INTRODUCTION

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The Shope papillomavirus or cottontail rabbit papillomavirus (CRPV) induces cutaneous papillomas (warts) in cottontail rabbits under natural conditions and in domestic rabbits under experimental conditions (51). Systemic regression of warts occurs in a variable proportion of rabbits (4, 30, 54) as a consequence of a specific cell-mediated immune response (15, 31). Persistent warts may progress into invasive carcinomas (46, 54). The natural history of CRPV-induced warts thus mimics that of cervical intraepithelial neoplasia associated with oncogenic human papillomaviruses (HPVs) (9). The organization of the CRPV genome is unique among papillomaviruses by the greater size of the E6 open reading frame (ORF) (21). Long E6 (LE6) and short E6 (SE6) proteins are translated in the same reading frame from transcripts initiated at two distinct promoters (2, 12). The two major transcripts detected in warts and carcinomas encode the SE6 and the E7 proteins (12, 19, 35). Like DNA extracted from warts (29), cloned CRPV DNA is infectious for domestic rabbits (20, 36). This has provided a model system to demonstrate that the E5 and L2 ORFs are dispensable for papilloma induction (7, 34).

Both wart evolution and the level of CRPV replication in warts have been shown or were suspected to depend on the genetic constitution of the host (3, 23, 30, 54) and on the genetic variability of the virus (18, 44, 46, 48, 52). Wart regression occurs in less than 10% of cottontail rabbits, and its frequency varies between 10 and 70% in domestic rabbits (9, 54). Progression into carcinomas is observed in approximately 25% of cottontail rabbits and in up to 75% of domestic rabbits with persistent warts (46, 54). Both regression and malignant conversion of domestic rabbit warts have been reported to be linked to class II DRA and DQA genes of the major histocompatibility complex (MHC), pointing to immunogenetic control of wart evolution (9, 23, 24). Warts of naturally infected cottontail rabbits usually contain large amounts of virions, in spite of a great variation in virus content observed among rabbits (3). In contrast, little, if any, infectious virus is recovered from domestic rabbit warts (3, 18, 51, 52), and vegetative viral DNA replication, late transcripts encoding L1 and L2 capsid proteins, and capsid antigens are seldom detected (35, 37, 41, 62).

The first evidence for a biological variation of CRPV has been the isolation of strains recoverable from domestic rabbit warts (18, 50, 52). The existence of cancer-producing variants was proposed to account for tumor progression (44) but could not be substantiated (43, 45, 53). Our recent observation that rabbits homozygous for a specific MHC class II DRA-DQA haplotype displayed an unusual mode of wart evolution (partial regression), characterized by the persistence of a few warts only, led us to suspect a genetic heterogeneity of CRPV (9, 48; J. Salmon, N. Chanteloup, F. Viard, P. Coudert, O. Croissant, G. Orth, and F. Breitburd, unpublished data). Available sequence data had disclosed only a low (about 0.5%) intratypic variability of CRPV (6, 20, 26, 57). Surprisingly, by cloning and partially sequencing the CRPV genomes present in our isolate obtained from pooled Kansas cottontail rabbit warts, we disclosed the existence of two CRPV strains, a variant of the CRPV prototype (CRPVa) and a CRPV subtype (CRPVb), characterized by strikingly divergent E6 and E7 oncoproteins (48).

The aim of this study has been first to fully characterize the genetic divergence between the CRPVa and CRPVb strains by establishing the complete nucleotide sequences of the two cloned genomes. We next investigated whether the CRPV variability influenced wart evolution and whether this involved the most variable genomic region containing the long regulatory region (LRR) and the E6 and E7 ORFs. The approach was to study the evolution of warts induced by CRPVa, CRPVb, and chimeric genomes in rabbits homozygous for the DRA-DQA haplotype associated with partial regression. Our last goal was to find out whether the two CRPV strains differed in their levels of viral replication in domestic rabbit warts by analyzing wart sections for vegetative viral DNA replication, transcription of the late L1 gene, and synthesis of capsid proteins.

