Papillomavirus Life Cycle Research Articles

Regulation of human papillomavirus type 31 gene expression during the differentiation-dependent life cycle through histone modifications and transcription factor binding

The life cycle of high-risk human papillomaviruses is linked to epithelial differentiation with virion production restricted to highly differentiated suprabasal cells. Two major viral promoters direct high-risk HPV gene expression and their activities are dependent upon differentiation. The early promoter controls initiation of transcripts at sites upstream of the E6 open reading frame and is active in both undifferentiated as well as differentiated cells. The late viral promoter directs transcription from a series of heterogeneous start sites in E7 and is activated upon differentiation. In this study, the state of histones as well as the spectrum of transcription factors bound to the two major HPV 31 viral promoters in undifferentiated and differentiated cells were examined using chromatin immunoprecipitation assays. Our studies indicate that, in undifferentiated cells, the chromatin surrounding both promoter regions is in an open, transcriptionally active state as indicated by the presence of dimethylated forms of histone H3 K4 as well as acetylated H3 and acetylated H4. Upon differentiation, there was an increase of four to six fold in the levels of dimethylated H3K4 and acetylated H3 respectively around both promoter regions as well as an increase of approximately nine fold in acetylated H4 at the early promoter. This suggests that nucleosomes of both promoter regions are further activated through histone modifications during differentiation. Chromatin immunoprecipitation assays were also used to examine the binding of transcription factors to the keratinocyte enhancer (KE)/early promoter region in the upstream regulatory region (URR) and late promoter sequences throughout differentiation. Our results suggest that a dynamic change in transcription factor binding occurs in both regions upon differentiation; most notably a significant increase in C/EBP-β binding to the KE/early promoter region as well as C/EBP-α binding to the late promoter region upon differentiation. These increases in binding cannot be solely explained by changes in the total cellular levels of these factors following differentiation, but instead reflect increased binding specific to HPV genomes. Finally, transient expression analyses confirmed that the KE/early promoter region of the URR contributes significantly to the activation of late gene expression and this is consistent with regulation through the combinatorial binding of multiple transcription factors.

Human papillomavirus: molecular and cytologic/histologic aspects related to cervical intraepithelial neoplasia and carcinoma

Cervical cancer is unique among human cancers because it was the first cancer discovered to be virtually solely attributable to the effects of an infectious agent. Numerous epidemiologic and laboratory studies have confirmed a strong causal association between human papillomavirus infection and the development of premalignant and malignant lesions of the uterine cervix, and human papillomavirus-mediated malignant transformation is an ideal model system for the study of virally mediated carcinogenesis. Neoplastic transformation of affected cervical epithelium appears to be a direct consequence of the unregulated overexpression of viral oncoproteins that have central roles in the normal viral replicative cycle. This review is focused on the mechanisms that regulate the normal papillomavirus life cycle and on the mechanisms that appear to have central roles in malignant transformation of the cervical mucosa.

The Regulatory Element in the 3′-Untranslated Region of Human Papillomavirus 16 Inhibits Expression by Binding CUG-binding Protein 1

The 3'-untranslated regions (UTRs) of human papillomavirus 16 (HPV16) and bovine papillomavirus 1 (BPV1) contain a negative regulatory element (NRE) that inhibits viral late gene expression. The BPV1 NRE consists of a single 9-nucleotide (nt) U1 small nuclear ribonucleoprotein (snRNP) base pairing site (herein called a U1 binding site) that via U1 snRNP binding leads to inhibition of the late poly(A) site. The 79-nt HPV16 NRE is far more complicated, consisting of 4 overlapping very weak U1 binding sites followed by a poorly understood GU-rich element (GRE). We undertook a molecular dissection of the HPV16 GRE and identify via UV cross-linking, RNA affinity chromatography, and mass spectrometry that is bound by the CUG-binding protein 1 (CUGBP1). Reporter assays coupled with knocking down CUGBP1 levels by small interfering RNA and Dox-regulated shRNA, demonstrate CUGBP1 is inhibitory in vivo. CUGBP1 is the first GRE-binding protein to have RNA interfering knockdown evidence in support of its role in vivo. Several fine-scale GRE mutations that inactivate GRE activity in vivo and GRE binding to CUGBP1 in vitro are identified. The CUGBP1.GRE complex has no activity on its own but specifically synergizes with weak U1 binding sites to inhibit expression in vivo. No synergy is seen if the U1 binding sites are made weaker by a 1-nt down-mutation or made stronger by a 1-nt up-mutation, underscoring that the GRE operates only on weak sites. Interestingly, inhibition occurs at multiple levels, in particular at the level of poly(A) site activity, nuclear-cytoplasmic export, and translation of the mRNA. Implications for understanding the HPV16 life cycle are discussed.

