ABSTRACT
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'.
Subject(s)
Cottontail rabbit papillomavirus/physiology , Host-Pathogen Interactions , Papillomaviridae/physiology , Papillomavirus Infections/virology , Animals , Disease Models, Animal , Humans , RabbitsABSTRACT
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.
Subject(s)
Papillomaviridae/physiology , Papillomavirus Infections/virology , Animals , Cottontail rabbit papillomavirus/physiology , Disease Models, Animal , Humans , Mice , Murinae , RabbitsABSTRACT
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.
Subject(s)
Amyloid Precursor Protein Secretases/physiology , Papillomaviridae/pathogenicity , Papillomavirus Infections/enzymology , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Animals , Cell Line , Cells, Cultured , Cottontail rabbit papillomavirus/genetics , Cottontail rabbit papillomavirus/pathogenicity , Cottontail rabbit papillomavirus/physiology , Enzyme Inhibitors/pharmacology , Female , Genitalia, Female/drug effects , Genitalia, Female/enzymology , Genitalia, Female/virology , HeLa Cells , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/physiology , Human papillomavirus 16/genetics , Human papillomavirus 16/pathogenicity , Human papillomavirus 16/physiology , Human papillomavirus 18/genetics , Human papillomavirus 18/pathogenicity , Human papillomavirus 18/physiology , Human papillomavirus 31/genetics , Human papillomavirus 31/pathogenicity , Human papillomavirus 31/physiology , Humans , Membrane Glycoproteins/physiology , Mice , Mice, Knockout , Papillomaviridae/genetics , Papillomaviridae/physiology , Papillomavirus Infections/virology , Presenilin-1/physiology , RabbitsABSTRACT
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.
Subject(s)
Cottontail rabbit papillomavirus , Matrix Metalloproteinase 9 , Mitogen-Activated Protein Kinase 3/metabolism , Promoter Regions, Genetic/genetics , Transcription Factor AP-1/metabolism , Transcription Factors/metabolism , Viral Proteins/metabolism , Animals , Anthracenes/pharmacology , Binding Sites , Carcinoma/virology , Cell Line , Cell Line, Tumor , Cottontail rabbit papillomavirus/genetics , Cottontail rabbit papillomavirus/physiology , DNA Mutational Analysis , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/virology , Flavonoids/pharmacology , Humans , Imidazoles/pharmacology , JNK Mitogen-Activated Protein Kinases/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Molecular Sequence Data , Pyridines/pharmacology , Rabbits , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
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.
Subject(s)
Cell Transformation, Viral , Cottontail rabbit papillomavirus , Keratinocytes/cytology , Keratinocytes/metabolism , Oncogene Proteins, Viral/metabolism , Retinoblastoma Protein/metabolism , Animals , Cell Line , Cell Nucleus/metabolism , Cottontail rabbit papillomavirus/genetics , Cottontail rabbit papillomavirus/physiology , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Gene Expression Regulation , Humans , Mitomycin/pharmacology , Oncogene Proteins, Viral/genetics , Polymerase Chain Reaction , Protein Binding , Proto-Oncogene Proteins c-mdm2/metabolism , Rabbits , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
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.
Subject(s)
Conjunctival Neoplasms/pathology , Cottontail rabbit papillomavirus/isolation & purification , Papilloma/pathology , Papillomavirus Infections/pathology , Animals , Conjunctival Neoplasms/surgery , Conjunctival Neoplasms/virology , Cottontail rabbit papillomavirus/genetics , Cottontail rabbit papillomavirus/physiology , DNA, Viral/analysis , Fatal Outcome , Female , Papilloma/surgery , Papilloma/virology , Papillomavirus Infections/surgery , Papillomavirus Infections/virology , RabbitsABSTRACT
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.
Subject(s)
Cottontail rabbit papillomavirus/physiology , Papillomavirus Infections/virology , Virus Replication , Animals , Cell Line , Disease Models, Animal , Humans , Papilloma/pathology , Papillomavirus Infections/pathology , Rabbits , Recombination, Genetic , Replication Origin/genetics , Simian virus 40/genetics , Virus Replication/geneticsABSTRACT
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.
Subject(s)
Cottontail rabbit papillomavirus/physiology , Cottontail rabbit papillomavirus/pathogenicity , Oncogene Proteins/physiology , Viral Proteins/physiology , Warts/virology , Amino Acid Sequence , Animals , Cadherins/metabolism , Cation Transport Proteins , Cell Line , Epidermal Growth Factor/physiology , Humans , MAP Kinase Signaling System/genetics , MAP Kinase Signaling System/physiology , Membrane Proteins/metabolism , Molecular Sequence Data , Neoplasm Proteins/metabolism , Oncogene Proteins/deficiency , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Protocadherins , Rabbits , Transcription Factor AP-1/metabolism , Two-Hybrid System Techniques , Viral Proteins/genetics , Viral Proteins/metabolism , Warts/enzymology , Warts/metabolism , Warts/prevention & controlABSTRACT
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.
