The glucocorticoid response element II is functionally homologous in rat and human insulin-like growth factor-binding protein-1 promoters.

In vivo, insulin-like growth factor-binding protein-1 (IGFBP-1) modulates the IGFs' bioavailability and may contribute to their delivery to peripheral tissues. In rat and human hepatocytes, glucocorticoids stimulate IGFBP-1 gene transcription through homologous glucocorticoid response units (GRU). Transfection experiments have shown that one of these, GRU2 (nucleotide (nt) -121 to -85 and nt -111 to -74 in human and rat promoters, respectively), was on its own able to mediate the glucocorticoid response in rat but not in human species (Suwanichkul, A., Allander, S., Morris, S. L. & Powell, D. R. (1994) J. Biol. Chem. 269, 30835-30841, Goswami, R., Lacson, R., Yang, E., Sam, R. & Unterman, T. (1994) Endocrinology 134, 736-743, and Suh, D. S., Ooi, G. T. & Rechler, M. M. (1994) Mol. Endocrinol. 8, 794-805). A close comparison of GRU2 sequences has pointed out a C to A transition in the underlying GREII, which creates a GATC tetranucleotide in rat species. This tetranucleotide is submitted to adenosyl methylation (dam methylation) in most Escherichia coli bacterial strains, but not in eucaryotic cells. We showed (i) that on its own, the unmethylated rat GRU2 (propagated in dam E. coli strains) was inactive, as is the case for its human counterpart (nonsignificant glucocorticoid inductions, 1.48 +/- 0.23 and 1.7 +/- 0.35-fold in Chinese hamster ovary cells, respectively) and (ii) that its adenosyl methylation in standard dam+ bacterial strains yielded a functional GRU (6.5 +/- 1. 1 and 13.1 +/- 3.9-fold glucocorticoid inductions in Chinese hamster ovary and HepG2 cells, respectively). Transient transfection in HepG2 hepatoma cells clearly showed that the interaction of liver-enriched trans-acting factor(s) with the 5'-overlapping insulin response element does not enable the unmethylated rat GRU2 or the human GRU2 to become responsive to glucocorticoids (nonsignificant 2.21 +/- 0.48 and 1.20 +/- 0.06-fold induction, respectively). Furthermore, we have correlated these functional data with in vitro DNA-protein interaction studies: the dam methylated rat GREII displayed a 2.8-fold higher affinity for the glucocorticoid receptor than its unmethylated counterpart.

Delimitation of two regions in the 90-kDa heat shock protein (Hsp90) able to interact with the glucocorticosteroid receptor (GR).

The role of the 90-kDa heat shock protein (Hsp90) as a chaperone and its regulatory functions for cellular proteins such as the glucocorticosteroid receptor (GR) depends on the direct interaction of the Hsp90 with the corresponding protein as part of a multiprotein complex. The search for the amino acid sequence(s) in Hsp90 involved in interaction with the human GR has been carried out by mutational deletion analysis in whole cells, studying the effects of interaction on the nucleocytoplasmic distributions of transiently expressed Hsp90 and GR derivatives in COS-7 cells. Using a recently developed confocal microscopic immunofluorescence method that allows quantification of the nucleocytoplasmic ratios of the proteins in individual cells and statistical comparison of cell populations, two subregions of the Hsp90 molecule have been defined that allow interaction with GR (residues 206-291 and 446-581). The latter region may play a fundamental role in the interaction, while the former may merely stabilize the binding to GR of the intact Hsp90 molecule. Moreover, the dissection of the Hsp90 molecule allowed us to define two regions displaying nuclear localization activity (residues 1-206 and 381-581), followed by two regions having a predominantly cytoplasmic localization activity (residues 287-381 and 581-728) and counteracting the nuclear localization activities.

Quantification of the nucleocytoplasmic distribution of wild type and modified proteins using confocal microscopy: interaction between 90-kDa heat shock protein (Hsp90 alpha) and glucocorticosteroid receptor (GR).