	MATERIALS AND METHODS

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Plasmids, virus, and rabbits. The CRPVa4 and CRPVb recombinant plasmids were described previously (48). The reference CRPVa1 clone (21) was a gift from M. Yaniv, Institut Pasteur, Paris, France. The pCMV--gal plasmid was made available to us by E. Meurs, Institut Pasteur. The CRPVa-L1 recombinant plasmid was constructed by inserting a CRPVa fragment from nucleotides (nt) 5865 to 7347 containing the L1 ORF into a pBluescript vector (Stratagene) at the SmaI restriction site. The CRPV suspension prepared from pooled Kansas cottontail rabbit warts was described previously (48). New Zealand White rabbits homozygous for the MHC DRA.D-DQA.B (PR) haplotype or the DRA.C-DQA.G (P) haplotype, as defined by a restriction fragment length polymorphism (RFLP) (23), were provided by P. Coudert, F. Viard, and N. Chanteloup (Centre de Recherche INRA, Tours-Nouzilly, France).

DNA sequencing. Cloned CRPVa and CRPVb DNAs (21, 48), CRPVb DNA extracted from the viral suspension (48), and CRPV genomes present in total DNA extracted from warts associated with CRPVa or CRPVb were used to establish the sequences of the 4.5-kb region between the 3' end of the E7 ORF and the 5' end of the L1 ORF (E1-L2 region) and the 0.6-kb fragment between the 3' end of the L1 ORF and the 5' end of the E6 ORF spanning the LRR. The strategy was that previously reported for sequencing the E6, E7, and L1 ORFs of the same CRPV DNAs (48). Briefly, nine overlapping fragments encompassing the E1-L2 region and three fragments covering the LRR were amplified by the PCR technique (47) using primers deduced from the published CRPV nucleotide sequence (21). Universal forward and reverse M13 sequences were added to the 5' ends of the forward and reverse primers, respectively, to allow direct sequencing of amplification products. Amplification experiments were performed with the Expand high-fidelity PCR system (Boehringer Mannheim) using an automated thermal cycler (GeneAmp 9600; Perkin-Elmer). PCR products were purified on Centricon-100 columns (Amicon) and sequenced on both strands by the dideoxynucleotide termination method (49), using fluorescent forward and reverse M13 primers and the ABI Prism Dye-Primer Cycle sequencing kit (Applied Biosystems). Labeled DNA fragments were purified in accordance with the manufacturer's instruction, separated in a 4% acrylamide gel under denaturing conditions, and analyzed with an ABI Prism 377 automated sequence analyzer (Applied Biosystems).

Chimeric CRPV genomes. The construction of chimeric CRPV genomes is depicted in Fig. 1. Briefly, a subgenomic 2.7-kb EspI-SacI fragment containing the LRR and the E6 and E7 ORFs was amplified from the cloned CRPVa4 and CRPVb DNAs (48) by using a forward primer located between nt 7027 and 7052 for CRPVa and nt 7020 and 7045 for CRPVb and designed to introduce a ClaI site in the 5' position (5'-CCATCGATGGGCACCCAACAATCATAGATAGATAATTGGC-3'). The nucleotide positions refer to the sequences reported in this paper. The reverse primer was chosen from the pBluescript plasmid sequence downstream of the SacI cloning site. Amplification experiments were carried out with the Expand high-fidelity PCR system (Boehringer Mannheim) using an automated thermal cycler (GeneAmp 2400; Perkin-Elmer). PCR products were inserted into a pBluescript plasmid between the ClaI and SacI restriction sites. The recombinant plasmids were double digested with SpeI and EcoNI endonucleases yielding a 1.9-kb fragment containing the variable LRR/E6/E7 region, extending from nt 7420 to 1439 for CRPVa (va) and from nt 7413 to 1431 for CRPVb (vb), and a 3.7-kb fragment containing the flanking CRPV E1 and L1 ORF sequences and the plasmid. The fragments were separated by agarose gel electrophoresis and purified using a Geneclean II kit (Bio 101, Inc.). Heterologous fragments were ligated to obtain chimeric recombinant plasmids, which were verified by sequencing. To obtain full-length chimeric CRPVa-vb and CRPVb-va genomes, the 2.7-kb EspI-SacI chimeric fragment was excised and ligated to the 5.2-kb SacI-EspI E1-L1 fragment of the CRPV subtype corresponding to the sequences flanking the va or vb insert. After insertion into a pBluescript plasmid at the SacI site, the recombinant plasmids were amplified in Escherichia coli XLI-Blue MRF' (Stratagene) and purified using the Nucleobond AX kit (PC 500; Macherey-Nagel).