Human papillomaviruses activate caspases upon epithelial differentiation to induce viral genome amplification

The life cycle of human papillomaviruses (HPVs) is linked to epithelial differentiation, with late viral events restricted to the uppermost stratified layers. Our studies indicated that HPV activates capases-3, -7, and -9 upon differentiation, whereas minimal activation was observed in differentiating normal keratinocytes. Activation occurred in the absence of significant levels of apoptosis, suggesting a potential role for caspases in the viral life cycle. In support of this, the addition of caspase inhibitors significantly impaired differentiation-dependent viral genome amplification. A conserved caspase cleavage motif was identified in the replication protein E1 ((46)DxxD(49)) that was targeted in vitro by both recombinant caspase-3 and caspase-7. Mutation of this site inhibited amplification of viral genomes, indicating that caspase cleavage is necessary for the productive viral life cycle. Our study demonstrates that HPV activates caspases upon differentiation to facilitate productive viral replication and represents a way by which HPV controls viral gene function in differentiating cells.

Papillomaviruses and the host SUMOylation system

SUMOylation of viral proteins is widespread and serves to modify or regulate the properties of those proteins. Papillomaviruses are a large group of small DNA viruses that infect the skin, leading to benign lesions (warts) that in some cases can progress to malignancy. The papillomavirus life cycle is intimately connected with the differentiation process of stratified epithelium, and several viral early proteins function to modulate the host cell environment. One of the critical early proteins is the E2 protein, which functions in both viral replication and transcription. In the present paper, we demonstrate that E2 proteins are SUMOylated and that overexpression of SUMOylation results in a dramatic increase in intracellular levels of the E2 protein. We have shown previously that there is increased SUMOylation during keratinocyte differentiation, suggesting that the levels of E2 protein may be tied to changes in the cellular SUMOylation state during differentiation. In addition to itself being regulated by SUMOylation, E2 appears to influence the SUMOylation state of one of its binding partners, the cellular transcription factor, C/EBP (CCAAT/enhancer-binding protein). Overall, these observations indicate a complex interplay between this viral protein and the host SUMOylation system.

The full-length E1^E4 protein of human papillomavirus type 18 modulates differentiation-dependent viral DNA amplification and late gene expression

Activation of the productive phase of the human papillomavirus (HPV) life cycle in differentiated keratinocytes is coincident with high-level expression of E1^E4 protein. To determine the role of E1^E4 in the HPV replication cycle, we constructed HPV18 mutant genomes in which expression of the full-length E1^E4 protein was abrogated. Undifferentiated keratinocytes containing mutant genomes showed enhanced proliferation when compared to cells containing wildtype genomes, but there were no differences in maintenance of viral episomes. Following differentiation, cells with mutant genomes exhibited reduced levels of viral DNA amplification and late gene expression, compared to wildtype genome-containing cells. This indicates that HPV18 E1^E4 plays an important role in regulating HPV late functions, and it may also function in the early phase of the replication cycle. Our finding that full-length HPV18 E1^E4 protein plays a significant role in promoting viral genome amplification concurs with a similar report with HPV31, but is in contrast to an HPV11 study where viral DNA amplification was not dependent on full-length E1^E4 expression, and to HPV16 where only C-terminal truncations in E1^E4 abrogated vegetative genome replication. This suggests that type-specific differences exist between various E1^E4 proteins.