Subject(s)
Matrix Metalloproteinase 9/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Papilloma/virology , Signal Transduction , Transcription Factor AP-1/metabolism , Transcription Factors/pharmacology , Viral Proteins/pharmacology , Animals , Cells, Cultured , Cottontail rabbit papillomavirus/pathogenicity , Cottontail rabbit papillomavirus/physiology , Disease Models, Animal , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/virology , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Keratinocytes/virology , Papilloma/physiopathology , Papillomavirus Infections/physiopathology , Papillomavirus Infections/virology , Promoter Regions, Genetic , Rabbits , Skin/cytology , Skin/metabolism , Skin/virology , Transcription Factors/genetics , Transcriptional Activation , Viral Proteins/geneticsABSTRACT
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.
Subject(s)
Cottontail rabbit papillomavirus/physiology , Oncogene Proteins, Viral/metabolism , Papilloma/virology , Papillomavirus Infections/virology , Amino Acid Sequence , Animals , Base Sequence , Cottontail rabbit papillomavirus/genetics , Cottontail rabbit papillomavirus/pathogenicity , DNA, Viral/analysis , DNA, Viral/genetics , DNA, Viral/metabolism , Molecular Sequence Data , Mutation , Oncogene Proteins, Viral/genetics , Papilloma/pathology , Papillomavirus Infections/pathology , Rabbits , Sequence Analysis, DNAABSTRACT
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.
Subject(s)
Cottontail rabbit papillomavirus/physiology , Transcription, Genetic/physiology , Virus Replication/physiology , Animals , Base Sequence , Cloning, Molecular , Cottontail rabbit papillomavirus/genetics , DNA Primers , DNA, Complementary , Mutation , RabbitsABSTRACT
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.
Subject(s)
Cottontail rabbit papillomavirus/pathogenicity , Papilloma/virology , Papillomavirus Infections/virology , Trans-Activators/metabolism , Transcription Factors/metabolism , Tumor Virus Infections/virology , Viral Proteins/metabolism , Animals , Cottontail rabbit papillomavirus/physiology , Disease Models, Animal , Female , Humans , Papilloma/physiopathology , Papillomavirus Infections/physiopathology , Rabbits , Trans-Activators/genetics , Transcription Factors/genetics , Transcriptional Activation , Tumor Virus Infections/physiopathology , Viral Proteins/genetics , Virus ReplicationABSTRACT
Animal papillomaviruses are widely used as models to study papillomavirus infection in humans despite differences in genome organization and tissue tropism. Here, we have investigated the extent to which animal models of papillomavirus infection resemble human disease by comparing the life cycles of 10 different papillomavirus types. Three phases in the life cycles of all viruses were apparent using antibodies that distinguish between early events, the onset of viral genome amplification, and the expression of capsid proteins. The initiation of these phases follows a highly ordered pattern that appears important for the production of virus particles. The viruses examined included canine oral papillomavirus, rabbit oral papillomavirus (ROPV), cottontail rabbit papillomavirus (CRPV), bovine papillomavirus type 1, and human papillomavirus types 1, 2, 11, and 16. Each papillomavirus type showed a distinctive gene expression pattern that could be explained in part by differences in tissue tropism, transmission route, and persistence. As the timing of life cycle events affects the accessibility of viral antigens to the immune system, the ideal model system should resemble human mucosal infection if vaccine design is to be effective. Of the model systems examined here, only ROPV had a tissue tropism and a life cycle organization that resembled those of the human mucosal types. ROPV appears most appropriate for studies of the life cycles of mucosal papillomavirus types and for the development of prophylactic vaccines. The persistence of abortive infections caused by CRPV offers advantages for the development of therapeutic vaccines.