View larger version (30K): [in this window] [in a new window]   FIG. 1.   Strategy for constructing chimeric genomes from cloned CRPVa4 and CRPVb DNAs. The subgenomic 2.7-kb fragments of CRPVa and CRPVb encompassing the LRR/E6/E7 region were amplified by PCR using a 5' primer introducing a ClaI restriction site and cloned into a pBluescript SK() [pBSK()] plasmid. The LRR/E6/E7 regions of CRPVa (va) and CRPVb (vb) were double digested by SpeI and EcoNI restriction enzymes (arrowheads) and exchanged to generate chimeric subclones. Chimeric subclones were double digested by EspI and SacI restriction enzymes (arrowheads) and ligated to the 5.2-kb SacI-EspI E1-L1 fragment corresponding to the E1 and L1 ORF sequences flanking the va or vb insert. The generated 7.9-kb CRPVa-vb or CRPVb-va full-length circular recombinant genomes were digested by SacI restriction endonuclease and cloned into a pBluescript SK() plasmid. Open and shaded boxes, CRPVa and CRPVb sequences, respectively; lines, plasmid sequences. The different ORFs and the LRR are represented. The positions of the last and first nucleotides of the CRPVa and CRPVb sequences are given. The localization of the restriction enzyme cleavage sites and their nucleotide positions on the CRPVa sequence (21) are indicated.

Inoculation of CRPV DNAs and virions. Cloned CRPV and chimeric genomes were excised from recombinant plasmids by SacI digestion, and linear viral DNA was purified by sedimentation through a 5 to 21% sucrose gradient in the presence of ethidium bromide (5 µg/ml) (20). After butanol extraction and dialysis (Slide-A-Lyzer cassettes; Pierce), CRPV DNAs were recircularized by self-ligation overnight at 4°C, at a low concentration of DNA (5 µg/ml), in the presence of T4 DNA ligase (0.5 Weiss unit per µg) and 1 mM ATP (Pharmacia Biotech.), that is, in conditions minimizing the formation of high-molecular-weight oligomers. Prior to inoculation of DNAs, the backs and flanks of the rabbits were shaved and treated twice every other day with a mixture of turpentine and acetone (1:1) to enhance the sensitivity of the bioassay (17, 20). Rabbits were anesthetized by intramuscular injection of 25 mg of ketamine (Imalgène 500; Rhône Mérieux, Lyon, France)/kg of body weight and 2 mg of xylazine (Rompun; 2%; Bayer)/kg. In the first experiment, CRPV genomes (10 µg per site) were inoculated by intradermal injection followed by repeated punctures of the sites of injection (20) at four sites on the right flanks (CRPVb) and the left flanks (CRPVa) of 5 P and 5 PR rabbits. In a second experiment, CRPVa, CRPVb, and chimeric genomes were targeted to rabbit skin with the Helios gene gun system (Bio-Rad) in conditions described by Xiao and Brandsma (59). After determining the optimal helium discharge pressure of 350 lb/in² for the intraepidermal expression of a -galactosidase reporter gene driven by a cytomegalovirus promoter (pCMV--gal plasmid), 1.6-µm gold particles were coated with viral DNAs (2 µg of DNA/mg of particles) as indicated by the manufacturer and were delivered at 1 µg per site at three sites per genome to the left flanks (CRPVa, CRPVb-va) and right flanks (CRPVb, CRPVa-vb) of 8 P and 8 PR rabbits. Wart development was photographed weekly for 24 weeks and monthly thereafter, up to 1 year. Warts were also induced by application of a suspension of CRPV virions on a 20-cm² area of shaved skin abraded with sandpaper (23) on the flanks of 4 PR rabbits (about 4 × 10¹⁰ particles per site) and 6 P rabbits (about 4 × 10⁹ particles per site).

Tissue specimens and CRPV DNA typing. Wart specimens were collected from PR rabbits (17 specimens) and P rabbits (31 specimens) infected with CRPV virions between 4 and 10 weeks after wart outgrowth, and biopsies from a single wart induced by CRPVa or by CRPVb DNA were taken from the same P rabbit 10 weeks after outgrowth. Fourteen warts showing signs of malignant conversion from nine rabbits infected with CRPVa or CRPVb-va DNA were biopsied. Biopsy specimens were fixed in buffered 10% formalin and embedded in paraffin. Serial 5- to 7-µm-thick sections were prepared for histological examination after hematoxylin and eosin staining and for in situ hybridization and immunohistochemistry experiments. To identify the CRPV subtype in virion-induced warts, DNA was extracted from three to five tissue sections as described previously (56) and the CRPV subtype was determined by amplification of a fragment of 132 (CRPVa) or 150 bp (CRPVb) using oligonucleotide primers flanking the 18-nt insertion present in the CRPVb E6 ORF (48).

