Cottontail Rabbit Papillomavirus Research Articles

Treatment of latent rabbit and human papillomavirus infections with 9-(2-phosphonylmethoxy)ethylguanine (PMEG)

The acyclic nucleotide PMEG was studied for effectiveness against Shope papillomavirus (CRPV) infection of rabbits and human papillomavirus type 11 (HPV-11) infections of human foreskin xenografts in athymic mice. PMEG given in the latent period strongly suppressed the subsequent growth rates of Shope papillomas. PMEG starting in the latent period and continuing for the duration of the experiment, inhibited HPV-11 infections of human skin, including condyloma growth, and synthesis of viral DNA and capsid antigen. Drug toxicity paralleled the therapeutic effects in rabbits but there was much less toxicity in athymic mice.

Biodistribution of Indium 111-Labeled Dihematoporphyrin Ether in Papillomas and Body Tissues: Relevance to Photodynamic Therapy

Hematoporphyrin derivative and its newly purified form, dihematoporphyrin ether, have been shown to localize selectively in malignant tissues and virally induced papillomas. Its use as a probe to distinguish tumors from normal tissues has been largely based on its fluorescence when activated by UV light. These findings are largely subjective, and a direct correlation to its use as a photosensitizing agent to selectively kill transformed cells when activated by an appropriate wavelength of light could not be made. The efficacy of dihematoporphyrin ether photodynamic therapy to selectively kill papillomavirus-transformed cells is based on the increased localization of dihematoporphyrin ether within these tissues as compared with normal tissues. Using cottontail rabbit papillomavirus, cutaneous papillomas were induced on the backs of Dutch belted rabbits. Dihematoporphyrin ether was labeled with indium 111 and intravenously injected into the rabbits. The animals were scanned twice daily for indium 111 activity on a large-field-of-view gamma camera. At 50 hours after injection, the rabbits were killed and papillomas, skin, and major organs collected for biodistribution studies. The results of this study and their relationship to dihematoporphyrin ether photodynamic therapy for the treatment of virally induced papilloma disease is discussed.

Genetic analysis of CRPV pathogenesis: The L1 open reading frame is dispensable for cellular transformation but is required for papilloma formation

Genetic studies to elucidate the role of papillomaviruses in the development and progression of tumors have been severely hampered because the viruses cannot be grown in tissue culture and therefore mutants are not available. We have employed recombinant DNA for papilloma induction to identify essential sequences involved in papillomavirus pathogenesis. Here, we demonstrated that deleting most of the open reading frame (ORF)L2 did not affect the potential of viral cottontail rabbit papillomavirus (CRPV) DNA to induce papillomas. The extrachromosomally maintained DNA in the papillomas was not rearranged and the major transcripts of 1.3 and 2.0 kb encoded E7 and E6, respectively. A recombinant DNA containing a larger deletion lacking the 3′ terminal half of ORF L2 and all of ORF L1 (pdl Bcll) did not induce papillomas. The results indicate that sequences in the late region not required for transformation of NIH 3T3 cells by bovine papillomavirus type-1 essential in CRPV for induction of papillomas.

Use of retroviral vectors for mapping of splice sites in cottontail rabbit papillomavirus.

Cottontail rabbit papillomavirus (CRPV) genomic sequences coding for virus early functions were introduced into a retroviral vector in order to produce cDNAs of the viral early region. Two constructs differing in the length of control sequences preceding the E6 open reading frame were transfected into Psi-2 cells and the released retroviral stock was used to infect NIH3T3 cells. The proviral sequences were rescued from antibiotic G418-resistant virus-infected cells after fusion with Cos cells, amplified as plasmids in Escherichia coli and analysed. Nucleotide sequencing showed that the splicing signals used in the construct containing only the early coding region are the same as in CRPV-expressing tumours, linking the beginning of E1 to the middle of E2. On the other hand, in a construct including most of the long control region a splice donor site located in the 5' end of this region, at position 7810, was very efficiently used, totally excluding the use of the donor site at position 1371. None of the constructs containing CRPV sequences transcribed from Moloney murine leukaemia virus promoter was able to transform mouse fibroblasts after DNA transfection.

E2 of cottontail rabbit papillomavirus is a nuclear phosphoprotein translated from an mRNA encoding multiple open reading frames.

The papillomavirus E2 protein is a transcription trans-activator and as such as a paramount effect on viral functions. We have identified and characterized the cottontail rabbit papillomavirus E2 protein expressed from the late simian virus 40 promoter in COS-7 cells. E2 was shown to be a highly phosphorylated 49-kilodalton protein. Subcellular fractionation and indirect immunofluorescent staining indicated that E2 was located in the nuclei. E2 was expressed from a vector which contained just the open reading frame. ORF E2 and also from vectors which extended farther upstream and also expressed E7 or the short E6 protein. However, the level of E2 was very low in cells transfected with a vector expressing the long E6 protein. Mapping of transcripts with nuclease S1 and exonuclease ExoVII in cells expressing the short E6 and E2 proteins showed that E2 was translated from an RNA which encoded the short E6, E7, and E2 proteins but served as mRNA for only the short E6 and E2 proteins.

