9 protein expression in HPV+when compared to HPV-OPSCC cells. TLR4 expression did not vary by HPV status in OPSCC cells, but TLR4 expression was significantly lower in HPV+OPSCC tissues. After stimulation with PGN, only one cell line (HPV+) did not secrete IL-6 or IL-8. Furthermore, HPV+OPSCC lines showed no IL-6 or IL-8 production on treatment with LPS/LPS-UP. The data suggest changes in TLR4 signaling in HPV+OPSCC, since we have shown lower tissue expression of TLR4 and no pro-inflammatory response after stimulation with LPS and LPS-UP. Also, it suggests that OPSCC may respond to HPV infection by increased expression of TLR9. This study demonstrates differences in expression and function of TLRs in OPSCC, which are dependent on HPV status, and may indicate subversion of the innate immune response by HPV infection

A cyclin-binding motif in human papillomavirus type 18 (HPV18) E1^E4 is necessary for association with CDK–cyclin complexes and G2/M cell cycle arrest of keratinocytes, but is not required for differentiation-dependent viral genome amplification or L1 capsid protein expression

Investigation into the effects the HPV E4 protein has in viral life cycleThe G2/M arrest function of human papillomavirus (HPV) E4 proteins is hypothesized to be necessary for viral genome amplification. Full-length HPV18 E1^E4 protein is essential for efficient viral genome amplification. Here we identify key determinants within a CDK-bipartite consensus recognition motif in HPV18 E1^E4 that are critical for association with active CDK–cyclin complexes and in vitro phosphorylation at the predicted CDK phosphorylation site (threonine 23). The optimal cyclin-binding sequence (43RRLL46) within this E4 motif is required for G2/M arrest of primary keratinocytes and correlates with cytoplasmic retention of cyclin B1, but not cyclin A. Disruption of this motif in the E4 ORF of HPV18 genomes, and the subsequent generation of stable cell lines in primary keratinocytes revealed that this motif was not essential for viral genome amplification or L1 capsid protein induction. We conclude that the HPV18 E4 G2/M arrest function does not play a role in early vegetative events

Human papilloma viruses and cervical tumours: mapping of integration sites and analysis of adjacent cellular sequences

BACKGROUND: In cervical tumours the integration of human papilloma viruses (HPV) transcripts often results in the generation of transcripts that consist of hybrids of viral and cellular sequences. Mapping data using a variety of techniques has demonstrated that HPV integration occurred without obvious specificity into human genome. However, these techniques could not demonstrate whether integration resulted in the generation of transcripts encoding viral or viral-cellular sequences. The aim of this work was to map the integration sites of HPV DNA and to analyse the adjacent cellular sequences. METHODS: Amplification of the INTs was done by the APOT technique. The APOT products were sequenced according to standard protocols. The analysis of the sequences was performed using BLASTN program and public databases. To localise the INTs PCR-based screening of GeneBridge4-RH-panel was used. RESULTS: Twelve cellular sequences adjacent to integrated HPV16 (INT markers) expressed in squamous cell cervical carcinomas were isolated. For 11 INT markers homologous human genomic sequences were readily identified and 9 of these showed significant homologies to known genes/ESTs. Using the known locations of homologous cDNAs and the RH-mapping techniques, mapping studies showed that the INTs are distributed among different human chromosomes for each tumour sample and are located in regions with the high levels of expression. CONCLUSIONS: Integration of HPV genomes occurs into the different human chromosomes but into regions that contain highly transcribed genes. One interpretation of these studies is that integration of HPV occurs into decondensed regions, which are more accessible for integration of foreign DNA