Papillomavirus Life Cycle Organization and Biomarker Selection

Human papillomaviruses (HPVs) are a diverse group of viruses that cause epithelial lesions of varying severity. Of the 100 or so types that have been identified, around 40 can infect the cervix, with a subset of these causing lesions that can progress to high-grade neoplasia and cervical cancer. These high-risk types are prevalent in the general population, and can predispose to the development of cancer in women who cannot resolve their infection. Virus infection usually leads to the establishment of productive flat warts, or to maintenance of the viral genome in an asymptomatic or latent state. Virus synthesis depends on the ordered expression of viral gene products as the infected basal cell migrates towards the epithelial surface. E7 is expressed in the lower epithelial layers, and is followed eventually by the expression of E4 and L1 closer to the epithelial surface. This ordered pattern changes in characteristic ways during neoplastic progression and latency, and can be irreversibly fixed following integration of the viral genome into the host cell chromosome. Our understanding of expression patterns and their significance, is beginning to explain the nature of disease progression, and offers a rational basis for the selection of biomarkers that may be used to predict disease status and prognostic outcome.

Bromodomain Protein 4 Mediates the Papillomavirus E2 Transcriptional Activation Function

The papillomavirus E2 regulatory protein has essential roles in viral transcription and the initiation of viral DNA replication as well as for viral genome maintenance. Brd4 has recently been identified as a major E2-interacting protein and, in the case of the bovine papillomavirus type 1, serves to tether E2 and the viral genomes to mitotic chromosomes in dividing cells, thus ensuring viral genome maintenance. We have explored the possibility that Brd4 is involved in other E2 functions. By analyzing the binding of Brd4 to a series of alanine-scanning substitution mutants of the human papillomavirus type 16 E2 N-terminal transactivation domain, we found that amino acids required for Brd4 binding were also required for transcriptional activation but not for viral DNA replication. Functional studies of cells expressing either the C-terminal domain of Brd4 that can bind E2 and compete its binding to Brd4 or short interfering RNA to knock down Brd4 protein levels revealed a role for Brd4 in the transcriptional activation function of E2 but not for its viral DNA replication function. Therefore, these studies establish a broader role for Brd4 in the papillomavirus life cycle than as the chromosome tether for E2 during mitosis.

Regulation of human papillomavirus type 31 late promoter activation and genome amplification by protein kinase C

The life cycle of papillomaviruses is tightly linked to differentiation of host keratinocytes, but the mechanisms and cues by which life cycle events are tied to differentiation remain obscure. We have begun a systematic study of the differentiation-dependent life cycle of HPV31. A variety of signaling pathways have been implicated in controlling keratinocyte differentiation, especially the protein kinase C (PKC) pathway. We have used pharmacological inhibitors to determine that genome amplification and late transcription depend on specific PKC isoforms, and that transcription and replication are independently controlled. We found that tyrosine kinases are necessary for viral amplification but not viral transcription. These studies indicate that the PKC pathway is an important regulator of differentiation-dependent HPV31 replication and transcription.

Methylation of the human papillomavirus-18 L1 gene: A biomarker of neoplastic progression?

Epigenetic transcriptional regulation plays an important role in the life cycle of human papillomaviruses (HPVs) and the carcinogenic progression of anogenital HPV associated lesions. We performed a study designed to assess the methylation status of the HPV-18 genome, specifically of the late L1 gene, the adjacent long control region (LCR), and part of the E6 oncogene in cervical specimens with a range of pathological diagnoses. In asymptomatic infections and infections with precancerous (precursor) lesions, HPV-18 DNA was mostly unmethylated, with the exception of four samples where hypermethylation of L1 was detected. In contrast, L1 sequences were strongly methylated in all cervical carcinomas, while the LCR and E6 remained unmethylated. HeLa cells, derived from a cervical adenocarcinoma, contain chromosomally integrated HPV-18 genomes. We found that L1 is hypermethylated in these cells, while the LCR and E6 are unmethylated. Treatment of HeLa cells with the methylation inhibitor 5-Aza-2′-deoxycytidine (5-Aza-CdR) led to the expected reduction of L1 methylation. After removal of 5-Aza-CdR, L1 methylation resumed and exceeded pretreatment levels. Unexpectedly, the LCR and E6 also became methylated under these conditions, albeit at lower levels than L1. We hypothesize that L1 is preferentially methylated after integration of the HPV genome into the cellular DNA, possibly since linearization prohibits its normal transcription, while the enhancer and promoter may be protected from methylation by transcription factors. Since our data suggest that HPV-18 L1 methylation can only be detected in carcinomas, except in some few precancerous lesions and asymptomatic infections, L1 methylation may constitute a powerful molecular marker for detecting this important step of neoplastic progression.