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 [ 35]S]cysteine but not from 32PO 4-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.

Study of the E2 gene product of the cottontail rabbit papillomavirus reveals a common mechanism of transactivation among papillomaviruses.

The long control region (LCR) of the cottontail rabbit papillomavirus (CRPV) harbors a transcriptional promoter which can be transactivated, as reflected by cat gene expression, by cotransfection with plasmids which express the intact E2 open reading frame of CRPV, human papillomavirus type 18 (HPV18), and bovine papillomavirus type 1 (BPV1). The E2 protein of CRPV can also transactivate the LCRs of BPV1, HPV1, and HPV18 inserted in front of the cat gene in enhancer or promoter configuration. Competition experiments in vivo and binding studies with CRPV E2 protein synthesized in vitro suggest that the different E2 proteins transactivate transcription by a common mechanism involving binding to the same ACCG-CGGT target sequence. The C-terminal part of the protein is necessary for its DNA-binding function. Analysis of the transactivation data and of the LCR sequences of these four viruses suggests that the two cutaneous viruses (CRPV and BPV1) present a similar pattern of promoter regulation but that the activity of the promoters of genital human viruses is less dependent on E2 regulation and is at least partially regulated by cellular factors.

Histological and molecular analysis of cottontail rabbit papillomavirus-induced papillomas treated with hematoporphyrin derivative photodynamic therapy.

The clinical regression of cottontail rabbit papillomavirus-induced cutaneous papillomas on Dutch-belted rabbits following treatment with hematoporphyrin derivative photodynamic therapy was recently reported. Those papillomas that underwent total regression failed to recur at 18 months. Tissues from totally regressed papillomas and adjacent sites failed to reveal the presence of papilloma on histological examination. These findings were confirmed by the absence of viral DNA following analysis by Southern blots for cottontail rabbit papillomavirus viral copy number and DNA restriction pattern. Partially regressed papillomas and untreated papillomas revealed histologically typical papillary features with approximately equal amounts of viral DNA.

Transcription of the cottontail rabbit papillomavirus early region and identification of two E6 polypeptides in COS-7 cells.

Cottontail rabbit papillomavirus (CRPV) early proteins are present at very low levels in virus-induced tumors and cannot be detected by immunological methods. Furthermore, cells in culture are not readily transformed by the virus. To overcome these difficulties in identifying and characterizing the putative transforming protein(s) coded by the E6 open reading frame, the early cottontail rabbit papillomavirus region was expressed under the control of the late simian virus 40 promoter. Mapping of the transcripts in transiently transfected COS-7 cells indicated that transcription was initiated in the late region of simian virus 40. Two E6-coded polypeptides were identified, representing translation products initiated at the first and second AUG codons.

Enhancers and trans-acting E2 transcriptional factors of papillomaviruses.

The upstream regulatory regions of human papillomavirus (HPV) types 1, 6b, 7, 11, 16, and 18, bovine papillomavirus type 1, and cottontail rabbit papillomavirus were cloned into transcriptional enhancer assay plasmids which carry the simian virus 40 early promoter lacking its own enhancer and the bacterial gene encoding chloramphenicol acetyltransferase (EC 2.3.1.28) (CAT). Enhancer activity, reflected by CAT gene expression, was detected in all of the upstream regulatory regions tested only when the recombinants were cotransfected with plasmids which express an intact E2 open reading frame of HPV types 1 and 11 or bovine papillomavirus type 1. Each E2 protein stimulated the enhancer from the same virus and, to somewhat lesser degrees, also those from the heterologous viruses. Hence, the enhancer and the E2 protein are functionally conserved among papillomaviruses. There was some nonreciprocity in the extent of trans-activation in heterologous E2-enhancer interactions. Primer extension analyses demonstrated that the E2 proteins increased the abundance of CAT gene mRNA. Tandem multiplication of the HPV type 11 enhancer sequence dramatically increased its response to E2 stimulation; this is possibly relevant to the pathogenicity of papillomaviruses.