The investigation of molecular interactions in whole cells by immunofluorescence was developed recently, based on the targeting of the protein partners to different cellular compartments and analysis of the modifications in their subcellular distribution resulting from their interaction. This paper describes the adaptation of the confocal microscopy to the quantification of the partitioning of transiently coexpressed proteins between nucleus and cytoplasm. We defined a nucleocytoplasmic ratio R, corresponding to the difference between nuclear and cytoplasmic fluorescence intensities divided by their sum (N - C/N + C), which does not refer to absolute fluorescence intensities. Interaction was detected by statistically comparing the distribution of R value frequencies in cell populations expressing one or both proteins. The convenience of this whole cell method was demonstrated by detecting and analyzing interaction between the human glucocorticosteroid receptor (GR) and the chick 90-kDa heat shock protein (Hsp90), using various combinations of wild-type and nuclear- or cytoplasmic-targeted GR and Hsp90. In addition, three Hsp90 deletion/ truncation mutants were tested: the C-terminal truncated mutant NC4 interacted slightly, indicating the contribution of this part of the molecule to the interaction with GR, while the shorter truncated mutant NC6 did not interact with GR, likely resulting from an incorrect folding of the molecule. No role for the first charged region (delta A') was found as shown by the strong interaction detected for the delta A'Hsp90. This method can fruitfully be applied to the delimitation of the amino-acid sequences involved in protein-protein interaction by mutational analysis, especially to seek confirmation of other methods or when other approaches have failed.

Stimulation of transcription in vitro from a liver-specific promoter by human glucocorticoid receptor (hGRalpha).

The rat tyrosine aminotransferase (TAT) gene is a liver-specific and glucocorticoid-inducible gene. Previous studies have shown that the TAT promoter (TAT0.35; nt -350 to +1) is able to sustain liver-specific gene expression both in transient transfection and in a transcription assay in vitro [Schweizer-Groyer, Groyer, Cadepond, Grange, Baulieu and Pictet (1994) Nucleic Acids Res. 22, 1583-1592]. Here we report that the basal transcriptional activity generated from TAT0.35 in the presence of crude liver nuclear extracts is enhanced by added human glucocorticoid receptor (hGRalpha), provided that TAT0.35 sequences were flanked (5') with a glucocorticoid responsive unit (GREII of the TAT gene, including its 5'-CCAAT flanking sequence). Two sources of hGRalpha were used: nuclear extracts prepared from Sf9 insect (Sf9-NEs) cells over-expressing hGRalpha, and hGRalpha from pRShGRalpha-transfected COS-7 cells, enriched by high-performance ion-exchange chromatography. The enhancement of transcription in vitro (1.5-4.5-fold) was dependent on the amount of added hGRalpha and independent of the nature (agonist or antagonist) of the ligand. Moreover, the hGRalpha-mediated stimulation of transcription was (i) dependent on GRE/progesterone response element (PRE) (it was inhibited by a 25-fold excess of GRE/PRE but not by a 100-fold excess of oestrogen response element) and (ii) receptor-dependent (Sf9-NEs prepared from uninfected Sf9 cells or from Sf9 cells infected with wild-type baculoviral DNA did not enhance transcription). Taken together, these experiments support the conclusions that in vitro the glucocorticoid receptor is able to enhance transcription from genomic, liver-specific, promoter sequences (those of the TAT gene), and that this enhancement of transcription from the liver-specific TAT0.35 promoter is dependent both on the glucocorticoid receptor and on the latter's interaction with its cognate response elements.

Glucocorticosteroid receptor dimerization investigated by analysis of receptor binding to glucocorticosteroid responsive elements using a monomer-dimer equilibrium model.

The aim of this study was to analyze the role of regions of the glucocorticosteroid receptor (GR) outside the DNA binding domain (DBD) in GR binding and homodimerization efficiencies by using a model according to which GR monomers and dimers are in equilibrium and able to bind to each half-palindromic motif of a GRE. We studied wild-type human GR (hGR), an N-terminal domain deleted mutant (lacking amino acids 1-417), a C-terminal deleted mutant (lacking amino acids 550-777, the main part of the ligand binding domain), and two rat GR derivatives limited to the DNA binding domain and proximal sequences. Specific GR monomer and dimer complexes with 33P-labeled palindromic or half-palindromic GREs were identified by gel-shift and methylation interference experiments. The different complexes were quantified, and the multiple equilibrium constants for their formation were determined. The affinity of the monomer for the GRE was not affected by the deletions of regions outside the DBD. However, the affinity of the dimer for the GRE was clearly increased by the presence of the N-terminal domain and, to a lesser extent, by that of the main part of the C-terminal domain. By using this model, we also obtained a GR dimerization constant in the absence of specific binding to GRE. Dimerization of the DBD was not increased by the presence of only one of the GR terminal domains, but an increase in dimerization efficiency was observed when both domains were present, suggesting a structural synergy between the N- and C-terminal domains in GR homodimerization.