In situ hybridization and immunohistochemistry. For in situ hybridization, sections mounted on pretreated slides (SuperFrost Plus; Mensel-Glaser) were incubated with proteinase K (20 to 50 µg/ml) in 100 mM Tris-HCl-50 mM EDTA, pH 8, for 10 min at 37°C, postfixed in 0.4% paraformaldehyde in diethyl pyrocarbonate (DEPC)-treated phosphate-buffered saline (PBS) for 20 min at 4°C, and washed in PBS containing 0.2% glycine.

Nucleotide sequence accession numbers. The complete CRPVa4 and CRPVb nucleotide sequences have been submitted to the EMBL/GenBank/DDBJ nucleotide sequence database and have been assigned accession No. AJ404003 and AJ243287, respectively.

	RESULTS

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Genetic divergence between CRPVa and CRPVb. We recently reported the cloning of the genomes and the nucleotide sequences of the E6/E8, E7, and L1 ORFs of a variant of the prototype CRPV (21), referred to as CRPVa, and of a highly divergent CRPV strain, referred to as CRPVb (48). The two cloned CRPV genomes were obtained from a viral suspension prepared from pooled Kansas cottontail rabbit warts that contained mostly (about 90%) CRPVb (48). We have now established the complete nucleotide sequences of these cloned CRPVa and CRPVb genomes (For accession numbers, see Materials and Methods). The data were confirmed by direct sequencing of overlapping PCR products obtained from the CRPVb DNA extracted from the viral suspension and from the CRPVa and CRPVb genomes present in the total DNA prepared from warts induced by each virus. We also sequenced the reference CRPV DNA clone (21) in view of the two errors previously found in the published sequence of the L1 ORF (48) and identified six additional errors (Table 1). Compared to the corrected sequence, our CRPVa strain showed five nucleotide substitutions, one in the E2 ORF (G4213A, Asp368Asn) and four shared with the CRPVb strain, three localized in the E1 and L2 ORFs (Fig. 2B) and one previously reported in the 5' untranslated part of the E7 ORF (48). Five sequence variants of CRPVa have been reported so far. Taking into account the chronological order of their description, they are referred to here as CRPVa1 for the sequenced CRPV prototype originating from Kansas cottontail rabbit warts (16, 21), CRPVa2 for the Washington B strain originating from naturally infected Whidbey Island cottontail rabbits (6, 15, 26, 57), CRPVa3 for the isolate harbored by the VX7 transplantable carcinoma (20, 43), CRPVa4 for the isolate further analyzed in this study (48), and CRPVa5 for the CRPV-HE isolate originating from Kansas cottontail rabbit warts (25).

View larger version (37K): [in this window] [in a new window]   FIG. 2.   Sequence variations in the E1-L2 regions of CRPVa and CRPVb. (A) Map of point mutations in the E1-L2 region. The organization of the different ORFs (boxes) of CRPVa1 (21) is given. Vertical dashed lines, positions of initiation codons; bars on the solid lines below the ORFs, synonymous (above) and nonsynonymous (beneath) nucleotide substitutions. The first and last nucleotide positions of each ORF are given. Nucleotide insertion (+2) and deletion (1) (arrows) and the resulting frameshift (fs), generated (star) or suppressed (star in parentheses) stop codons, and mutations downstream of the E5b stop codon (dashed bars) are represented. (B) Nucleotide and amino acid variations in the E1-L2 region. Nucleotide positions refer to the CRPVa1 sequence (21) as corrected in this paper. Arrows, beginning of the ORFs. Nucleotide substitutions in CRPVb are given beneath the corresponding CRPVa1 nucleotides. c, mutations common to CRPVb and CRPVa4. Conserved nucleotides (dashes) are given for the regions flanking the variable E4 stop codons (shaded), the frameshift and the premature E5 stop codon (shaded) generated by the T4342 deletion, and the CA insertion in the E5-L2 overlap. Variable nucleotide positions within the same codon are underlined. Amino acid changes are given beneath the corresponding nucleotides, and stop codons suppressed or generated (shaded) by mutations are indicated (stars).

