Gérard Orth

Plurality of genital human papillomaviruses: characterization of two new types with distinct biological properties

The genomes of two new genital human papillomavirus (HPV) types, tentatively named HPVs 39 and 42, have been cloned from biopsy specimens of penile Bowenoid papules and vulvar papillomas, respectively. Blot hybridization experiments, performed under stringent conditions (Tm -10 degrees), have revealed no cross-hybridization between the DNAs of HPVs 39 and 42, and between these DNAs and those of other genital and cutaneous HPVs. A significant cross-hybridization has been observed between the DNA of HPV42 and that of HPV32, the latter being associated with oral focal epithelial hyperplasia. The fraction of HPV32 and HPV42 hybrid molecules resistant to nuclease S1 treatment after hybridization in liquid phase at saturation has been evaluated to 20%, supporting the view that these HPVs constitute distinct types. In addition to HPV42 DNA, a 6.8-kb BamHI fragment, cross-hybridizing with HPV39 DNA, has been cloned from the vulvar papilloma DNA preparation. The cross-hybridization has been evaluated to 16%, pointing to the existence of an additional HPV39-related type. Electron microscope analysis of heteroduplex molecules formed between HPV32 and HPV42 DNAs showed paired regions over about 60 and 87% of their genome lenghts under stringent (Tm -18 degrees) and nonstringent (Tm -42 degrees) conditions, respectively. The 6.8-kb HPV DNA and HPV39 DNA formed paired regions over about 63 and 95% of the 6.8-kb fragment length at Tm -18 degrees and Tm -26 degrees, respectively. These data point to greater DNA sequence homologies than anticipated from the percentages of nuclease S1 resistance. Heteroduplex mapping has allowed the alignment of the physical maps of HPV39 and 42 DNAs and of the 6.8-kb HPV DNA with the map of the open reading frames of the HPV16 genome. So far, HPV42 has been detected only in benign genital lesions showing usually no cell atypia. HPV39 has been detected in a few cases of intraepithelial neoplasias and invasive carcinomas of the uterine cervix. The viral DNA sequences have been found integrated into the cell genome in all four HPV39-associated cervical cancers of our series. It seems most likely that HPV42 belongs to the low-risk group of genital HPVs, while HPV39 represents a potentially oncogenic genital HPV type.

Evidence for antigenic determinants shared by the structural polypeptides of (Shope) rabbit papillomavirus and human papillomavirus type 1

Abstract In contrast to antivirion antisera raised against (Shope) rabbit papillomavirus (RPV) and human plantar wart papillomavirus (HPV-1), sera of rabbits bearing Vx7 carcinoma, a transplantable tumor deriving from a RPV-induced papilloma, permit the detection of an antigenic relationship between these viruses. Sera containing anti-RPV antibodies were collected between the 116th and 165th transplant from 10 rabbits selected for their large intramuscular tumors of long duration or for their purposely grafted tumors (both intramuscularly and intraperitoneally). When tested by immunofluorescence using plantar wart sections, seven of these sera detected an antigen whose location corresponds to that of HPV-1 capsid antigens. The reactivity of fluorescein-conjugated IgG, obtained from the most cross-reactive serum, was abolished by incubation with alkali-disrupted HPV-1 virions but not with intact HPV-1 particles. In contrast to an anti-HPV-1 virion antiserum, this Vx7 serum reacted with the main HPV-1 structural polypeptide (MW, 54,000) after SDS disruption and iodination of HPV-1 particles, as shown by radioimmunoprecipitation. Furthermore, one of the two anti-disrupted HPV-1 virion antisera and the two-anti-HPV-1 polypeptide antisera studied reacted with RPV antigens, as shown by immunofluorescence using cottontail rabbit wart sections. Moreover, one of the cross-reactive Vx7 sera gave a precipitin line continuous to that given by an anti-HPV-1 virion antiserum when tested by immunodiffusion using HPV-1 virions as antigens. This was further observed for four of the 110 additional Vx7 sera tested. The specificity of this reaction was confirmed by immune electron microscopy experiments. Similarly, an anti-HPV-1 polypeptide antiserum reacted with RPV virions. The results indicate the existence of two kinds of antigenic determinants shared by RPV and HPV-1 structural polypeptides; some are masked in intact particles and others are located on the virion surface but in a form usually unable to elicit the formation of antibodies.