Multiple liver-enriched trans-acting factors interact with the glucocorticoid- (GRU) and cAMP-(CRU) responsive units within the h-IGFBP-1 promoter.

In response to hormonal control, serum concentrations of insulin-like growth factor-binding protein-1 (IGFBP-1) may vary as much as 10-fold, owing to strict control of its gene's expression in hepatocytes. IGFBP-1 gene transcription is increased by glucocorticoids and cAMP and inhibited by insulin. The effect of insulin is dominant since it suppresses constitutive and both glucocorticoid- and cAMP-stimulated transcription. Close examination of the human (h)IGFBP-1 promoter sequences showed that the glucocorticoid (GRE, nt -88 to -102) and cAMP (CRE, nt -259 to -264) response elements are 5'-flanked by an A/T-rich imperfect palindrome (nt -102 to -117 and -265 to -285, respectively). These A/T-rich motifs are putative cis-elements for liver-enriched trans-acting factors. Competition experiments in electrophoretic mobility shift assay were carried out using rat liver nuclear extracts and a set of synthetic oligonucleotides designed from hIGFBP-1 Glucorticoid and cAMP Response Units (GRU and CRU), the rat transthyretin HNF3 cis-element and the "D-site' of the mouse albumin promoter. The nucleotide motifs located between nt -108 and -121 of the GRU, interacted with the HNF3 family of trans-acting factors (alpha, beta, gamma), whereas those encompassing nt -81 to -104 bound DBP and/or nuclear proteins sharing similar sequence specificity (i.e. from the C/EBP family of bZIP proteins). We have also shown that the hIGFBP-1-GRE binds glucocorticoid receptor homodimers. In the case of the CRU, the cis-elements located between nt -249 and -285 bound DBP and/or nuclear proteins sharing similar sequence specificities. In addition, the nucleotide stretch lying between nt -256 and -275 was able to interact with the HNF3 family of trans-acting factors. Our results support the view that the dominant inhibitory effect of insulin over glucocorticoid- and cAMP- enhanced transcription may be mediated by different target sequences located 5'- of the GRE and CRE. In both cases, the mechanism would involve the interplay of common trans-acting factor(s), some of which are liver-enriched [HNF3, DBP or C/EBP related bZIP proteins] with their cognate target sequence.

Selective deletions in the 90 kDa heat shock protein (hsp90) impede hetero-oligomeric complex formation with the glucocorticosteroid receptor (GR) or hormone binding by GR.

We have developed an in vivo system using coexpression of human glucocorticosteroid receptor (hGR) and chick hsp90 alpha (chsp90) in recombinant virus-infected Sf9 cells to study the formation of hetero-oligomeric complexes. We detected, in the cytosol, hGR complexes containing chsp90 as shown by the displacement of the [3H]triamcinolone acetonide bound hGR "8S" peak on glycerol/sucrose gradients by specific antibodies directed against chsp90 (BF4 and D7 alpha). We took advantage of this system and of the immunoadsorption of hGR containing complexes with anti-hGR antibody to analyze the effect of deletions introduced into the hsp90 molecule on the formation of complexes with the hGR. Deletion of the hydrophilic region "A", between amino-acids 221 and 290, abolished the formation of hGR/chsp90 complexes. Deletion of the hydrophilic region "B" (between amino-acids 530 and 581) or deletion of a leucine repeat region "Z" in the middle of the molecule (amino-acids 392 to 419) still allowed formation of hetero-oligomeric complexes detected by immunoadsorption but the hGR complexes formed with mutated chsp90s were devoid of steroid binding properties. These results are consistent with (1) the possible involvement of the "A" region in the interaction of hsp90 with steroid receptors and (2) a role of B and Z regions in the hsp90 structure for maintaining the steroid binding property of the hGR.