Identification of the major cottontail rabbit papillomavirus late RNA cap site and mapping and quantitation of an E2 and minor E6 coding mRNA in papillomas and carcinomas

The capsite of the 2.6- and 4.8-kb major late transcripts of cottontail rabbit papillomavirus (CRPV) has been mapped by primer extension. A leader exon of about 300 nucleotides common to both RNAs is located in the untranslated region of the genome upstream of the capsites for early transcripts. In contrast to the early capsites which are all preceded by TATA boxes, no such sequence is present 30 nucleotides upstream of the late capsite. These data indicate that the switch from early to late transcription involves recognition of a new promoter and suppression of transcription termination at the early polyadenylation site. We have also identified a minor exon with a coding potential for a putative E2 transactivating protein. Quantitation by S1 mapping of the E2 coding exon and a minor exon coding for a full-sized E6 protein unique in size to the highly oncogenic CRPV did not reveal differences in the level of transcription between papillomas and carcinomas.

Promoters and processing sites within the transforming region of bovine papillomavirus type 1.

The mRNAs present in bovine papillomavirus type 1 (BPV-1)-transformed C127 cells were studied by primer extension. The results show that two internal promoters are present in the E region of BPV-1 in addition to the previously identified promoter at coordinate 1 (H. Ahola, A. Stenlund, J. Moreno-López, and U. Pettersson, Nucleic Acids Res. 11:2639-2650, 1983). One, located at coordinate 31, generated a set of mRNAs with heterogeneous 5' ends, which may encode the major transforming protein of BPV-1, the E5 protein. The second promoter, which is located at coordinate 39, generates colinear mRNAs which encode either the E4 protein or a truncated form of the E2 protein. Unlike the cottontail rabbit papillomavirus (O. Danos, E. Georges, G. Orth, and M. Yaniv, J. Virol. 53:735-741, 1985), BPV-1 appears to lack a separate promoter for expression of the E7 protein. The major splice sites in the transforming region (E region) of the BPV-1 genome were also identified by nucleotide sequence analysis.

Structure and Genetic Expression of Papillomaviruses

This article reviews the physical structures, genetic organization, expression, and regulation of papillomaviruses. In view of the extensive characterization of cellular transformation and viral functions that have been achieved in experimentally infected animals and in transformed cell cultures, the bovine papillomavirus type 1 (BPV-1) and Shope cottontail rabbit papillomavirus (CRPV) are compared and contrasted with the human papillomaviruses.

Hematoporphyrin derivative therapy of papillomas. Experimental study.

Hematoporphyrin derivative has been shown to selectively localize in malignant tissues and chemically induced animal papillomas. It is a powerful photo-sensitizing agent that can cause destruction and death of tissues in which it has localized by the generation of singlet oxygen when activated by light of the appropriate wavelength (photodynamic therapy [PDT]). Laryngeal papillomas are rapidly growing benign epithelial neoplasms with a clinical course marked by multiple recurrences after surgical removal. Before considering PDT as a therapeutic modality in the treatment of this debilitating disease we developed an animal model for experimentation. Using the cottontail rabbit papillomavirus, sometimes referred to as the Shope papillomavirus, large cutaneous papillomas were induced on the backs of Dutch Belted rabbits. Following intravenous administration of hematoporphyrin derivative, PDT was delivered with white light. Marked regression of the papillomas was noted with replacement by normal hair-bearing skin. The clinical implications of this work to the control of laryngeal papillomatosis is discussed.

Regression of flat warts and common warts

Wart (human papilloma) is such a common disease that even a child can make a diagnosis at a glance. Furthermore, from our long experience, we are aware that warts often disappear in a short period of time, spontaneously or after various magical rituals that are prevalent in different parts of the world. In Japan, we can find some shrines or temples in every district where ardent worshipers make pilgrimages in search of quick answers to their prayers to cure their warts. Evaluation of clinical effectiveness of a new therapeutic modality is most difficult because of its high incidence of spontaneous wart resolution. The lesion can be cured even by hypnosis. Recently, the phenomenon of spontaneous regression of warts was reviewed from the viewpoint of tumor immunology. We now know that in the body, tumors may be rejected by an immune “surveillance” mechanism analogous to resistance to microbial pathogens. Various modalities of immunization procedure or enhancement of host immunity can induce partial or complete resolution of established tumors in both experimental animals and humans. Even malignant tumors, such as melanoma or choriocarcinoma, may disappear spontaneously. Wart is one of the most common tumors in humans and probably the tumor that most commonly shows the phenomenon of spontaneous regression. Advances in molecular biology and biotechnology have disclosed that, as there are various clinical types of wart, there are many distinct HPV types and that the distinct HPV types are associated with distinct cutaneous and mucosal papillomas. 1 Analogous to animals with the Shope papillomavirus or bovine papillomavirus infections, it is known that some human papillomavirus lesions may undergo malignant transformation. 2,3

