The study of the human pathogen papillomaviruses (HPVs) has been hampered by the inability to propagate the virus in tissue culture. The addition of 12-O-tetradecanoyl phorbol-13-acetate to the media of organotypic (raft) cultures increased expression of physiological markers of keratinocyte differentiation and concomitantly induced production of virions. Capsid production was detected in differentiated suprabasal cells. Virions approximately 54 nanometers in size were observed by electron microscopy in raft tissue cross sections in the suprabasal layers. Virions purified through isopycnic gradients were found to contain type 31b DNA and exhibited an icosahedral shape similar to that of papillomaviruses found in clinical samples.

Primary human keratinocytes are useful for studying the pathogenesis of many different diseases of the cutaneous and mucosal epithelia. In addition, they can form organotypic tissue equivalents in culture that can be used as epidermal autografts for wound repair as well as for the delivery of gene therapy. However, primary keratinocytes have a finite lifespan in culture that limits their proliferative capacity and clinical use. Here, we report that treatment of primary keratinocytes (originating from 3 different anatomical sites) with Y-27632, a Rho kinase inhibitor, greatly increased their proliferative capacity and resulted in efficient immortalization without detectable cell crisis. More importantly, the immortalized cells displayed characteristics typical of primary keratinocytes; they had a normal karyotype and an intact DNA damage response and were able to differentiate into a stratified epithelium. This is the first example to our knowledge of a defined chemical compound mediating efficient cell immortalization, and this finding could have wide-ranging and profound investigational and medical applications. IntroductionSomatic cells have a limited lifespan, gradually slow in growth, and stop dividing, a process known as cellular senescence. This process is thought to limit the vulnerability of aging cells to disease. Human keratinocytes are invaluable for the study of skin biology and the pathogenesis of skin-related diseases, but their short lifespan in culture is a limitation. Different conditions have been developed to optimize the culture of keratinocytes; for example, the presence of fibroblast feeder cells increases the proliferative capacity of primary keratinocytes from approximately 20 to 40-60 population doublings (1, 2). Spontaneously immortalized keratinocyte lines, for example HaCaT (3) and NIKS (4), have been used for skin-related research. However, these cell lines have genetic abnormalities, such as mutations in p53 (5) or isochromosomes (4).Continuous replication of primary human cells is blocked by 2 separate events: mortality stage 1 (M1; replicative senescence) and mortality stage 2 (M2; crisis). At M1, signaling by shortened telomeres results in activation of the p53 and pRB pathways. M2 represents a critical period of genomic instability, with extremely eroded telomeres, resulting in chromosomal fusions and translocations. In retinal pigment epithelial cells and foreskin fibroblasts (which have decreased expression of components of the p16INK4A/pRB pathway), telomerase expression is sufficient to bypass both M1 and M2 and stabilize and elongate chromosome ends (6). However, telomerase expression is not sufficient for immortalization of keratinocytes, and p16INK4A function must also be disrupted (7,8).Keratinocytes expressing the E6 and E7 proteins encoded by high-risk human papillomavirus (HPV) types bypass both M1 and M2 blocks and become immortal (reviewed in ref. 9). E7 inactivates and degrades the pRB retinoblastoma tumor suppressor protein to induce G 1 /S phase progression of th...

Cellular and viral microRNAs (miRNAs) are the transcriptional products of RNA polymerase II and are regulated by transcriptional factors for their differential expression. The altered expression of miRNAs in many cancer types has been explored as a marker for possible diagnosis and therapy. We report in this study that oncogenic human papillomaviruses (HPVs) induce aberrant expression of many cellular miRNAs and that HPV18 infection produces no detectable viral miRNA. Thirteen abundant host miRNAs were specifically regulated by HPV16 and HPV18 in organotypic raft cultures of foreskin and vaginal keratinocytes as determined by miRNA array in combination with small RNA sequencing. The increase of miR-16, miR-25, miR-92a, and miR-378 and the decrease of miR-22, miR-27a, miR-29a, and miR-100 could be attributed to viral oncoprotein E6, E7, or both, all of which are known to target many cellular transcription factors. The examination of 158 cervical specimens, including 38 normal, 52 cervical intraepithelial neoplasia (CIN), and 68 cervical cancer (CC) tissues, for the expression of these eight miRNAs showed a remarkable increase of miR-25, miR-92a, and miR-378 with lesion progression but no obvious change of miR-22, miR-29a, and miR-100 among the HPV-infected tissues. Further analyses indicate that an expression ratio ≥1.5 of miR-25/92a group over miR-22/29a group could serve as a cutoff value to distinguish normal cervix from CIN and from CIN to CC. oncogenes E6 and E7 | noncoding RNAs | regulatory RNAs | virol oncogenesis | DNA tumor viruses