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Sequence variations in the LRR. The alignment of the region extending from the stop codons of the L1 ORFs to the initiation codons of the LE6 ORFs of CRPVa1 (a) (21) and CRPVb (b) (48) is represented, except for the conserved sequence between nt 7347 and 7426. Dashes, conserved nucleotides. The two 32-bp direct repeats found in the CRPVa LRR are overlined by arrows. The TATA boxes for the LE6 and SE6 transcripts (12) and the putative promoter for the late transcripts (55) (shaded), the cap sites of the late and LE6 mRNAs (12, 55) (bent arrows), and the binding motifs for the viral E1 (dotted box) and E2 (solid boxes) proteins are indicated. Putative binding motifs for Tef-2, NF1, Sp1, AP1 (39), and c-myb (27) cellular transcription factors identified in the CRPVa LRR are overlined by brackets, and an ets-binding motif (38) found in the CRPVb LRR is underlined by a bracket. Start and stop codons are in boldface.

CRPV variation and genetic constitution of host as related to wart evolution. Our aim was to find out whether CRPV genetic variability influenced wart evolution towards regression or persistence and malignant conversion. Our approach has been to use rabbits homozygous for two MHC class II DRA-DQA RFLP haplotypes, characterized by a highly divergent antigen-binding 1 domain of DQ molecules (23, 24). Rabbits homozygous for each haplotype had been found previously to show distinct modes of wart evolution when inoculated with a viral suspension containing the two CRPV subtypes (48; Salmon et al., unpublished data). The DRA.D-DQA.B haplotype was found to be preferentially associated with the early regression of most warts and the persistence of a few warts in the majority of the rabbits (partial regression [PR] haplotype), whereas the DRA.C-DQA.G haplotype was found to be preferentially linked to wart persistence (P haplotype) (9, 48; Salmon et al., unpublished data).

View larger version (28K): [in this window] [in a new window]   FIG. 4.   Course of the disease induced by CRPVa, CRPVb, or chimeric genomes in rabbits homozygous for two distinct MHC class II DRA-DQA RFLP haplotypes. CRPVa () and CRPVb (----) DNAs and CRPVa-vb (----) and CRPVb-va () chimeric genomes obtained by exchanging the LRR/E6/E7 region, as described for Figure 1, were excised from recombinant plasmids, circularized, and inoculated by intradermal injection (A and B) (Table 3, experiment 1) or by using a gene gun (C and D) (Table 3, experiment 2) into five (A) and eight (C) rabbits homozygous for the DRA.D-DQA.B (PR) haplotype and to five (B) and eight (D) rabbits homozygous for the DRA.C-DQA.G (P) haplotype, as described in Materials and Methods. The total numbers of sites with warts observed at different times after inoculation during an 18-week follow-up are represented. Both regression occurring as early as 1 week after outgrowth and delayed outgrowth of some warts account for the fluctuations in the numbers of positive sites.

View larger version (90K): [in this window] [in a new window]   FIG. 5.   Evolution of warts induced by CRPVa and CRPVb DNAs in rabbits homozygous for two different MHC class II DRA-DQA RFLP haplotypes. CRPVa (a) and CRPVb (b) DNAs were excised from recombinant plasmids, recircularized, and inoculated by intradermal injection (10 µg/site) as described in Materials and Methods into four sites not evenly spaced on the left (CRPVa) and right (CRPVb) flanks of rabbits homozygous for the DRA.D-DQA.B (PR) or DRA.C-DQA.G (P) haplotype. Warts (one to three per site) developed at 5 or 6 weeks after infection (p.i.) at three sites (P rabbits) or four sites (PR rabbits). CRPVb warts showed early regression in PR rabbits and persisted in P rabbits, whereas CRPVa warts persisted in both P and PR rabbits. Bars, 15 mm.