Specificity of cytopathic effect of cutaneous human papillomaviruses

Papillomaviruses are highly tissue-specific, and are characterized by a specific mode of interaction with the squamous epithelia they infect. 1 It has been proposed that this interaction presents two successive steps that depend on the stage of differentiation of the host cell in the wart. In the basal germinal cells the viral replication is never observed. Based on the well-studied model of the papillomas induced by the Shope cottontail rabbit papillomavirus, it is most likely that the viral genome is present in the basal cells as a small number (10 to 50) of extrachromosomal copies. 2,3 The viral messenger RNAs expressed correspond to nonstructural viral proteins, as yet unidentified, but most probably involved in the altered response of basal cells to the mechanisms regulating their mitotic rate and the size of the germinal cell population, thus resulting in the formation of a papilloma. 4 Vegetative viral DNA replication is triggered upon the onset of the terminal differentiation process in the suprabasal layers, followed by the synthesis of viral structural proteins and the assembly of viral particles in more superficial layers. 1,4

Shope papillomavirus transcription in benign and malignant rabbit tumors

Shope papillomavirus induced benign and malignant tumors from both wild and domestic rabbits were analyzed by the Northern blotting technique for the presence of viral-specific RNAs. Virus-producing benign tumors of wild cottontail rabbits are shown to contain two major RNA species approximately 5000 and 3000 bases in length that originate in the early region and include the majority of the L1 and L2 late open reading frames. Non-productive tumors including wild-rabbit carcinomas, and benign warts and primary and metastatic carcinomas of domestic rabbits uniformly are shown to contain two major viral-specific RNA species, 2400 and 1400 bases in length. These transcripts map entirely within the early region of the viral genome. In addition, Southern blot analysis of metastatic tumors indicates that the viral DNA remains exclusively extrachromosomal and apparently unrearranged supporting previous findings with epithelial malignancies.

Two Shope papillomavirus-associated VX2 carcinoma cell lines with different levels of keratinocyte differentiation and transplantability

Two cell lines, named VX2T and VX2R, were isolated from the transplantable VX2 carcinoma, a wholly anaplastic tumor established from a carcinoma induced by the Shope cottontail rabbit papillomavirus (CRPV) (J.G. Kidd and P. Rous, J. Exp. Med. 71:813-838, 1940). The CRPV genome was found to be maintained and transcribed in both cell lines, as in the VX2 carcinoma. The VX2T cells retained the tumor-producing capacity in the rabbit and the low expression of epidermal keratinocyte differentiation of the VX2 tumor cells. The VX2R cells, although tumorigenic for nude mice, were no longer serially transplantable in the rabbit and expressed differentiated functions of keratinocytes. These data indicate that the anaplastic characteristic and the transplantability of VX2 carcinoma cells to immune competent allogenic hosts may be lost without any detectable modification of the physical state and transcription of the CRPV genome.

Genomic structure of the cottontail rabbit (Shope) papillomavirus.

The nucleotide sequence of the cottontail rabbit papillomavirus (CRPV or Shope papillomavirus) genome has been determined. The overall organization of the genome is similar to that of the other three papillomaviruses already sequenced. The amino acid sequences of the putative viral proteins were compared to the available protein data banks. Of particular interest is the homology found between the COOH terminus of the E2 putative protein of the different papillomaviruses and the viral or cellular mos oncogene product. This analysis also reveals a specific feature of the rabbit virus that may be related to its natural oncogenic potential. One of its open reading frames (E6) shows significant homologies with the beta subunit of a family of ATP synthases from mitochondria, chloroplasts, and bacteria, including the conservation of amino acids residues involved in nucleotide binding. The viral noncoding region includes a highly A+T-rich segment and shows a complex array of repetitions and inverted repeats that may act as control elements for gene expression and genome replication.

Fine structure of the cottontail rabbit papillomavirus mRNAs expressed in the transplantable VX2 carcinoma.

We analyzed the polyadenylated mRNAs transcribed from the cottontail rabbit papillomavirus genomes present in the domestic rabbit transplantable carcinoma line VX2, employing a combination of S1 nuclease mapping and primer extension techniques with vector M13-based single-stranded DNA probes. Each of the two major mRNA species (1,860 and 1,110 bases long) contained two exons which corresponded to the E6-E7 and E2-E4 open reading frames. The splice donor site for these transcripts was located at position 1371 at the beginning of the E1 open reading frame. Consequently, the splicing event did not lead to the fusion of the E region proximal (E6 and E7) and distal (E2, E4, or E5) open reading frames. The translation of the polycistronic RNA could result in the production of E6 and E7 proteins alone or of an additional E1-E4 fusion product if translation reinitiation can occur after the E7 stop codon. A heterogeneity was detected in the 5' ends of the longer transcript; E6 transcripts could thus yield a full-length or a truncated E6 protein. We also detected a minor subset of mRNAs covering the E2-E4 coding region and including at least three species with estimated sizes of 4.2, 2.8, and 1.8 kilobases. All the viral transcripts detected in the VX2 tumor cells were polyadenylated at the same site (position 4367) 20 base pairs beyond the first AATAAA signal which borders the E region.