MicroRNAs (miRNA) play pivotal roles in controlling cell proliferation and differentiation. Aberrant miRNA expression in human is becoming recognized as a new molecular mechanism of carcinogenesis. However, the causes for alterations in miRNA expression remain largely unknown. Infection with oncogenic human papillomavirus types 16 (HPV16) and 18 (HPV18) can lead to cervical and other ano-genital cancers. Here, we have demonstrated that cervical cancer tissues and cervical cancerderived cell lines containing oncogenic HPVs display reduced expression of tumor-suppressive miR-34a. The reduction of miR34a expression in organotypic tissues derived from HPV-containing primary human keratinocytes correlates with the early productive phase and is attributed to the expression of viral E6, which destabilizes the tumor suppressor p53, a known miR-34a transactivator. Knockdown of viral E6 expression in HPV16 + and HPV18 + cervical cancer cell lines by siRNAs leads to an increased expression of p53 and miR-34a and accumulation of miR-34a in G 0 /G 1 phase cells. Ectopic expression of miR-34a in HPV18+ HeLa cells and HPV À HCT116 cells results in a substantial induction of cell growth retardation and a moderate cell apoptosis. Together, this is the first time a viral oncoprotein has been shown to regulate cellular miRNA expression. Our data have provided new insights into mechanisms by which high-risk HPVs contribute to the development of cervical cancer.

Human papillomavirus type 18 (HPV18) is the second most common oncogenic HPV genotype, responsible for ϳ15% of cervical cancers worldwide. In this study, we constructed a full HPV18 transcription map using HPV18-infected raft tissues derived from primary human vaginal or foreskin keratinocytes. By using 5 rapid amplification of cDNA ends (RACE), we mapped two HPV18 transcription start sites (TSS) for early transcripts at nucleotide (nt) 55 and nt 102 and the HPV18 late TSS frequently at nt 811, 765, or 829 within the E7 open reading frame (ORF) of the virus genome. HPV18 polyadenylation cleavage sites for early and late transcripts were mapped to nt 4270 and mainly to nt 7299 or 7307, respectively, by using 3 RACE. Although all early transcripts were cleaved exclusively at a single cleavage site, HPV18 late transcripts displayed the heterogeneity of 3 ends, with multiple minor cleavage sites for late RNA polyadenylation. HPV18 splice sites/splice junctions for both early and late transcripts were identified by 5 RACE and primer walking techniques. Five 5 splice sites (donor sites) and six 3 splice sites (acceptor sites) that are highly conserved in other papillomaviruses were identified in the HPV18 genome. HPV18 L1 mRNA translates a L1 protein of 507 amino acids (aa), smaller than the 568 aa residues previously predicted. Collectively, a full HPV18 transcription map constructed from this report will lead us to further understand HPV18 gene expression and virus oncogenesis.Cervical cancer is a leading cause of death for women in the developing world, with about 493,000 new cases and nearly 273,000 deaths each year (www.who.int/hpvcenter/en/). Oncogenic human papillomavirus (HPV) infection is widely recognized as a principal cause of cervical, penile, and anal cancers (62). Among over 120 genotypes with human origin (3), infection with HPV16 and HPV18, the two most common oncogenic HPV types, leads to the development of ϳ70% of all cervical and other anogenital cancers (55, 62). HPV18 alone accounts for more than 15% of all cervical cancer cases worldwide (14). Persistent HPV16 infection is responsible for development of both cervical squamous cell carcinoma and adenocarcinoma. In contrast, persistent HPV18 infection is a preferential risk factor for the development of cervical adenocarcinoma (7, 10).HPVs are a group of small DNA viruses which have a high degree of conservation with respect to genome structure and organization as well as gene expression (44, 91). The circular, double-stranded viral genome is approximately 8 kb in size and contains eight open reading frames (ORFs) which are all transcribed from the same strand. In general, the viral genome can be divided into three major regions: an early region, a late region, and a long control region (LCR) or a noncoding region (NCR). The early region is positioned in the 5Ј half of the virus genome and encodes six common ORFs (E1, E2, E4, E5, E6, and E7) with regulatory functions in viral replication, gene expression, and pathogenesis. The late region lies dow...

Epstein-Barr virus is a ubiquitous human herpesvirus associated with epithelial and lymphoid tumors. EBV is transmitted between human hosts in saliva and must cross the oral mucosal epithelium before infecting B lymphocytes, where it establishes a life-long infection. The latter process is well understood because it can be studied in vitro, but our knowledge of infection of epithelial cells has been limited by the inability to infect epithelial cells readily in vitro or to generate cell lines from EBV-infected epithelial tumors. Because epithelium exists as a stratified tissue in vivo, organotypic cultures may serve as a better model of EBV in epithelium than monolayer cultures. Here, we demonstrate that EBV is able to infect organotypic cultures of epithelial cells to establish a predominantly productive infection in the suprabasal layers of stratified epithelium, similar to that seen with Kaposi's-associated herpesvirus. These cells did express latency-associated proteins in addition to productive-cycle proteins, but a population of cells that exclusively expressed latency-associated viral proteins could not be detected; however, an inability to infect the basal layer would be unlike other herpesviruses examined in organotypic cultures. Furthermore, infection did not induce cellular proliferation, as it does in B cells, but instead resulted in cytopathic effects more commonly associated with productive viral replication. These data suggest that infection of epithelial cells is an integral part of viral spread, which typically does not result in the immortalization or enhanced growth of infected epithelial cells but rather in efficient production of virus.Epstein-Barr virus | epithelial | organotypic culture | productive replication A lthough the association between Epstein-Barr virus and epithelial malignancies has been known for more than three decades, the EBV life cycle within the epithelial milieu is still only poorly understood. In contrast, our broad understanding of the biology of EBV within the B-cell compartment has been facilitated by the ability of EBV to infect and immortalize primary B cells in vitro and by the ability of some EBV-positive B-cell tumors to give rise to cell lines that maintain restricted programs of latency gene expression similar to those seen in vivo. Although in primary EBV infection the entire complement of EBV latencyassociated nuclear proteins (EBNAs 1, 2, 3A, 3B, 3C, and LP) and membrane proteins (LMPs 1, 2A, and 2B) promote cellular proliferation and survival (Latency III), EBV gene expression must be progressively silenced (Latency II; EBNA1 and LMPs 1 and 2) so that the most restricted program, Latency 0 (in which EBV gene expression is believed to be completely silenced