Variability of the LRR/E6/E7 region and wart evolution. The most variable region of the CRPV genome includes the LRR and the E6 and E7 ORFs (on the whole, 7% variable nucleotide positions). This variability may affect both the level of expression and the biologic and antigenic properties of the viral E6 and E7 oncoproteins, which are likely targets involved in regression (9). Therefore, we constructed chimeric CRPV genomes by exchanging the SpeI-EcoNI fragment encompassing this region (Fig. 1). The exchanged fragment did not include the invariant first 75 nt of the LRR and contained the first 84 nt of the E1 ORF. Depending on the origin of the variable (v) region, the chimeric genomes were referred to as CRPVa-vb or CRPVb-va. Recircularized chimeric and parental CRPV genomes were each delivered at three sites onto the skin of eight PR and eight P rabbits using a gene gun (57). Warts developed in most rabbits between 3 and 7 weeks (Fig. 4C and D; Table 3). Positive sites ranged from 50 to 79.2% for chimeric DNAs and from 45.8 to 75% for parental genomes. In PR rabbits, all warts induced by CRPVa-vb and over 90% of those induced by CRPVb showed early regression, whereas most positive sites inoculated with CRPVb-va and CRPVa persisted for 6 months after transfection (Table 3; Fig. 4C). In P rabbits, the rates of early regression of warts induced by the four genomes (from 38.9 to 68.4% of the positive sites) were higher than those observed for CRPVa and CRPVb in the first experiment (Table 3; Fig. 4B and D). Differences among the four genomes were not found statistically significant. Six months after infection, all CRPVb-induced warts had regressed and warts persisted at less than 25% of the CRPVa-, CRPVa-vb-, or CRPVb-va-positive sites (Table 3). The influence of the variability of the LRR/E6/E7 region on wart evolution as related to the DRA-DQA haplotype is illustrated in Fig. 6. Cancers arose between 19 and 42 weeks after wart outgrowth at about 40% of the sites positive for CRPVa (six cancers) or CRPVb-va (five cancers) in a total of six PR rabbits (Table 3; Fig. 6) and from a persistent CRPVb-va-induced wart in one P rabbit (Table 3). Taken together, the data indicate that the LRR/E6/E7 region plays a major part in wart evolution and that both sequence variations in this region and the genetic constitution of the host influence the outcome of the disease.

View larger version (74K): [in this window] [in a new window]   FIG. 6.   Evolution of warts induced by chimeric and parental genomes in rabbits homozygous for the PR or the P DRA-DQA RFLP haplotype. CRPVa (a), CRPVb (b), and chimeric genomes obtained by exchanging the LRR/E6/E7 region of CRPVa (va) and CRPVb (vb) were excised from recombinant plasmids, recircularized, and inoculated (1 µg/site) with a gene gun apparatus as described in Materials and Methods into three sites on the left (CRPVa, CRPVb-va) and right (CRPVb, CRPVa-vb) flanks of rabbits. CRPVb- and CRPVa-vb-induced warts regressed in PR rabbits by 11 weeks after inoculation (p.i.). Carcinomas that arose from CRPVa- and CRPVb-va-induced warts are also illustrated. Bars, 5 (data at 6 and 11 weeks p.i.) and 15 mm (data at 36 weeks p.i.).

CRPV duality and level of expression of late viral functions. CRPV vegetative DNA replication, late transcripts, and capsid antigens are seldom detected in domestic rabbit warts (37, 41, 62). CRPV strains recoverable from domestic rabbit warts have been reported, however (18, 50, 52), suggesting a genetic heterogeneity of the virus. This prompted us to compare the expression of the late viral functions in warts induced by the two CRPV subtypes by using in situ methods. Single CRPVa- and CRPVb-induced wart biopsy specimens were taken 10 weeks after wart outgrowth from the P rabbit illustrated in Fig. 5 and analyzed for viral DNA replication, transcription of the L1 ORF, and presence of capsid antigens. As illustrated for the detection of viral DNA in CRPVb- and CRPVa-induced warts (Fig. 7A and B), an unusually high level of expression of late viral functions was observed for the CRPVb specimen (Fig. 7A, C, and D). Compared to those in the CRPVa-induced specimen, the numbers of positive cells per square millimeter in the CRPVb-induced wart tissue sections analyzed were six- to eightfold greater for viral DNA (60.1 versus 7.9 cells/mm²), L1 transcripts (12.5 versus 2.2 cells/mm²), and capsid antigens (25.8 versus 3.9 cells/mm²).

