Papillomavirus infection requires gamma secretase.

The mechanism by which papillomaviruses breach cellular membranes to deliver their genomic cargo to the nucleus is poorly understood. Here, we show that infection by a broad range of papillomavirus types requires the intramembrane protease γ secretase. The γ-secretase inhibitor (S,S)-2-[2-(3,5-difluorophenyl)-acetylamino]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-propionamide (compound XXI) inhibits infection in vitro by all types of papillomavirus pseudovirions tested, with a 50% inhibitory concentration (IC(50)) of 130 to 1,000 pM, regardless of reporter construct and without impacting cellular viability. Conversely, XXI does not inhibit in vitro infection by adenovirus or pseudovirions derived from the BK or Merkel cell polyomaviruses. Vaginal application of XXI prevents infection of the mouse genital tract by human papillomavirus type 16 (HPV16) pseudovirions. Nicastrin and presenilin-1 are essential components of the γ-secretase complex, and mouse embryo fibroblasts deficient in any one of these components were not infected by HPV16, whereas wild-type and ß-secretase (BACE1)-deficient cells were susceptible. Neither the uptake of HPV16 into Lamp-1-positive perinuclear vesicles nor the disassembly of capsid to reveal both internal L1 and L2 epitopes and bromodeoxyuridine (BrdU)-labeled encapsidated DNA is dependent upon γ-secretase activity. However, blockade of γ-secretase activity by XXI prevents the BrdU-labeled DNA encapsidated by HPV16 from reaching the ND10 subnuclear domains. Since prior studies indicate that L2 is critical for endosomal escape and targeting of the viral DNA to ND10 and that γ secretase is located in endosomal membranes, our findings suggest that either L2 or an intracellular receptor are cleaved by γ secretase as papillomavirus escapes the endosome.

AP-1 and ERK1 but not p38 nor JNK is required for CRPV early protein 2-dependent MMP-9 promoter activation in rabbit epithelial cells.

Human papillomaviruses (HPV) are the known cause for a variety of cancers including cervical and epithelial cancers. The cottontail-rabbit papillomavirus (CRPV) serves as a suitable animal model to study the development of these cancers in vivo. We have previously demonstrated that CRPV-induced skin carcinomas express high levels of MMP-9, a metalloproteinase contributing to cancer progression by extracellular matrix remodelling. Based on our previously reported finding that CRPV early protein 2 can activate a truncated human 670bp MMP-9 promoter fragment, we hypothesized that MMP-9 expression in the rabbit carcinomas is a consequence of activation of the rabbit MMP-9 promoter in-trans by CRPV early protein 2. Further elucidation of the mechanism revealed the requirement for both a proximal and distal AP-1 transcription factor binding site in the rabbit MMP-9 promoter and the AP-1 complex as demonstrated by the inhibitory effect of TAM67, a trans-activation deficient c-jun mutant. The characterization of signal-transduction requirements revealed predominantly ERK1 to be required for CRPV early protein 2-dependent MMP-9 promoter activation, but not JNK nor p38. In summary CRPV early protein 2 activates the expression of MMP-9 in-trans through AP-1 and ERK1 and may contribute to cancer development and progression via this mechanism within the animal model.

The rabbit papillomavirus model: a valuable tool to study viral-host interactions.

Cottontail rabbit papillomavirus (CRPV) was the first DNA virus shown to be tumorigenic. The virus has since been renamed and is officially known as Sylvilagus floridanus papillomavirus 1 (SfPV1). Since its inception as a surrogate preclinical model for high-risk human papillomavirus (HPV) infections, the SfPV1/rabbit model has been widely used to study viral-host interactions and has played a pivotal role in the successful development of three prophylactic virus-like particle vaccines. In this review, we will focus on the use of the model to gain a better understanding of viral pathogenesis, gene function and host immune responses to viral infections. We will discuss the application of the model in HPV-associated vaccine testing, in therapeutic vaccine development (using our novel HLA-A2.1 transgenic rabbits) and in the development and validation of novel anti-viral and anti-tumour compounds. Our goal is to demonstrate the role the SfPV1/rabbit model has played, and continues to play, in helping to unravel the intricacies of papillomavirus infections and to develop tools to thwart the disease. This article is part of the theme issue 'Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses'.

Papillomavirus E2 protein induces expression of the matrix metalloproteinase-9 via the extracellular signal-regulated kinase/activator protein-1 signaling pathway.