Human papillomaviruses (HPVs) are etiologic agents of anogenital cancers. The lack of an efficient in vitro system with which to study the differentiation-dependent viral life cycle has impeded most investigations of viral transcription and gene expression. The CIN-612 clone 9E cell line latently maintains episomal copies of HPV type 31b (HPV31b). The complete replicative life cycle of HPV31b can be studied by using the organotypic (raft) culture system. A number of spliced HPV31b early gene transcripts and two late gene transcripts have been described in studies using the raft system. An HPV31b early promoter, P 97 , and a differentiation-induced promoter, P 742 , have been characterized by using this system. In this study, we used the raft system to analyze the temporal expression patterns of HPV31b late gene transcripts during the viral life cycle. The expression of late RNAs peaked at day 12 after lifting to the air-liquid interface; the levels then declined dramatically by day 16. The peak of late RNA expression was coincident with the appearance of virus particles in the raft tissues. We characterized transcripts with the potential to encode late gene products, including 19 RNAs containing the L1 region and 4 RNAs containing the E5b and L2 open reading frames. We also found evidence for two novel promoters. Transcription of both L1-and L2-containing RNAs initiated at a region upstream of the early promoter. In addition, late gene RNAs were also transcribed by using a promoter in the E4 reading frame.

The viral early-to-late switch of papillomavirus infection is tightly linked to keratinocyte differentiation and is mediated in part by alternative mRNA splicing. Here, we report that SRp20, a cellular splicing factor, controls the early-to-late switch via interactions with A/C-rich RNA elements. An A/C-rich SE4 element regulates the selection of a bovine papillomavirus type 1 (BPV-1) late-specific splice site, and binding of SRp20 to SE4 suppresses this selection. Expression of late BPV-1 L1 or human papillomavirus (HPV) L1, the major capsid protein, inversely correlates with SRp20 levels in the terminally differentiated keratinocytes. In HPV type 16, a similar SRp20-interacting element also controls the viral early-to-late switch. Keratinocytes in raft cultures, which support L1 expression, make considerably less SRp20 than keratinocytes in monolayer cultures, which do not support L1 expression. Conversely, abundant SRp20 in cancer cells or undifferentiated keratinocytes is important for the expression of the viral early E6 and E7 by promoting the expression of cellular transcription factor SP1 for transactivation of viral early promoters.Papillomaviruses are small DNA tumor viruses that infect cutaneous or mucosal epithelial cells and cause benign tumors and sometimes malignant neoplasms, including cervical cancer in women (36). Papillomavirus infections are transmitted mainly by close skin-to-skin or mucosa-to-mucosa contact. Infecting viral particles reach the keratinocytes in the basal layer of the squamous epithelium via microwounds that expose the basal keratinocytes to incoming virus. After infection of the basal keratinocytes, viral-gene expression and replication proceed in a tightly controlled fashion regulated by keratinocyte differentiation (25, 34).Although we do not fully understand how keratinocyte differentiation regulates papillomavirus gene expression and virus production, different parts of the viral life cycle occur at different stages of keratinocyte differentiation. The early stage of virus infection takes place in undifferentiated or intermediately differentiated keratinocytes in basal or parabasal layers; at this stage, the viral early genes (E1, E2, E5, E6, and E7) are expressed from the early region of the viral genome and encode all five viral regulatory nonstructural proteins. In contrast, the expression of two structural viral capsid proteins (L1 and L2) from the late region of the virus genome at the late stage of viral infection occurs only in keratinocytes undergoing terminal differentiation in the granular and cornified layers of the epithelium (34, 41). Although the early-to-late switch of viral-gene expression involves a switch in the use of viral promoters during the viral life cycle (21, 48, 49), strict regulation of viral-RNA processing, including alternative RNA splicing and polyadenylation, is absolutely necessary for expression of the viral genes at the appropriate times (42, 57).Alternative RNA splicing and polyadenylation occur during RNA processing in most eukaryotic...