Cottontail Rabbit Papillomavirus Research Articles

A simplified ‘coupled’ probing approach for the detection of proteins on dot and western blots

Western blotting is one of the most commonly and widely used methods for identifying the presence of an antigen in biochemical studies. The present study describes a novel ‘coupled blotting’ approach that allows antigen on dot and Western blots to be simultaneously probed with primary and secondary probing reagents. The study utilized the highly sensitive enhanced chemiluminescence (ECL) detection system, purified E7 protein of cottontail rabbit papillomavirus (CRPV) and E7-specific antibodies. Comparison of the abilities of sequential primary antibody followed by secondary reagent and coupled treatments to detect E7 protein on blots showed no reduction in signal strength after coupled probing. The new ‘coupled’ probing has the advantage of saving hands-on time and buffer solutions over the standard procedure. Overall, the results suggest that the ‘coupled blotting’ is useful for the rapid detection of proteins without compromising quality.

High efficiency, long-term clinical expression of cottontail rabbit papillomavirus (CRPV) DNA in rabbit skin following particle-mediated DNA transfer.

The ability of skin to support long lasting expression of genes delivered with a particle-mediated system was evaluated in rabbits inoculated with cottontail rabbit papillomavirus (CRPV) DNA. The optimal delivery force for maximal gene expression in rabbit skin was determined in transient beta-galactosidase assays. Forty-five sites in four rabbits were then inoculated at 350-400 p.s.i. with CRPV DNA. All sites (100%) formed papillomas with multiple papillomas at most sites. These results support the feasibility of using a particle-mediated delivery system for gene therapy and suggest that some papillomavirus features, such an origin of replication, may be well suited for use in vectors to target long term expression to skin.

Conserved Features in Papillomavirus and Polyomavirus Capsids

Capsids of papilloma and polyoma viruses (papovavirus family) are composed of 72 pentameric capsomeres arranged on a skewed icosahedral lattice (triangulation number of seven, T=7). Cottontail rabbit papillo mavirus (CRPV) was reported previously to be a T=7 laevo(left-handed) structure, whereas human wart virus, simian virus 40, and murine polyomavirus were shown to be T=7 dextro(right-handed). The CRPV structure determined by cryoelectron microscopy and image reconstruction was similar to previously determined structures of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 1 (HPV-1). CRPV capsids were observed in closed (compact) and open (swollen) forms. Both forms have star-shaped capsomeres, as do BPV-1 and HPV-1, but the open CRPV capsids are ≈2 nm larger in radius. The lattice hands of all papillomaviruses examined in this study were found to be T=7 dextro. In the region of maximum contact, papillomavirus capsomeres interact in a manner similar to that found in polyomaviruses. Although papilloma and polyoma viruses have differences in capsid size (≈60 versus≈50 nm), capsomere morphology (11 to 12 nm star-shaped versus8 nm barrel-shaped), and intercapsomere interactions (slightly different contacts between capsomeres), papovavirus capsids have a conserved, 72-pentamer, T=7 dextrostructure. These features are conserved despite significant differences in amino acid sequences of the major capsid proteins. The conserved features may be a consequence of stable contacts that occur within capsomeres and flexible links that form among capsomeres.

Transforming properties of the cottontail rabbit papillomavirus oncoproteins Le6 and SE6 and of the E8 protein