Subject(s)
Bovine papillomavirus 1/physiology , Capsid Proteins , Cottontail rabbit papillomavirus/physiology , Papillomaviridae/physiology , Papillomavirus Infections/virology , Tumor Virus Infections/virology , Animals , Antigens, Viral/metabolism , Bovine papillomavirus 1/metabolism , Capsid/metabolism , Cottontail rabbit papillomavirus/metabolism , Disease Models, Animal , Genes, Viral , Humans , Oncogene Proteins, Viral/metabolism , Papillomaviridae/metabolism , Papillomavirus Infections/metabolism , Papillomavirus Infections/pathology , Rabbits , Time Factors , Tumor Virus Infections/metabolism , Tumor Virus Infections/pathology , Viral Structural Proteins/metabolismABSTRACT
The papillomavirus E2 protein plays an important role in viral transcriptional regulation and replication. We chose to study the cottontail rabbit papillomavirus (CRPV) E2 protein as a transcriptional regulator because of the availability of an animal model for papilloma formation, which may be relevant for human papillomavirus (HPV) infection and replication. We studied the effect of expression levels of E2 on the long control region, which contains transcriptional promoter and enhancer elements, and synthetic E2-dependent artificial promoters in which the E2 was the dominant factor in the transcriptional activation. These experiments indicated that high levels of E2 were inhibitory and low levels were stimulatory for transactivation. In addition, we showed that the complex formed between CRPV E2 and the cognate binding site was less stable than the complex formed between HPV E2 and the same cognate binding site. Furthermore, we showed that CRPV E2 binding to its transcriptional regulatory sequence was stabilized by other proteins such as E1, which produced increments in transcriptional activation of E2-dependent genes. The data may be used to define conditions in which the rabbit model can be used for the screening of drugs which are inhibitory to the HPV and CRPV replication and gene expression.
Subject(s)
Cottontail rabbit papillomavirus/genetics , Gene Expression Regulation, Viral , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Animals , Base Sequence , Binding Sites , Cell Line , Cottontail rabbit papillomavirus/physiology , Genes, Reporter , Molecular Sequence Data , Polymerase Chain Reaction , Promoter Regions, Genetic , Rabbits , Recombinant Proteins/metabolism , Regulatory Sequences, Nucleic Acid , Spodoptera , Transcription, Genetic , Transcriptional Activation , TransfectionABSTRACT
We previously reported the partial characterization of two cottontail rabbit papillomavirus (CRPV) subtypes with strikingly divergent E6 and E7 oncoproteins. We report now the complete nucleotide sequences of these subtypes, referred to as CRPVa4 (7,868 nucleotides) and CRPVb (7,867 nucleotides). The CRPVa4 and CRPVb genomes differed at 238 (3%) nucleotide positions, whereas CRPVa4 and the prototype CRPV differed by only 5 nucleotides. The most variable region (7% nucleotide divergence) included the long regulatory region (LRR) and the E6 and E7 genes. A mutation in the stop codon resulted in an 8-amino-acid-longer CRPVb E4 protein, and a nucleotide deletion reduced the coding capacity of the E5 gene from 101 to 25 amino acids. In domestic rabbits homozygous for a specific haplotype of the DRA and DQA genes of the major histocompatibility complex, warts induced by CRPVb DNA or a chimeric genome containing the CRPVb LRR/E6/E7 region showed an early regression, whereas warts induced by CRPVa4 or a chimeric genome containing the CRPVa4 LRR/E6/E7 region persisted and evolved into carcinomas. In contrast, most CRPVa, CRPVb, and chimeric CRPV DNA-induced warts showed no early regression in rabbits homozygous for another DRA-DQA haplotype. Little, if any, viral replication is usually observed in domestic rabbit warts. When warts induced by CRPVa and CRPVb virions and DNA were compared, the number of cells positive for viral DNA or capsid antigens was found to be greater by 1 order of magnitude for specimens induced by CRPVb. Thus, both sequence variation in the LRR/E6/E7 region and the genetic constitution of the host influence the expression of the oncogenic potential of CRPV. Furthermore, intratype variation may overcome to some extent the host restriction of CRPV replication in domestic rabbits.
Subject(s)
Cottontail rabbit papillomavirus/physiology , Genetic Variation , Papillomavirus Infections/virology , Tumor Virus Infections/virology , Warts/virology , Animals , Base Sequence , Cloning, Molecular , Cottontail rabbit papillomavirus/classification , Cottontail rabbit papillomavirus/genetics , DNA, Viral/genetics , Molecular Sequence Data , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Papillomavirus Infections/pathology , Rabbits , Recombinant Fusion Proteins , Tumor Virus Infections/pathology , Virus Replication , Warts/pathologyABSTRACT
Latent human papillomavirus (HPV) infections are widespread in the genital and respiratory tracts and are a source of recurrent disease. This study used a cottontail rabbit papillomavirus (CRPV) model to determine the presence of E1, E6, and E7 transcripts in latent infection and to determine the temporal change in transcripts following UV activation. We found E1 transcripts in all latently infected sites but no detectable E6 and E7 transcripts, consistent with our earlier studies of HPV6/11 latency. These results suggest that this transcription pattern is broadly characteristic of latent papillomavirus infections. E6/E7 transcripts were detectable within 1 week of irradiation, with maximal induction (approximately 40% of sites) at 2 weeks postirradiation. Papillomas were induced in approximately 26% of irradiated sites after a 3- to 5-week lag. Sites that did not form papillomas by 3 months after irradiation were CRPV DNA positive but E6/E7 RNA negative. Thus, only a subset of latent infections can be induced to express E6/E7 transcripts and form papillomas. We propose that CRPV can be used to study the molecular processes regulating papillomavirus activation.