View larger version (111K): [in this window] [in a new window]   FIG. 7.   In situ detection of CRPV DNA, L1 transcripts, and capsid antigens in warts induced in the same rabbit by cloned CRPVa or CRPVb DNAs. Two warts were collected 10 weeks after wart outgrowth, fixed in formalin, and embedded in paraffin, and 5-µm-thick sections were prepared for in situ hybridization and immunochemistry experiments. Representative positive fields are illustrated. (A and B) Detection of CRPV DNA in a section of the warts induced by CRPVb (A) and CRPVa (B) using a digoxigenin-labeled CRPV DNA probe. CRPV genomes were detected in the nuclei of upper terminally differentiating granular cells (gr), where vegetative viral DNA replication occurs (41), and in keratinized superficial cells (k), where viral DNA is encapsidated (11). b, basal cell layer; dp, dermal papilla. (C) Detection of L1 transcripts in an adjacent section of a CRPVb-induced wart using a 35S-labeled antisense CRPV L1 RNA probe (dark-field illumination). Late transcripts were observed in the upper granular cells but not in the nonliving keratinized cells. (D) Immunochemical detection of CRPV capsid antigens in an adjacent section of a CRPVb-induced wart using antibodies raised against disrupted CRPV virus-like particles (8) and an alkaline phosphatase-conjugated secondary antibody. Viral antigens were detected in the nuclei of some of the uppermost granular cells and in keratinized cells, as expected (37, 41). Bars, 50 µm.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The complete nucleotide sequence reported here for the genomes of the CRPVa4 and CRPVb isolates disclosed both an intratype variability unusual among papillomaviruses and the great genetic stability of the CRPVa subtype, in agreement with the conclusions drawn from our previous partial sequence data (48). Our study also provided evidence for variations in the CRPV genome affecting the interaction of the virus with the host.

The CRPVa4 and CRPVb strains were found to differ in 3.1% of the nucleotide positions, whereas the CRPVa4 and the prototypical CRPVa1 strains differed by 5 (0.06%) nt only. Compared to those for CRPVa4, the sequence data available for the Washington B strain or CRPVa2 (4,136 nt in the E6-E2 region) (6, 26, 57) showed about 0.3% variable nucleotides, which further substantiates the genetic stability of the CRPVa subtype. The LRR/E6/E7 region was found to be the most divergent (Table 2). In the CRPVb LRR, a nucleotide substitution in the putative promoter sequence for the late transcripts (55), an additional binding site for the viral E2 protein, and the deletion of a 32-bp sequence containing the cap site of CRPVa LE6 transcripts (12, 55) could affect the transcription of the viral genome. The divergence in the coding sequences involved point mutations only (E1, E2, E8, L2, and L1 ORFs) or point mutations and nucleotide insertions or deletions and mutations modifying stop codons (E6, E7, E4, and E5 ORFs). This resulted in amino acid changes ranging from 0.8 (L1) to 16.2% (LE6) and in variations in the sizes of the E6, E7, E4, and E5 proteins (Table 2). It is worth stressing that the putative E5 proteins encoded by CRPVa1 (21) or CRPVa4, CRPVa2 (7), CRPVa5 (25), and CRPVb vary greatly in amino acid sequences and in size (from 25 to 101 amino acids). CRPVa2 E5 mutant DNAs encoding only the 7 or 61 amino-terminal residues were found to induce warts in domestic rabbits, although at frequencies lower than that for wild-type DNA (7, 32). In our experiments, we found no significant difference between the infectivities of CRPVa4 and CRPVb genomes, which encode 101- and 25-amino-acid E5 proteins, respectively. This supports the conclusion drawn from mutant studies (7, 32) that the E5 protein is dispensable for papilloma production.

The probability of wart regression and the risk for developing a cancer were found to depend on the genetic constitution of the host and on the CRPV strain, specifically, on the sequence variation of the LRR/E6/E7 region. Of 25 rabbits inoculated with both CRPVa and CRPVb genomes and, for 16 of them, also with chimeric genomes containing the LRR/E6/E7 region of CRPVb (CRPVa-vb) or CRPVa (CRPVb-va), warts developed in all inoculated with CRPVa genomes and in most inoculated with CRPVb (80%) and chimeric genomes (87.5%). In rabbits homozygous for the MHC class II PR haplotype, warts induced by CRPVb or CRPVa-vb showed an early regression, whereas most warts induced by CRPVa or CRPVb-va persisted with similarly high rates. In contrast, in rabbits homozygous for the P haplotype, the rates of early wart regression were similar for CRPVa and CRPVb (about 35% of positive sites) and for CRPVa-vb and CRPVb-va (68.4 and 58.8% of positive sites, respectively). Due to a higher rate of late regression of CRPVb-induced warts, about 2.5-fold more CRPVa sites remained positive 6 months after inoculation. It is worth stressing that regression was higher in P rabbits for gene gun DNA delivery than for intradermal inoculation. This could be due to a direct transfection of the epidermal antigen-presenting Langerhans cells, which may have resulted in a greater stimulation of T-cell responses (42).