Cottontail rabbit papillomavirus induces on cottontail and domestic rabbits papillomas which progress at a high frequency to carcinoma. The virus encodes three transforming proteins; one is translated from open reading frame (ORF) E7 and binds the retinoblastoma protein, and two, LE6 and SE6, are translated from the first and second ATGs of ORF E6, respectively. Here we show that neither of the E6 proteins coprecipitated with p53 in vitro, nor did they bind to a recently identified E6-binding protein (J. J. Chen, C. E. Reid, V. Band, and E. Androphy, Science 269:529-531, 1995). This protein was shown to bind to the E6 proteins of the high-risk human papillomairus types 16 and 18 but not to the low-risk human papillomavirus types VI and II. In-frame deletions cloned into the pZipNeo vector were used to identify structural features of SE6 and LE6 important for transformation of NIH 3T3 cells. Three deletions covering the amino-terminal half of SE6 did not transform cells. In two of the three deletions, two Cys-X-X-Cys motifs were deleted, each deletion preventing the formation of one of the potential small Zn fingers of SE6. Among the LE6 deletions, only one had a reduced transformation efficiency, while seven transformed cells at least as efficiently as wild-type LE6. In each of three of these seven mutants, two Cys-X-X-Cys motifs were deleted. None of the three amino acid deletions which abolished transformation by SE6 reduced transformation by LE6. Furthermore, transformation did not correlate with the level of SE6 or LE6 proteins detectable. ORF E8 colinear with ORF E6, which could generate a 50-amino-acid protein with a hydrophobic segment, did not transform cells when cloned into the pZipNeo vector. However, mutation of the E8 ATG, which did not alter the amino acid sequence of LE6, increased transformation by LE6 without affecting the level of LE6 expression. The data suggest that transformation by the E6 proteins is not mediated by interfering with p53 function or through binding to the E6-binding protein. Furthermore, different structural features are important to maintain transformation functions and protein stability of LE6 and SE6. Finally, E8 seems not to be a transforming protein but rather appears to modulate transformation bv LE6.

Particle-mediated delivery of recombinant expression vectors to rabbit skin induces high-titered polyclonal antisera (and circumvents purification of a protein immunogen).

Polyclonal antibodies were generated in rabbits by delivery to skin of gold particles coated with mammalian expression vectors encoding a cytoplasmic (beta-galactosidase) or a nuclear (L1 capsid of cottontail rabbit papillomavirus) protein. One primary and one booster immunization of 30 micrograms DNA per rabbit yielded specific antisera with titers from 1:24 000 to 1:120 000 in each of eight rabbits, as detected by ELISA and Western blot analysis. Genetic immunization requires relatively small amounts of DNA, eliminates the need to purify the protein immunogen, and does not require irritating adjuvants.

The primary target cells of the high-risk cottontail rabbit papillomavirus colocalize with hair follicle stem cells.

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.

Rapid, efficient, large-scale purification of unfused, non-denatured E7 protein of cottontail rabbit papillomavirus.

Cottontail rabbit papillomavirus (CRPV) E7 protein is one of the :high risk' papillomavirus E7 oncoproteins that are partially insoluble in aqueous solution. An Escherichia coli expression system was used for purification of CRPV E7 protein in quantities sufficient for immunologic studies and structural analysis. A glutathione S-transferase (GST)-CRPV E7 fusion protein was solubilized in the presence of non-ionic and ionic detergents, and isolated on an affinity column of glutathione Sepharose beads. The CRPV E7 portion was cleaved from the column with thrombin at a thrombin cleavage site between the fused partners. Thrombin was removed subsequently by adsorption to benzamidine. This method is rapid, requiring just one week, and efficient, yielding 3 mg of pure CRPV E7 protein per liter of bacterial culture. It produced a protein product that was about 95% pure. High-titered polyclonal antisera generated to the product recognized CRPV E7 but not GST. Purified CRPV E7 protein exhibited the ability to bind pRB, making it the first unfused, non-denatured CRPV E7 product reported to do so. This attribute could facilitate structure-function studies of CRPV E7-pRB interactions.

Immunization with viruslike particles induces long-term protection of rabbits against challenge with cottontail rabbit papillomavirus

Rabbits were immunized with recombinant baculovirus-produced virus-like particles (VLPs) of cottontail rabbit papillomavirus (CRPV) to determine whether these antigens could induce long-term protection against experimental challenge with CRPV. Infectious CRPV and human papillomavirus type 11 L1 VLPs were used as positive and negative control immunogens, respectively. Three groups of immunized animals were challenged with 10-fold serial dilutions of infectious CRPV at 2 weeks, 6 months, and 12 months after immunizations. Antibody titers in serum reached 1:10,000 immediately after the final booster immunization and then decayed to 1:150 at 6 months and 1:100 at 12 months in unchallenged rabbits. Serum neutralization titers followed similar kinetics. Papillomas grew on control-immunized rabbits at sites challenged with 10(-1) (100% of sites), 10(-2) (96% of sites), 10(-3) (63% of sites), and 10(-4) (13% of sites) dilutions of virus. At 2 weeks after CRPV L1 VLP immunizations, the rabbits were completely protected against virus challenge. At both 6 and 12 months after CRPV L1 VLP immunizations, strong protection was also observed. In the last two groups, three of seven rabbits were completely protected and only 4 of 14 or 29% of sites challenged with 10(-1 dilution of virus grew papillomas. Papillomas growing at these four sites were also reduced in size (3.5 +/- 0.7 mm) at 50 days postchallenge compared with sites challenged with 10(-1) dilution on control-immunized rabbits (13.2 +/- 4.2 mm). The results demonstrate that strong and long-lasting protection against experimental challenge with papillomaviruses can be achieved with VLP immunogens.