Papillomaviruses are involved in the development of cancers of the female cervix, head and neck, and skin. An excellent model to study papillomavirus-induced tumor induction and progression is the New Zealand White rabbit, where the skin is infected with the cottontail rabbit papillomavirus (CRPV). This leads to the formation of benign tumors that progress into invasive and metastasizing carcinomas without the need for cofactors. We have shown previously that specific mutations in the transactivation domain of the transcription/replication factor E2 cause a dramatic loss in the tumor induction efficiency of the viral genome and a major deficiency in tumor progression as we show now. By comparing wild-type (WT) and mutant E2-induced skin tumors, we found high levels of matrix metalloproteinase-9 (MMP-9) protein and transcripts in WT CRPV-E2-induced tumors in contrast to certain mutant CRPV-E2-induced papillomas and normal uninfected skin. Stable cell lines and reporter assays revealed that E2 from different papillomavirus types is able to transactivate the MMP-9 promoter via the promoter-proximal activator protein-1 (AP-1) site as shown in reporter gene assays with mutant MMP-9 promoter constructs. Furthermore, WT E2 but not mutant E2 strongly transactivated a minimal promoter reporter construct with multiple AP-1 sites. The MMP-9 protein induced in cells expressing E2 degrades collagen matrices as measured in Matrigel-based invasion/mobility assays. E2-induced MMP-9 expression can be blocked by a chemical inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase 1 (PD 098059), suggesting that E2 activates the MAPK/ERK signaling pathway, which is further supported by the induction of ERK1 in CRPV-E2-transfected cells.

Rodent Papillomaviruses.

Preclinical infection model systems are extremely valuable tools to aid in our understanding of Human Papillomavirus (HPV) biology, disease progression, prevention, and treatments. In this context, rodent papillomaviruses and their respective infection models are useful tools but remain underutilized resources in the field of papillomavirus biology. Two rodent papillomaviruses, MnPV1, which infects the Mastomys species of multimammate rats, and MmuPV1, which infects laboratory mice, are currently the most studied rodent PVs. Both of these viruses cause malignancy in the skin and can provide attractive infection models to study the lesser understood cutaneous papillomaviruses that have been frequently associated with HPV-related skin cancers. Of these, MmuPV1 is the first reported rodent papillomavirus that can naturally infect the laboratory strain of mice. MmuPV1 is an attractive model virus to study papillomavirus pathogenesis because of the ubiquitous availability of lab mice and the fact that this mouse species is genetically modifiable. In this review, we have summarized the knowledge we have gained about PV biology from the study of rodent papillomaviruses and point out the remaining gaps that can provide new research opportunities.

Establishment from Shope carcinoma induced in an inbred rabbit of culture cell lines with various potentials for differentiation and tumorigenicity.

Shope papillomas induced by cottontail rabbit papilloma-virus (CRPV) in domestic rabbits frequently regress spontaneously or, failing to do so, convert into squamous cell carcinomas at a high rate. This papilloma-carcinoma complex in rabbits provides an experimental model for human papillo-mavirus-associated malignancies. The aim of this study was to prepare an experimental system in inbred rabbits by establishing culture cell lines of the tumor. Squamous cell carcinoma developed from a Shope papilloma that had been induced 6 months previously by inoculating CRPV into an inbred B/J rabbit. By in vitro culturing of the tumor cells, cell lines with potentials for terminal differentiation and tumorigenicity were established. Cloning yielded sublines that varied in these potentials and possessed episomal and integrated CRPV genomes as revealed by Southern hybridization in both one- and two-dimensional electrophoresis. Major CRPV-specific transcripts were similarly observed both in well-differentiated and in poorly differentiated sublines. Immunofluorescence with syngeneic rabbit antibody against tumor-specific antigens localized such antigens mainly in the nuclei of the cells of these sublines. This experimental system allows experiments that were not feasible in randomly bred rabbits.

High efficiency induction of papillomas in vivo using recombinant cottontail rabbit papillomavirus DNA.

Plasmids containing cottontail rabbit papillomavirus (CRPV) DNA can induce papillomas in vivo, but efficiency has been low. The aim of the present investigation was to explore some of the technical variables involved in inoculation of rabbits with recombinant CRPV DNA in attempts to improve both yield and consistency of papilloma induction. It was found that induction of epidermal hyperplasia, with either a mixture of turpentine and acetone or phorbol esters, produced a marked increase in papilloma yield. An additional powerful factor was the use of very vigorous, cutaneous scarification, sufficient to penetrate the papillary dermis and produce bleeding. When used in combination, papilloma yields were consistent and often reached 90-100% of inoculated sites. A number of other variables which did not consistently affect papilloma yield were tested. These included bleb and puncture injections, plasmid dose, vector type, occlusive dressings, lipofection reagent, carrier DNA, and different methods for plasmid DNA extraction and purification. It is concluded that the most important variables in improving papilloma yields were prior induction of epidermal hyperplasia and vigorous cutaneous scarification.