Spontaneous regression of CRPV-induced warts precludes tumor progression and thus represents a crucial event in host control of the oncogenic potential of the virus (9). Regression is the consequence of a specific cell-mediated immune response (15, 31), and our data support the hypothesis that antigenic peptides derived from LE6, SE6, and/or E7 proteins are major targets. The different regression rates of warts induced by the four genomes, especially in PR rabbits, are likely to be due to distinct immunogenic properties of the viral oncoproteins, in view of their great amino acid variations. Different levels of expression of the LE6 and SE6 proteins could also contribute to the phenomenon. The DQA alleles defining the PR and P haplotypes differ at 22.2% of the amino acids in their 1 peptide-binding domains (24). This suggests that an allelic restriction of the binding of CRPVa and CRPVb E6- and/or E7-derived epitopes by MHC molecules and of their presentation to specific T lymphocytes plays a major role in determining wart regression.

Cancer developed in a total of nine (36%) PR or P rabbits, in eight (32%) from warts induced by CRPVa and in six (42.9%) from warts induced by CRPVb-va. It is worth stressing that cancers did not arise from persistent CRPVb- or CRPVa-vb-induced warts in P rabbits. Because only two P rabbits developed a CRPVa- or a CRPVb-va-associated cancer, further study is required before concluding that variations in the E6 and E7 oncoproteins affect their transforming properties. It is worth mentioning that a CRPVb-induced wart biopsy specimen taken 4 months after outgrowth from a P rabbit showed histologic features of an invasive carcinoma 8.5 months later, after two passages in athymic nude mice. This indicates that CRPVb-induced warts have a potential to progress to malignancy (F. Breitburd, unpublished data).

In addition to its influence on wart evolution, CRPV intratype variation was found to affect the host restriction of the expression of late viral functions in domestic rabbits. Domestic rabbit warts usually yield little, if any, infectious virus (3, 18, 51, 52). Vegetative viral DNA replication and capsid proteins were detected in a significantly greater proportion of warts induced by CRPVb, and the mean number of positive cells (expressed per square millimeter of wart section) was found greater by 1 order of magnitude for CRPVb-positive specimens. A similar difference in the level of expression of late viral functions was found also for warts induced in the same rabbit by CRPVa and CRPVb DNAs. That virions are produced in CRPVb-induced warts has been shown by the recovery of infectious particles from a CRPVb-induced wart grafted to a nude mouse (F. Breitburd, unpublished results). The somewhat alleviated host restriction of CRPVb replication might result from a higher capacity of the E6 and E7 oncoproteins to overcome the switch-off of the host DNA replication machinery that occurs in keratinocytes committed to terminally differentiate (28). It may also involve a higher activity or level of expression of the E1 and E2 proteins that are required for viral DNA replication (28). It is worth stressing that the CT transition affecting the putative promoter for the late transcripts in CRPVb was previously identified in CRPVa DNA in the transplantable VX7 carcinoma, a tumor derived from a wart induced by a CRPV strain recoverable in domestic rabbits (20, 43). CRPV strains obtained from cottontail rabbits have been shown to differ in their abilities to be recovered from domestic rabbit warts (3, 18). Our data strongly suggest that this old observation, unexplained as yet, is a consequence of an intratype variability of CRPV.

An intratypic variation has been described for HPV5 associated with the skin carcinomas of epidermodysplasia verruciformis (13) and for HPV16 and HPV18 associated with the carcinomas of the uterine cervix (33). There is some evidence that the variability of HPV16 could correspond to different oncogenic potentials (14, 58, 60). The characterization of two CRPV subtypes showing a striking divergence in the regulatory region of their genomes and in their E6 and E7 oncogenes has allowed us to demonstrate unequivocally, for the first time, that intratype variation influences the evolution of potential cancer precursor lesions. It has allowed us also to show that intratype variability is able to overcome to some extent the host restriction of viral replication. By using various chimeric CRPV genomes, our current experiments are aimed at understanding the molecular basis of the distinct biologic properties of CRPV subtypes.