Subject(s)
Cottontail rabbit papillomavirus/physiology , Gene Expression Regulation, Viral/radiation effects , Genes, Viral/genetics , Oncogene Proteins, Viral/genetics , Papillomavirus Infections/virology , Virus Activation , Virus Latency/genetics , Animals , Cottontail rabbit papillomavirus/genetics , Cottontail rabbit papillomavirus/radiation effects , DNA, Viral/analysis , DNA, Viral/genetics , Gene Expression Regulation, Viral/genetics , In Situ Hybridization , Papilloma/pathology , Papilloma/virology , Papillomavirus Infections/pathology , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Viral/analysis , RNA, Viral/genetics , Rabbits , Time Factors , Transcription, Genetic/genetics , Transcription, Genetic/radiation effects , Transcriptional Activation/genetics , Transcriptional Activation/radiation effects , Tumor Virus Infections/pathology , Tumor Virus Infections/virology , Ultraviolet Rays , Virus Activation/genetics , Virus Latency/radiation effectsABSTRACT
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.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cottontail rabbit papillomavirus/genetics , Cottontail rabbit papillomavirus/immunology , DNA, Viral , Papilloma/virology , Papillomavirus Infections/virology , Tumor Virus Infections/virology , Animals , Cottontail rabbit papillomavirus/physiology , Gene Expression , Papilloma/pathology , Papillomavirus Infections/pathology , RNA, Messenger , RNA, Viral , Rabbits , Tumor Virus Infections/pathology , Virus LatencyABSTRACT
Papillomaviruses are small DNA tumor viruses with a life cycle inseparably linked to the differentiation of the pluristratified epithelium. The infection of epithelial layers of the skin may remain latent or may result in the development of benign tumors. A certain number of distinct papillomavirus types, however, cause lesions which have a high risk of progression into carcinomas, and extensive efforts have been made to understand this process. comparatively little is known about the initial events during the establishment of a persistent infection and papilloma development. Although it is generally accepted that the growth of a papilloma requires the infection of cells in the basal layer of the epithelium, it remains unknown which cells perform this task. We have analyzed by in situ hybridization biopsy samples taken at various time points after infection of domestic rabbits with cottontail rabbit papillomavirus. The positive cells detected at a low frequency in biopsy samples taken after 11 days predominantly expressed high levels of E6 and E7 mRNA and were localized in the outer epithelial root sheath and in the bulbs of hair follicles. A clonal analysis of keratinocytes isolated from different subfragments of individual rabbit hair follicles demonstrated a clear colocalization of cottontail rabbit papillomavirus mRNA-positive cells with clonogenic cells in hair follicles. These data suggest that the cells competent to establish papillomatous growth represent a subpopulation of keratinocytes in hair follicles with properties expected of epithelial stem cells.
Subject(s)
Cottontail rabbit papillomavirus/physiology , Hair Follicle/virology , Papillomavirus Infections/virology , Stem Cells/virology , Tumor Virus Infections/virology , 3T3 Cells , Animals , Cottontail rabbit papillomavirus/genetics , Cottontail rabbit papillomavirus/isolation & purification , Hair Follicle/cytology , Hair Follicle/metabolism , Mice , Papillomavirus Infections/pathology , Papillomavirus Infections/veterinary , RNA, Viral , Rabbits , Skin/pathology , Skin/virology , Stem Cells/cytology , Stem Cells/metabolism , Time Factors , Tumor Virus Infections/pathology , Tumor Virus Infections/veterinary , Viral Proteins/geneticsABSTRACT
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.
Subject(s)
Cottontail rabbit papillomavirus/physiology , DNA, Viral/metabolism , Papilloma/virology , Skin Neoplasms/virology , Acetone/chemistry , Animals , Cell Transformation, Neoplastic , Cell Transformation, Viral , Cottontail rabbit papillomavirus/genetics , Female , Hyperplasia/chemically induced , Male , Plasmids , Rabbits , TurpentineABSTRACT
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.