Induction of productive human papillomavirus type 11 life cycle in epithelial cells grown in organotypic raft cultures

The study of the human papillomavirus (HPV) life cycle was hampered for more than 50 years by the lack of a conventional cell culture system for propagating HPV. Considerable progress has been made in the production of several HPV types using either organotypic rafts or human epithelial xenografts in immunocompromised mice. In this study, we demonstrated episomal maintenance of HPV-11 DNA in N-Tert cells. HPV-11 episomal DNA containing cell populations grown in raft culture showed induction of the productive viral life cycle. HPV-11 DNA amplification and viral capsid antigen synthesis were detected in differentiated layers of epithelia. The viruses generated were able to infect keratinocytes in vitro, which indicate that viruses generated were infectious. The demonstration of the productive HPV-11 life cycle in raft culture from cloned HPV-11 DNA will facilitate genetic analyses of viral gene functions that was not possible using the human xenograft athymic mouse model.

Interactions with Pocket Proteins Contribute to the Role of Human Papillomavirus Type 16 E7 in the Papillomavirus Life Cycle

Human papillomaviruses (HPVs), most commonly the HPV16 genotype, are the principle etiological determinant for cervical cancer, a common cancer worldwide resulting in over 200,000 deaths annually. The oncogenic properties of HPVs are attributable in part to the virally encoded protein E7, best known for its ability to bind to and induce the degradation of the retinoblastoma tumor suppressor, pRb, and related "pocket proteins" p107 and p130. Previously, we defined a role for E7 in the productive stage of the HPV16 life cycle, which takes place in stratified squamous epithelia. HPV perturbs the normal processes of cell growth and differentiation of stratified squamous epithelia. HPVs reprogram cells to support continued DNA synthesis and inhibit their differentiation in the suprabasal compartment of the epithelia, where cells normally have withdrawn from the cell cycle and initiated a well-defined pattern of terminal differentiation. These virus-induced perturbations, which contribute to the production of progeny HPVs, are dependent on E7. In this study, we define the mechanism of action by which E7 contributes to the productive stage of the HPV16 life cycle. We found that the ability of HPV16 to reprogram suprabasal cells to support DNA synthesis correlates with E7's ability to bind pocket proteins but not its ability to induce their degradation. In contrast, the ability of HPV16 to perturb differentiation correlated with both E7's binding to and degradation of pocket proteins. These data indicate that different hallmarks of the productive stage of the HPV16 life cycle rely upon different sets of requirements for E7.

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

The life cycle of human papillomaviruses (HPVs) is tightly linked to the differentiation program of the host's stratified epithelia that it infects. E1(circumflex)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(circumflex)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(circumflex)E4 contributes to the replication of the viral plasmid genome as a nuclear plasmid in basal cells, in which we also found E1(circumflex)E4 protein to be expressed at low levels. In the suprabasal compartment of organotypic raft cultures harboring E1(circumflex)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(circumflex)E4 protein-encoding requirements for these various processes differed. These data support the hypothesis that E1(circumflex)E4 is a multifunctional protein and that the different properties of E1(circumflex)E4 contribute to different processes in both the early and late stages of the virus life cycle.

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

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.