Ashley's hypothesis revisited.

Nowadays it is widely accepted that cervical cancer is a virus related, sexually transmitted disease, but no complete agreement exists about its etiological factors. They may be identified in HSV or in HPV or in both. Moreover, its is accepted that cervical cancer does not arises ex abrupto from normal epithelium, but often arises from the epithelia with well codified morphological lesions now defined as "Cytohistological Cervical Cancer Precursors". Epidemiological data available at the moment, also concerning risk age, suggest that two different risk classes do exist. They range respectively between 20 and 40 years and between 50 and 70 years of age. When considering sexual promiscuity, younger patients refer a higher and more intensive level than that referred by older; the latter often claim for a low level of sexual activity. Starting from this observation, as well as from clinical and histological evidence, Ashley first suggested in 1965 the existence of two different types of cervical cancer. In the Ashley hypothesis the first type, slow-growing, is often preceded by a precursor, non-invasive stage, while the second, fast-growing, rapidly invades, often without evidence of a previnvasive stage. In our series of 1,010 invasive and 210 in situ cervical cancers, indeed, two different ranges of risk-ages exist, fulfilling Ashley's hypothesis. Morphological evaluation of lesions shows that in the younger patients, invasive cancer is, in a high percentage, associated with HPV-VCE and CIN in the surroundings, while in the older, this feature is less evident.(ABSTRACT TRUNCATED AT 250 WORDS)

The E7 protein of the cottontail rabbit papillomavirus immortalizes normal rabbit keratinocytes and reduces pRb levels, while E6 cooperates in immortalization but neither degrades p53 nor binds E6AP.

Human papillomaviruses (HPVs) cause cervical cancer and are associated with the development of non-melanoma skin cancer. A suitable animal model for papillomavirus-associated skin carcinogenesis is the infection of domestic rabbits with the cottontail rabbit papillomavirus (CRPV). As the immortalizing activity of CRPV genes in the natural target cells remains unknown, we investigated the properties of CRPV E6 and E7 in rabbit keratinocytes (RK) and their influence on the cell cycle. Interestingly, CRPV E7 immortalized RK after a cellular crisis but showed no such activity in human keratinocytes. Co-expressed CRPV E6 prevented cellular crisis. The HPV16 or CRPV E7 protein reduced rabbit pRb levels thereby causing rabbit p19(ARF) induction and accumulation of p53 without affecting cellular proliferation. Both CRPV E6 proteins failed to degrade rabbit p53 in vitro or to bind E6AP; however, p53 was still inducible by mitomycin C. In summary, CRPV E7 immortalizes rabbit keratinocytes in a species-specific manner and E6 contributes to immortalization without directly affecting p53.

Persistent conjunctival papilloma due to oral papillomavirus infection in a rabbit in New Zealand.

A 3-yr-old female Flemish Giant pet rabbit developed a papilloma on the right nictitating membrane. Although the papilloma was excised surgically, it promptly recurred. Examination of the eye 10 wk after surgery revealed that in addition to the initial mass, 2 smaller papillomas were present on the lower eyelid. All 3 masses were excised, and histology revealed papillomatous hyperplasia of the conjunctival epithelium, koilocytosis, and intranuclear viral inclusions. Polymerase chain reaction amplified papillomaviral DNA from the largest papilloma. Sequencing of the amplicon revealed 99.3% homology with rabbit oral papillomavirus (ROPV). All 3 masses recurred after removal. In addition, the rabbit was noted to be losing weight. Weight loss continued until the rabbit died 3 mo later. All 3 papillomas persisted until death. This article provides the fi rst description of ROPV causing conjunctival papillomas and is the fi rst report of ROPV from the southern hemisphere. The persistence of the papillomas in this case is also unusual and may suggest that ROPV-induced conjunctival papillomas are less likely than oral papillomas to spontaneously regress. Alternatively, the death of this rabbit may indicate a compromised immune system that allowed papillomaviral persistence.

Papillomavirus particles assembled in 293TT cells are infectious in vivo.

Papillomaviruses (PVs) demonstrate both tissue and species tropisms. Because PVs replicate only in terminally differentiating epithelium, the recent production of infectious PV particles in 293 cells marks an important breakthrough. In this article, we demonstrate that infectious PV particles produced in 293TT cells can cause papillomatous growths in the natural host animal. Moreover, we show that species-matched PV genomes can be successfully delivered in vivo by a heterologous, species-mismatched PV capsid. Additionally, our results indicate that the addition of the simian virus 40 origin of replication to the papillomavirus genome increases the production of infectious papillomavirus particles by increasing genome amplification in the transfected 293TT cells.