Protection against Papillomavirus with a Polynucleotide Vaccine

Genital infections with human papillomavirus (HPV) are increasingly recognized as a significant source of human disease; HPV is now implicated in up to 90% of cervical carcinomas. Neutralizing antibodies against papillomaviruses recognize conformational epitopes formed when viral capsid proteins assemble into virions or virus-like particles. Immunization with plasmid DNA encoding the major viral capsid protein L1 was studied as a means of inducing neutralizing antibodies and protection against virus challenge. In a cottontail rabbit papillomavirus (CRPV) model, immunization with plasmid DNA encoding L1 elicited conformationally specific neutralizing antibodies and provided immunity against papilloma formation upon challenge with CRPV. Immunization with DNA encoding the capsid protein may provide a means of protecting humans against HPV and would simplify the production of multivalent vaccines by combining plasmids that encode the viral capsid proteins of different strains. This may be of importance given the multiplicity of HPV types capable of causing disease.

E2 represses the late gene promoter of human papillomavirus type 8 at high concentrations by interfering with cellular factors.

The late gene promoter P7535 of the epidermodysplasia verruciformis-associated human papillomavirus type 8 (HPV8) is regulated by the viral E2 protein. Transfection experiments performed with the human skin keratinocyte cell line RTS3b and P7535 reporter plasmids revealed transactivation at low amounts and a repression of basal promoter activity at high amounts of E2 expression vector. This repression was promoter specific and correlated with the amount of transiently expressed E2 protein. Mutational analyses revealed that the negative regulation of P7535 activity is mediated by the low-affinity E2 binding site P2, which is separated by one nucleotide from the P7535 TATA box. Biochemical and genetic analyses suggested that repression is due to a displacement of the TATA-box binding protein by E2 and an interference of E2 with promoter-activating cellular factors that specifically recognize the P2 sequence. The high conservation of the P2 sequence among several papillomaviruses (epidermodysplasia verruciformis-associated HPVs, HPV1, cottontail rabbit papillomavirus, and bovine papillomavirus type 1) in the vicinity of the late gene promoter cap site suggests that an interplay of E2 and cellular factors at this sequence element is important for the expression of structural proteins.

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.

Papillomavirus L1 capsids agglutinate mouse erythrocytes through a proteinaceous receptor

Virus-like particles (VLPs) composed of L1 derived from bovine papillomavirus type 1 (BPV-1), several human papillomavirus types, or cottontail rabbit papillomavirus (CRPV) agglutinated mouse but not human or rat erythrocytes. Treatment of mouse erythrocytes with trypsin prevented hemagglutination (HA) by BPV-1. Sera from rabbits immunized with native CRPV VLPs, which protect against experimental CRPV infection, exhibited high titers of antibodies that inhibited CRPV VLP HA activity, while sera from rabbits immunized with denatured CRPV VLPs or native BPV VLPs, which do not protect against CRPV infection, were not inhibitory. Testing for HA inhibition is a rapid and simple method for examining the serological relatedness of papillomaviruses and measuring protective antibody titers after VLP vaccination.