In vivo functional protein-protein interaction: nuclear targeted hsp90 shifts cytoplasmic steroid receptor mutants into the nucleus.

In target tissue extracts, heat shock protein hsp90 has been found associated to all unliganded steroid receptors. Modulation of important functions of these receptors, including prevention of DNA binding and optimization of transcriptional activity, has been attributed to hsp90. However no unequivocal in vivo demonstration of interaction between receptors and hsp90 has been presented. We targeted chicken hsp90, a mainly cytoplasmic protein, with the nucleoplasmin nuclear localization signal (90NLS). After transfection into COS-7 cells, 90NLS was found in the nucleus with specific immunofluorescence and confocal microscopy techniques. A human glucocorticosteroid receptor mutant devoid of NLS sequence was also expressed in COS-7 cells and found exclusively cytoplasmic. Coexpression of 90NLS and of the cytoplasmic human glucocorticosteroid receptor mutant led to complete nuclear localization of the receptor, indicating its piggyback transport by 90NLS and thus physical and functional interaction between the two proteins in the absence of hormone. The same nuclear localization was obtained after cotransfection of 90NLS and a cytoplasmic rabbit progesterone receptor mutant. Finally, coexpression of wild-type rabbit progesterone receptor (nuclear) and wildtype hsp90 (cytoplasmic) into COS-7 cells provoked partial relocalization of hsp90 into the nucleus. These experiments lay the groundwork on which to study hsp90 as a chaperone, regulating activities of steroid receptors and possibly participating in their nuclear-cytoplasmic shuttling.

Interaction of glucocorticosteroid receptor and wild-type or mutated 90-kDa heat shock protein coexpressed in baculovirus-infected Sf9 cells.

Coexpression of the human glucocorticosteroid receptor (hGR) and chicken 90-kDa heat shock protein alpha (chsp90) in recombinant baculovirus-infected Sf9 cells is a system that provides a large quantity of wild-type chsp90-hGR complexes able to bind hormone ([3H]triamcinolone acetonide; TA), sedimenting at 8 S, and displaceable to 11 S by BF4 and D7 alpha anti-chsp90 monoclonal antibodies. Thus, we were able to examine the effects of selective chsp90 mutations on hetero-oligomeric complex formation. Two deletions involved hydrophilic regions, A between amino acids 221 and 290 and B between amino acids 530 and 581, and the third, Z, removed a central leucine heptad repeat region (amino acids 392-419). When these chsp90 mutants were expressed, the lack of displacement of [3H]TA receptor complexes on sucrose gradient by specific chsp90 antibodies was consistent with the formation of [3H]TA receptor complexes containing only endogenous insect hsp90. By using an immunoadsorption method and sedimentation analysis, we found that the deletion of region A precluded the interaction of chsp90 with the hGR, while B and Z deletions led to formation of abnormal complexes with the hGR, which displayed large forms (> 10 S), were unable to bind hormone, and apparently formed only small amounts of tightly bound nuclei hGR upon in vivo hormone treatment. As a whole, the data are consistent with distinct roles of hsp90 regions in hGR function.

Hormonal regulation of the nuclear localization signals of the human glucocorticosteroid receptor.

Nuclear localization of the rat glucocorticosteroid receptor (rGR) transiently expressed in COS-7 cells appears to be mediated by two nuclear localization signals, NL1 and NL2, in a hormone-dependent mechanism. We investigated the intracellular distribution of the human GR (hGR) expressed in COS-7 cells, by a different immunohistochemical technique involving immunostaining of cell pellet sections, thus avoiding the use of cell permeabilizing agents and allowing rigorous comparison between successive experiments. With a large set of hGR mutants, we could define determinants of the hGR nuclear localization and compare them with those previously reported for rGR. Our study demonstrated two hormone-dependent nuclear localization signals. NL1 activity, overlapping the DNA-binding domain (DBD)-hinge boundary, was repressed by the unliganded ligand-binding domain (LBD), even if the repressed NL1 retained a residual potency to target hGR in the nucleus. Structure/function analysis suggested a bipartite structure of NL1, analogous to that of other nuclear targeting signals (the carboxy-terminal part of DBD between amino acids 478 and 487 and the beginning of the hinge region which includes a basic amino acid stretch between 491 and 498). Upon hormone binding, NL2, located in the LBD, was activated, but was unable by itself to sustain full nuclear localization, which required the derepressed NL1 activity. Only two sequences in the LBD, localized between amino acids 600 and 626 and from amino acid 696 up to the carboxyl-terminal amino acid 777, respectively, were found to inhibit NL1 activity. As previously reported, efficient nuclear retention, mandatory for gene expression, did not required DNA-binding activity. The controversial intracellular localization of the unliganded form of hGR and the role of hsp90 in cytoplasmic localization are further discussed.