Variant Upstream Regulatory Region Sequences Differentially Regulate Human Papillomavirus Type 16 DNA Replication throughout the Viral Life Cycle

While the central role of the viral upstream regulatory region (URR) in the human papillomavirus (HPV) life cycle has been well established, its effects on viral replication factor expression and plasmid replication of HPV type 16 (HPV16) remain unclear. Some nonprototypic variants of HPV16 contain altered URR sequences and are considered to increase the oncogenic risk of infections. To determine the relationship between viral replication and variant URRs, hybrid viral genomes were constructed with the replication-competent HPV16 prototype W12 and analyzed in assays which recapitulate the different phases of normal viral replication. The establishment efficiencies of hybrid HPV16 genomes differed about 20-fold among European prototypes and variants from Africa and America. Generally, European and African genomes exhibited the lowest replication efficiencies. The high replication levels observed with American variants were primarily attributable to their efficient expression of the replication factors E1 and E2. The maintenance levels of these viral genomes varied about fivefold, which correlated with their respective establishment phenotypes and published P(97) activities. Vegetative DNA amplification could also be observed with replicating HPV16 genomes. These results indicate that efficient E1/E2 expression and elevated plasmid replication levels during the persistent stage of infection may comprise a risk factor in HPV16-mediated oncogenesis.

Induction of the Human Papillomavirus Type 31 Late Promoter Requires Differentiation but Not DNA Amplification

The human papillomavirus (HPV) life cycle is linked to the differentiation state of the host cell. In virus-infected undifferentiated basal epithelial cells, HPV genomes are maintained as episomes at low copy number. Upon differentiation, a concomitant increase in viral copy number and an induction of late gene expression from a differentiation-specific promoter is seen. To investigate whether late gene expression was dependent on the amplification of the viral genome, inhibitors of DNA replication and in vitro systems for epithelial differentiation were used in conjunction with cells that stably maintain HPV31 episomes. Treatment of cells induced to differentiate in methylcellulose with the DNA synthesis inhibitor cytosine beta-arabinofuranoside (AraC) blocked viral DNA amplification but did not prevent induction of late transcription. This suggests that late gene expression does not strictly require amplification of the viral genome and that differentiation signals alone are sufficient to activate transcription from the late promoter. However, DNA amplification does appear to be necessary for maximal induction of the late promoter. In order to examine the cis-acting elements that contribute to the activation of the late promoter, a transient reporter assay was developed. In these assays, an induction of late gene expression was seen upon differentiation that was specific to the late promoter. Mapping studies localized important regulatory elements to the E6/E7 region and identified short sequences that could serve as binding sites for transcription factors. Elements within the upstream regulatory region were also found to positively and negatively influence transcription from the late promoter. These results identify mechanisms important for the differentiation-dependent activation of late gene expression of high-risk papillomaviruses.

The positively charged termini of L2 minor capsid protein required for bovine papillomavirus infection function separately in nuclear import and DNA binding.

During the papillomavirus (PV) life cycle, the L2 minor capsid protein enters the nucleus twice: in the initial phase after entry of virions into cells and in the productive phase to mediate encapsidation of the newly replicated viral genome. Therefore, we investigated the interactions of the L2 protein of bovine PV type 1 (BPV1) with the nuclear import machinery and the viral DNA. We found that BPV1 L2 bound to the karyopherin alpha2 (Kap alpha2) adapter and formed a complex with Kap alpha2beta1 heterodimers. Previous data have shown that the positively charged termini of BPV1 L2 are required for BPV1 infection after the binding of the virions to the cell surface. We determined that these BPV1 L2 termini function as nuclear localization signals (NLSs). Both the N-terminal NLS (nNLS) and the C-terminal NLS (cNLS) interacted with Kap alpha2, formed a complex with Kap alpha2beta1 heterodimers, and mediated nuclear import via a Kap alpha2beta1 pathway. Interestingly, the cNLS was also the major DNA binding site of BPV1 L2. Consistent with the promiscuous DNA encapsidation by BPV1 pseudovirions, this DNA binding occurred without nucleotide sequence specificity. Moreover, an L2 mutant encoding a scrambled version of the cNLS, which supports production of virions, rescued the DNA binding but not the Kap alpha2 interaction. These data support a model in which BPV1 L2 functions as an adapter between the viral DNA via the cNLS and the Kaps via the nNLS and facilitates nuclear import of the DNA during infection.