Induction of Neutralizing Antibodies to Papillomaviruses by Anti-idiotypic Antibodies

Anti-idiotypic antibodies (anti-Ids) were generated against three mouse monoclonal antibodies (MAbs) which neutralized three different papillomaviruses. The neutralizing MAbs (N-MAbs) were generated against infectious human papillomavirus type 11 (HPV-11), cottontail rabbit papillomavirus (CRPV), and bovine papillomavirus type 1 (BPV-1), and all recognized surface conformational epitopes that were lost upon denaturation of the virions. The polyclonal anti-Ids were screened in an ELISA using a panel of 10 N-MAbs (4 HPV-11 neutralizing, 5 CRPV neutralizing, and 1 BPV-1 neutralizing), and cross-reactive idiotypic reactivity was observed. Affinity-purified anti-H11.B2 (HPV-11 N-MAb) cross-reacted with the 3 other HPV-11 N-MAbs, was unreactive to a nonneutralizing HPV-11 MAb and all 5 CRPV N-MAbs, but surprisingly was reactive to the BPV-1 N-MAb. In contrast, affinity-purified anti-CRPV-1A (CRPV N-MAb) reacted with all 5 CRPV N-MAbs, but not with any other MAb. Affinity-purified anti-B1.A1 (BPV-1 N-MAb) also showed unexpected cross-reactivity with the 4 HPV-11 N-MAbs, but not with any of the CRPV N-MAbs. Two of the three polyclonal rabbit anti-Ids induced strong anti-vital antibody responses (including virus-neutralizing antibodies) in BALB/c mice following immunization. The results indicate that within a mouse strain, papillomavirus-neutralizing antibodies share cross-reactive idiotopes that group the N-MAbs into several subsets. One group includes the CRPV N-MAbs, and another group includes N-MAbs directed to apparently unrelated papillomaviruses, including HPV-11, BPV-1, and possibly HPV-6.

Immunization with viruslike particles from cottontail rabbit papillomavirus (CRPV) can protect against experimental CRPV infection

We tested the ability of vaccination with virus-like particles (VLPs) to protect domestic rabbits against papillomas induced by the cottontail rabbit papillomavirus (CRPV). A recombinant baculovirus system that expressed only the L1 major papillomavirus structural protein or L1 plus the minor L2 protein was used in insect cells as the source of VLPs. Groups of 10 rabbits were immunized with native or denatured VLPs from CRPV or type 1 bovine papillomavirus by using Freund's adjuvant. Alum was used as the adjuvant for an additional group immunized with CRPV L1-L2 VLPs. Animals were challenged with 5 x 10(10) and 2 x 10(11) particles on opposing flanks. No protection was seen in rabbits immunized with native or denatured bovine papillomavirus L1-L2 or with denatured CRPV L1-L2. In these groups, the lower and higher challenge doses resulted in 27 of 30 animals with extensive papillomas, with each of the remaining animals having a smaller number of persistent papillomas. Progression to carcinoma developed in 20 rabbits. Animals inoculated with native CRPV VLPs composed of L1 alone or L1-L2 developed many fewer lesions; the lower and higher challenge doses resulted in 17 of 29 and 5 of 29 rabbits, respectively, with no lesions, and the remainder developed only one to eight papillomas, which all regressed except for those on 1 rabbit. None developed cancer within 1 year of infection. Rabbits vaccinated with native CRPV VLPs developed high-titer antibodies in an enzyme-linked immunosorbent assay based on native VLPs, and passive transfer of serum or immunoglobulin G from rabbits immunized with CRPV VLPs protected against CRPV challenge. We conclude that native VLPs can induce antibody-mediated, type-specific protection against experimental papillomavirus infection.

Tumor Regression Is Associated with a Specific Immune Response to the E2 Protein of Cottontail Rabbit Papillomavirus

Cottontail rabbit papillomavirus is the major papillomavirus animal model with which to study host-virus interactions. As with human papillomaviruses, papillomas may spontaneously regress, persist, or progress to carcinoma. Here we show that the majority (88%) of regressor rabbits had antibody to the nonstructural protein E2 compared to 29% in animals with persisting papilloma. The antibody response to other nonstructural viral proteins was the same for rabbits with regressing and persisting papilloma. The cellular immune response was measured by an in vitro proliferation assay. The responses to E6 and E7 were infrequent and similar in papilloma-bearing and in regressor rabbits and no rabbits responded to E1. In contrast, the response to E2 was more frequent in regressor rabbits. These data suggest that E2-specific immune responses may play a role in tumor regression.