Mutations in the "zinc fingers" or in the N-terminal region of the DNA binding domain of the human glucocorticosteroid receptor facilitate its salt-induced transformation, but do not modify hormone binding.

While the effects of the ligand (hormone) binding domain (LBD) on other receptor domain functions are known, the effects of other domains on LBD functions have not been studied. In this work, we examined the importance of the structural integrity of other domains of the human glucocorticosteroid receptor (hGR) on LBD activity (stability of 8S complexes, binding of hormone, and transformation from the 8S to the 4S form). Several mutations introduced outside the LBD affect neither the formation of stable 8S heterooligomeric complexes nor the hGR binding affinity for the agonist triamcinolone acetonide (TA) or the antagonist RU486. However, some of them led to an easier salt-induced transformation of the 8S-hGR into a 4S form. Deletion of the second zinc finger of the DNA binding domain (DBD) facilitated 8S dissociation whether the ligand was TA or RU486. Deletion of the first zinc finger facilitated dissociation only in the presence of RU486, while replacement of PRO 416 (in the N-terminal region of the DBD) by ARG destabilized the 8S form only in the presence of TA. Variations in the salt-sensitivity of the mutated 8S GR complexes as a function of the ligand suggest that the DBD may interact functionally (if not physically) with the LBD. This interaction (possibly mediated by hsp90) could be influenced by minor structural differences between agonist and antagonist-GR complexes.

Heat shock protein 90 as a critical factor in maintaining glucocorticosteroid receptor in a nonfunctional state.

Previous work demonstrated that the ligand binding domain (LBD) was required to determine the formation of the cytosolic, untransformed, inactive, 8 S, heterooligomeric form of the human glucocorticosteroid receptor (hGR) which includes the 90-kDa heat shock protein (hsp90) (Pratt, W.B., Jolly, D.J., Pratt, D.V., Hollenberg, S.M., Giguère, V., Cadepond, F.M., Schweizer-Groyer, G., Catelli, M.G., Evans, R.M., and Baulieu, E.E. (1988) J. Biol. Chem. 263, 267-273). Truncations of hGR deleting all or almost all of the LBD give GR derivatives in the non-hsp90-interacting 4 S form able to stimulate transcription in a hormone-independent manner. To identify the LBD subregion(s) involved in 8 S formation, we analyzed the sedimentation behavior of hGR mutants with various LBD internal deletions and/or truncations transiently expressed in cells that contain hsp90 but very low levels of endogenous GR, and we correlated the results with their transcriptional activity. LBD has been divided into three subregions: two of them, LBD1 (between amino acids 551 and 626) and LBD2 (between amino acids 627 and 696), include amino acid sequences highly conserved in the steroid receptor superfamily, and LBD3 consists of the carboxyl-terminal part of the molecule (amino acids 697-777). Each of these subregions can be deleted without impeding 8 S heterooligomer formation, and the corresponding receptors do not have transcriptional activity in the absence as well as in the presence of hormone. When linked to hGR mutants truncated after amino acids 532 or 550, each subregion does separately promote 8 S heterooligomeric complex formation and repress the intrinsic constitutive transcriptional activity of the truncated receptors. These 8 S complexes contain hsp90. In a control experiment, the linkage of 1,017 amino acids of beta-galactosidase to the carboxyl-terminal of 1-532 hGR gave a hybrid receptor still constitutively transcriptionally active which did not bind hsp90. These results provide evidence that there is a strong correlation between the association with hsp90 and the loss of GR functional properties and that hsp90 may play a critical role in maintaining the receptor in a nonfunctional state.