Cottontail rabbit papillomavirus E8 protein is essential for wart formation and provides new insights into viral pathogenesis.

The cottontail rabbit papillomavirus (CRPV) a and b subtypes display a conserved E8 open reading frame encoding a 50-amino-acid hydrophobic protein, with structural similarities to the E5 transmembrane oncoprotein of genital human PVs (HPVs). CRPV E8 has been reported to play a role in papilloma growth but not to be essential in papilloma formation. Here we report that the knockout of E8 start codon almost prevented wart induction upon biobalistic inoculation of viral DNA onto rabbit skin. The scarce warts induced showed very slow growth, despite sustained expression of E6 and E7 oncogenes. This points to an essential role of E8 in disturbing epidermal homeostasis. Using a yeast two-hybrid screen, we found that E8 interacted with the zinc transporter ZnT1, protocadherin 1 (PCDH1), and AHNAK/desmoyokin, three proteins as yet unrelated to viral pathogenesis or cell transformation. HPV16 E5 also interacted with these proteins in two-hybrid assay. CRPV E8 mainly localized to the Golgi apparatus and the early endosomes of transfected keratinocytes and colocalized with ZnT1, PCDH1, and AHNAK. We showed that ZnT1 and PCDH1 formed a complex and that E8 disrupted this complex. CRPV E8, like HPV16 E5, increased epidermal growth factor (EGF)-dependent extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and both the EGF-dependent and the EGF-independent activity of activating protein-1 (AP-1). Competition experiments with a nonfunctional truncated ZnT1 protein showed that E8-ZnT1 interaction was required for AP-1 activation. Our data identify CRPV E8 as a key player in papilloma induction and unravel novel cellular targets for inducing the proliferation of keratinocytes.

Identification of the E9/E2C cDNA and functional characterization of the gene product reveal a new repressor of transcription and replication in cottontail rabbit papillomavirus.

Cottontail rabbit papillomavirus (CRPV) genomes mutated in the trans-activation domain of the E2 protein, which stimulates both viral DNA replication and transcription, are severely impaired in their ability to induce tumors in New Zealand White rabbits. A number of papillomaviruses encode, in addition to full-length E2, a shortened E2 protein or an E2 protein fused to a short stretch of amino acids derived from the small E8 open reading frame that counteract the activities of E2. We identified and cloned the novel cDNA E9/E2C of CRPV from papillomas of New Zealand White and cottontail rabbits and characterized the functions of the encoded gene product. E9/E2C was shown to be a bona fide repressor of minimal viral promoters, with the E9 domain being essential for this activity, and to repress E1/E2-dependent replication of a CRPV origin construct. In addition, E9/E2C counteracted the transactivation effect of the full-length E2 on minimal promoters containing several E2 binding sites. To investigate the role of E9/E2C in tumorigenesis, we constructed two CRPV genomes mutated in E9/E2C. One, designated CRPV-E9atgmut-pLAII, contained a mutation in the unique start codon in the E9 open reading frame, and the second E9/E2C mutant was constructed by the introduction of a stop codon close to the splice donor site at nucleotide 3714 that additionally prevented the correct splicing of the transcript. When we infected New Zealand White rabbits with these constructs, we surprisingly noted no differences in tumor induction efficiency, viral genome copy number, and viral transcription in comparison to wild-type CRPV.

Identification and characterization of the CRPV E7 protein expressed in COS-7 cells.

The papillomavirus E7 protein may play an important role in oncogenesis as it is the major viral protein present in human cervical cancer-derived cell lines. Because of the relevance of the cottontail rabbit papillomavirus (CRPV) system for the study of viral-induced malignancies, we characterized its E7 protein expressed by a heterologous strong promoter. An E7-specific antiserum was obtained by immunizing rabbits with a Trp E-E7 fusion protein containing the 88 carboxyl-terminal amino acids of E7. This antiserum specifically precipitated from [35S]cysteine but not from 32PO4-labeled transiently transfected COS-7 cells a 14-kDa protein. The protein was present only in the soluble cytoplasmic fraction and exhibited a heterogeneous sedimentation rate in nondenaturing glycerol gradients.

Immunization with nonstructural proteins E1 and E2 of cottontail rabbit papillomavirus stimulates regression of virus-induced papillomas.