Characterization of a new type of human papillomavirus (HPV) related to HPV5 from a case of actinic keratosis

Human papillomavirus (HPV) DNA sequences, related to the genomes of HPVs associated with epidermodysplasia verruciformis (EV), were detected in DNA samples extracted from biopsied lesions in 2 of 24 cases of actinic keratosis found in the general population. An HPV DNA was molecularly cloned from one of these samples. Blot hybridization experiments, performed under stringent conditions, revealed a significant cross-hybridization only between this HPV DNA and the DNAs of HPV5 and of the HPV5-related types. The extent of homology between them ranged from 7 to 30%, as evaluated by hybridization in liquid phase at saturation followed by nuclease S1 analysis. This showed that the cloned HPV represented a new type, tentatively named HPV36. HPV36 was not found in the other 22 cases of actinic keratosis, but was detected in scrapings of benign lesions of 7 of 18 (39%) EV patients.

Restriction mapping and physical characterization of the cottontail rabbit papillomavirus genome in transplantable VX2 and VX7 domestic rabbit carcinomas

The transplantable VX2 and VX7 carcinomas were analyzed for the presence, the nature, and the physical state of cottontail rabbit papillomavirus (CRPV) DNA sequences. Two CRPV DNA preparations, obtained from pooled cottontail rabbit papillomas, were found highly homogeneous as to their sensitivities to seven restriction endonucleases and were, therefore, considered as wild-type CRPV DNA. A cleavage map of the wild-type genome was constructed by locating 14 restriction sites. The carcinoma cells contained multiple copies of apparently whole viral genomes (42 and 444 genome equivalents per diploid VX2 and VX7 cell DNA contents, respectively), as determined by reassociation kinetic experiments and blot hybridization analyses of restricted VX2 and VX7 cell DNAs. Viral DNA molecules bearing a deletion of about 10% of the genome length, as well as minor sequence rearrangements, were also detected in VX7 carcinoma cells (about 25 copies per diploid cell DNA content). In both VX2 and VX7 cell DNAs, either unrestricted or treated with a no-cut endonuclease for the CRPV genome (PvuI), viral DNA sequences were present exclusively as slow-migrating species with similar electrophoretic mobilities. The conversion of these species into one-genome-unit-length linear DNA molecules, by a one-cut endonuclease for both wild-type and deleted DNA molecules (SmaI), shows that the viral sequences are not integrated in the cell genome. After cleavage of VX7 cell DNA by a no-cut endonuclease for deleted DNA molecules (EcoRI), the deleted molecules migrated with a mobility corresponding to form I and II DNA molecules. This suggests that the viral genomes may be present in carcinoma cells as free catenated DNA molecules.

Molecular and biological characterization of cottontail rabbit papillomavirus variant DNA sequences integrated in the VX7 carcinoma

The transplantable VX7 carcinoma was derived from a tumor induced by a recoverable strain of cottontail rabbit papillomavirus (CRPV) able to replicate in domestic rabbits. Low levels of late viral gene expression have been retained through serial propagation in rabbits. We have cloned and characterized the three major types of CRPV sequences integrated in this tumor, a genome-length 8-kb DNA molecule and two rearranged 9- and 3.8-kb molecules. The VX7 8-kb DNA displays only a few differences in its restriction map, when compared to the wild-type (wt) CRPV DNA. The VX7 9- and 3.8-kb DNAs derive from the VX7 8-kb DNA since they share the same restriction site polymorphism. The VX7 9-kb DNA contains a duplication of the E6 open reading frame. The VX7 3.8-kb DNA results from the deletion of most of the E region and the insertion, between the borders of the deletion, of 174-nucleotide-long segment of the long control region potentially driving the expression of a truncated L2 protein. Both VX7 9- and 3.8-kb species potentially allow the expression of abnormal E6 fusion proteins. Nineteen point mutations were detected in the 3.8-kb DNA, compared to the wt CRPV DNA. None of these molecules were able to induce warts in domestic rabbits, in contrast to wt CRPV DNA. Furthermore, when cloned VX7 DNAs were inoculated together with wt CRPV DNA, none of the VX7 CRPV sequences, as identifiable by their specific restriction enzyme cleavage patterns, could be detected in the resulting warts. This suggests that CRPV sequences integrated in the VX7 carcinoma are no longer able to replicate as episomes, which might be a prerequisite for the production of warts.