[Human papillomaviruses in the epithelial cells of the cervix uteri: frequency of types 16 and 18. Preliminary results of a clinical, cytologic and viral study]. / Recherche de papillomavirus humains dans des cellules épithéliales du col utérin: fréquence des types 16 et 18. Résultats préliminaires d'une étude clinique, cytologique et virologique.

Between April 1 and June 30 1984, cervical scrapes were taken from 381 women attending the Gynecology Department of the Anticancer Center René-Huguenin. The scrapes were examined for the presence of human papillomavirus (HPV) DNA, by a molecular hybridization method, at the Pasteur Institute. The four HPV types involved in genital pathology, HPV 6, HPV 11, HPV 16 and HPV 18, were studied. Twenty four specimens (6.3%) were found positive: 19 for HPV 16, 3 for HPV 18, 2 for HPV 6 or HPV 11. Results of molecular hybridization were compared with cytological findings. HPV 6 or HPV 11 were detected in cases of mild dysplasia. HPV 16 or HPV 18 were mainly detected in cases diagnosed as severe dysplasia or carcinoma in situ (9 out of 14, i.e. 64.3%), and invasive carcinoma (3 out of 5 cases). The results were further confirmed when virological data obtained with cervical scrapes were compared with the histological diagnosis on biopsies: 14 out of 15 cases of severe dysplasia or carcinoma in situ (93%) and 3 out of 6 cases of invasive squamous carcinoma had been found positive for HPV 16 or HPV 18. Interestingly, four "normal" women (1.3%) with a negative cytology were found positive for HPV 16 or HPV 18. The data obtained by this sensitive and reliable method are useful to the clinician to identify women presenting a high risk of subsequent cervical intraepithelial neoplasia or invasive carcinoma, and, thus, to adapt the treatment and the follow-up of these patients.

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.

Humoral and cell-mediated immunity to human papillomavirus type 1 (HPV-1) in human warts.

The humoral and cell-mediated immune response to human papillomavirus type 1 (HPV-1) has been studied in 162 patients carrying papillomas of various clinical types: deep plantar wart or myrmecia, common wart, flat wart, and anogenital wart. Circulating antibodies were detected by immunodiffusion and microcomplement fixation, using purified HPV-1 particles as type-specific antigen. A significant association between myrmecia and anti-HPV-1 antibodies was found (39% of the cases). Cell-mediated immunity was evaluated by a study of delayed hypersensitivity (DH). The main capsid components of HPV-1 (HPV-1 CP), consisting mostly of a polypeptide of molecular weight 54,000, were injected intradermally. In addition to the type-specific antigens, HPV-1 CP contain other antigenic determinants shared by various types of human papilloma-viruses and masked in intact viral particles. The DH tests to HPV-1 CP showed no differences between the carriers of different papilloma types, confirming the presence of common antigenic determinants. Moreover, they gave rise to an increase or to new anti-HPV-1 antibody production mostly in myrmecia carriers (78% and 33% of the cases, respectively), and to new DH to HPV-1 CP in all groups of papilloma carriers (33% to 56%, depending on the clinical papilloma type).

Identification of papillomaviruses in butchers' warts.

We have studied the papillomaviruses found in the hand warts of 60 butchers, most of them from 2 distant slaughterhouses. Warts differing in morphology and location were studied separately. The viruses were identified by molecular hybridization, restriction enzyme analysis and immunofluorescence. Four known human papillomaviruses (HPV-1, HPV-2, HPV-3, HPV-4) were detected and one hitherto unknown papillomavirus was identified in 9 butchers. The DNA of the latter virus did not anneal with any of the RNAs complementary to either HPV-1 to HPV-5 or bovine papillomavirus type 1 (BPV-1) DNAs, and showed a Hind II + III restriction enzyme cleavage pattern distinct from those of known HPVs and BPVs. This virus showed distinct antigenic properties, as shown by immunofluorescence, using HPV-1, -2, -3, -5, and BPV-1 antisera. It may represent a new type of human papillomavirus (HPV-7) or a yet unidentified animal papillomavirus. In addition, 6 butchers were found to be infected with a papillomavirus, distinct from the known skin HPVs and from BPV-1, which could not be characterized by restriction enzyme analysis. Eleven butchers were found to be infected by 2 viruses. A characteristic histological pattern was found to be associated with the different papillomaviruses.