Cottontail rabbit papillomavirus is the major animal model for cancer-associated papillomaviruses. Here we show that vaccination with the nonstructural proteins E1 and E2 induces the regression of virus-induced papillomas and that vaccination is equally effective when proteins are given with and without adjuvant. There was no correlation between antibody levels and regression, suggesting that tumor regression may be due to a cell-mediated response.

Regression of papillomas induced by cottontail rabbit papillomavirus is associated with infiltration of CD8+ cells and persistence of viral DNA after regression.

Cottontail rabbit papillomavirus (CRPV) is a highly oncogenic papillomavirus and has been successfully used as a model to develop protective vaccines against papillomaviruses. Papillomas induced by the virus may spontaneously regress, suggesting that CRPV can also serve as a model to develop therapeutic vaccines. As a first step toward this goal, we have analyzed immunologic and viral aspects associated with papilloma regression and have identified several features unique to regression. Immunohistochemical staining of biopsies from growing and regressing papillomas and from sites after complete regression showed infiltration of CD8+ cells into the basal and suprabasal layers of the epidermis only during active regression. In situ hybridizations with mRNA-specific probes were strongly positive for E6 and E7 mRNAs during regression, but no late mRNA was present. Viral DNA was detected by in situ hybridization during regression but not after regression. However, analysis by PCR revealed persistence of viral DNA for several months at the majority of regression sites. The results suggest that stimulation of a strong CD8+ response to virus-infected cells is important for an effective therapeutic vaccine and that special attention should be given to the suppression of latent infection.

A transactivator function of cottontail rabbit papillomavirus e2 is essential for tumor induction in rabbits.

Infection of domestic rabbits with cottontail rabbit papillomavirus (CRPV) causes local papillomas which progress to carcinomas in more than 80% of cases. This animal model system therefore allows the identification of molecular mechanisms required for the induction and progression of epithelial tumors. The viral E2 protein stimulates both viral DNA replication and transcription, and these functions can be genetically separated. We introduced the respective mutations into CRPV E2 and found, in line with published data for other papillomavirus E2 proteins, that mutation of the highly conserved amino acid 37 or 73 resulted in replication-competent but transactivation-deficient E2 proteins, whereas E2 proteins with mutations at residue 39 were replication deficient and transactivation competent. The R37A, I73L, and I73A E2 mutants, showing a loss of transactivation function, and the R37K E2 mutant, which is still transactivation competent, were introduced into the whole genome of CRPV, which was then injected into the skin of rabbits. Strikingly, the ability to induce tumors within 6 weeks was abolished by each of the E2 mutations, in contrast to the tumor induction rate (93%) obtained with wild-type CRPV DNA. Two small papillomas induced by mutant E2 I73A CRPV DNA appeared as late as 12 or 24 weeks postinjection, were significantly smaller, and showed no further extension of growth. These data suggest that functionally conserved amino acids in the transactivation domain of E2 are also required for the induction and growth of epithelial tumors in rabbits infected with CRPV.

The viral E4 protein is required for the completion of the cottontail rabbit papillomavirus productive cycle in vivo.

Expression of the papillomavirus E4 protein correlates with the onset of viral DNA amplification. Using a mutant cottontail rabbit papillomavirus (CRPV) genome incapable of expressing the viral E4 protein, we have shown that E4 is required for the productive stage of the CRPV life cycle in New Zealand White and cottontail rabbits. In these lesions, E4 was not required for papilloma development, but the onset of viral DNA amplification and L1 expression were abolished. Viral genome amplification was partially restored when mutant genomes able to express longer forms of E4 were used. These findings suggest that efficient amplification of the CRPV genome is dependent on the expression of a full-length CRPV E4 protein.

Postattachment neutralization of papillomaviruses by monoclonal and polyclonal antibodies.

Postattachment neutralization of papillomaviruses (PVs) was analyzed in three PV-infectivity models: (i) the BPV-1-induced focus-forming assay using C127 cells; (ii) in vitro abortive infection of rabbit RK-13 and Sf1Ep cells with CRPV; and (iii) HPV-11-induced morphological transformation of human foreskin chips in the athymic mouse xenograft system. In each assay system, aliquots of infectious virus were added to the appropriate target cells and incubated at 37 degrees, followed at various postinfection time intervals with neutralizing monoclonal antibodies (N-MAbs) that target surface conformational epitopes. In all three model systems, the N-MAbs were able to neutralize PV infection when added as late as 8 hr after addition of infectious PV to host cells. These results imply that papillomaviruses attach to but do not penetrate inside host cells for a significant period of time and that the bound virus is thus still susceptible to neutralization by neutralizing antibodies.