The human papillomavirus type 8 E2 protein suppresses beta4-integrin expression in primary human keratinocytes.

Human papillomaviruses (HPVs) infect keratinocytes of skin and mucosa. Homeostasis of these constantly renewing, stratified epithelia is maintained by balanced keratinocyte proliferation and terminal differentiation. Instructions from the extracellular matrix engaging integrins strongly regulate these keratinocyte functions. The papillomavirus life cycle parallels the differentiation program of stratified epithelia, and viral progeny is produced only in terminally differentiating keratinocytes. Whereas papillomavirus oncoproteins can inhibit keratinocyte differentiation, the viral transcription factor E2 seems to counterbalance the impact of oncoproteins. In this study we show that high expression of HPV type 8 (HPV8) E2 in cultured primary keratinocytes leads to strong down-regulation of beta4-integrin expression levels, partial reduction of beta1-integrin, and detachment of transfected keratinocytes from underlying structures. Unlike HPV18 E2-expressing keratinocytes, HPV8 E2 transfectants did not primarily undergo apoptosis. HPV8 E2 partially suppressed beta4-integrin promoter activity by binding to a specific E2 binding site leading to displacement of at least one cellular DNA binding factor. To our knowledge, we show for the first time that specific E2 binding contributes to regulation of a cellular promoter. In vivo, decreased beta4-integrin expression is associated with detachment of keratinocytes from the underlying basement membrane and their egress from the basal to suprabasal layers. In papillomavirus disease, beta4-integrin down-regulation in keratinocytes with higher E2 expression may push virally infected cells into the transit-amplifying compartment and ensure their commitment to the differentiation process required for virus replication.

Propagation of human papillomavirus 16 using a novel adenovirus and cre/loxp mechanism

Problem: Human papillomavirus type 16 (HPV16) infection is a major risk factor for the development of squamous cell cancers of the cervix and of the head and neck. A major barrier to understanding the progression from initial infection to cancer has been the lack of in vitro models that allow infection, replication, and persistence of the viral genome as an episome in differentiated epithelial cells. Methods: To overcome this barrier, we designed an adenoviral delivery vector that contained a full HPV16 genome flanked by LoxP homologous recombination sites plus a fluorescent reporter that was expressed only after the HPV genome was excised by Cre recombinase. Results: This system delivered circular HPV16 genomes to cervical epithelial cells and well-differentiated human airway epithelia. Following delivery, the HPV16 genome replicated and persisted as an episome in cervical keratinocytes. These cells developed an immortalized phenotype and a dysplastic epithelial appearance. Moreover, induction of differentiation led to the expression of late genes and production of infectious HPV16 virions. Conclusion: This work provides a novel means of introducing biologically active HPV genomes into epithelial cells, which are normally difficult to transfect. These methods will allow the study of HPV genome replication and gene expression in the earliest stages of HPV genome establishment and may provide a means with which to study non-oncogenic HPV viral types. Significance: Understanding the papillomavirus life cycle may aid in developing novel treatments for HPV-related head and neck diseases. Support: None reported.

Propagation, infection, and neutralization of authentic HPV16 virus

Despite the prevalence of HPV16 in invasive cervical cancers, an in vitro system capable of producing infectious HPV16 is lacking. The organotypic (raft) culture system has allowed for the study of the entire differentiation-dependent life cycle of human papillomaviruses (HPVs). However, the use of this system with the prototype HPV16-containing cell line, W12, has failed to yield infectious virus. Our laboratory has introduced clinically derived HPV16(114/B) genomic DNA into primary keratinocytes, where it subsequently recircularized and maintained episomally at 50–100 copies per cell. Virion morphogenesis occurred after epithelial stratification and differentiation in raft culture. HPV16 virions were isolated that were able to infect keratinocytes in vitro. Infection was neutralized by monoclonal antibodies raised against HPV16 but not by monoclonal antibodies known to neutralize other HPV types.