Variable-region sequences for T-cell receptor-γ and -δ chains of rabbit killer cell lines against Shope carcinoma cells

A rabbit γδ killer T-cell line against Shope carcinoma cells was established from peripheral blood lymphocytes (PBL) of a human T-lymphotropic virus type I (HTLV-I)-infected rabbit bearing Shope papilloma and carcinoma. Southern hybridization analysis of this cell line with an HTLV-I probe showed that the cell line contained multiple clones of HTLV-I-transformed cells, and three sublines with different integration patterns of the HTLV-1 genome were isolated by cloning of the cell line. In all these sublines T-cell receptor (TCR)-γ and -δ genes were rearranged and expressed. A PCR-based analysis of the expression of variable (V) genes showed that the killer cell line preferentially expressed Vγ1.1 and Vδ1 genes, whereas Vγ2 and Vδ1 genes were dominantly expressed in normal PBL. Analysis of the junctional sequences of TCR-γ and -δ genes which dictate the fine specificities of epitope recognition revealed that all three sublines expressed Vγ1.1/Vδ1 genes without the nucleotide diversity at the V-J junctions.

Vaccination with yeast-expressed cottontail rabbit papillomavirus (CRPV) virus-like particles protects rabbits from CRPV-induced papilloma formation

Papillomaviruses infect epithelia of the skin and mucous membranes and cause benign or malignant tumours in animals and in humans. The viruses are highly species-specific, and cell culture systems for propagating human papillomaviruses (HPVs) do not exist. However, there are several animal papillomavirus models. In the cottontail rabbit papillomavirus (CRPV) system, we demonstrated that recombinant CRPV virus-like particles (VLPs) consisting of the capsid proteins L1 or L1+L2 can be produced in the yeast Saccharomyces cerevisiae. Three immunizations with L1 VLPs formulated on aluminum adjuvant at 1–100 μg dose −1 efficiently protected rabbits from challenge with CRPV. Sera of immunized rabbits were shown to contain high-titered serum antibodies to CRPV L1 VLPs and to neutralize CRPV in vitro. Our results suggest that recombinant yeast-derived VLPs could be the basis for a candidate HPV vaccine.

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.

Methylation of cottontail rabbit papillomavirus DNA and tissue-specific expression in transgenic rabbits

Transgenic rabbits carrying multiple copies of CRPV genomic DNA were described previously (Peng et al. (1993) J. Virol. 67, 1698–1701). CRPV DNA was detectable in all tissues of the transgenic rabbits, howeverm the transcripts of CRPV genes only were found in skin and skin tumors. Tumor development was also restricted to skin. To study the mechanism involving tissue-specific expression of CRPV genes in rabbits, cellular DNAs, isolated from different normal tissues and skin tumors, were digested with the two isoschizomeric restriction endonucleases Msp I (methylation resistant) and Hpa II (methylation sensitive), respectively, and analyzed by Southern blot. CRPV DNA, especially its upstream regulatory region (URR), was extensively methylated in all normal tissues, but methylation was remarkably reduced in skin tumors. These data suggest that extensive methylation of CRPV genome, especially in the URR, might be a factor in controlling its tissue-specific expression.

Varied expression of major histocompatibility complex and oncogenes in Shope carcinoma cell lines derived from a single tumor.

The cellular gene expression was compared in four Shope carcinoma cell lines, which were derived from a single tumor and possess various potentials for differentiation and tumorigenicity. The E6 and E7 transforming genes of cottontail rabbit papillomavirus were expressed in all these cell lines, highest level of expression being in the most tumorigenic and undifferentiated cell line, where the major histocompatibility complex (MHC) class I expression was the lowest. The MHC class II antigen, which is not expressed on normal epithelial cells, was detected in all the cell lines, but hardly, if at all, on the surface of these cells. The surface expression of the MHC class II antigen could not be induced by the culture supernatant of phytohaemagglutinin-stimulated splenocytes, which increased the surface expression level of the MHC class I antigen of the same cells. These findings suggest that the aberrant expression of the MHC class II antigen in these cells could not be implicated in the immune response against tumors. The c-fos, c-myc and c-H-ras oncogenes were variably expressed in these cell lines, but there was no correlation with tumorigenicity.