Fusarium species complex and mycotoxins in grain maize from maize hybrid trials and from grower's fields.

AIMS: To quantify and to compare the occurrence of Fusarium species in maize kernels and stalk pieces, to analyse mycotoxins in kernels and maize crop residues, to evaluate two approaches to obtain kernel samples and to compare two methods for mycotoxin analyses. METHODS AND RESULTS: The occurrence of Fusarium species in maize kernels and stalk pieces from a three-year maize hybrid trial and 12 kernel samples from grower's fields was assessed. Nine to 16 different Fusarium species were detected in maize kernels and stalks. In kernels, F. graminearum, F. verticillioides and F. proliferatum were the most prevalent species whereas in stalks, they were F. equiseti, F. proliferatum and F. verticillioides. In 2006, 68% of the kernel samples exceeded the recommended limit for pig feed for deoxynivalenol (DON) and 42% for zearalenone (ZON), respectively. Similarly, 75% of the samples from grower's fields exceeded the limits for DON and 50% for ZON. In maize crop residues, toxin concentrations ranged from 2.6 to 15.3 mg kg(-1) for DON and from 0.7 to 7.4 mg kg(-1) for ZON. Both approaches to obtain maize kernel samples were valid, and a strong correlation between mycotoxin analysis using ELISA and LC-MS/MS was found. CONCLUSIONS: The contamination of maize kernels, stalk pieces and remaining crop residues with various mycotoxins could pose a risk not only to animal health but also to the environment. With the hand-picked sample, the entire Fusarium complex can be estimated, whereas combine harvested samples are more representative for the mycotoxin contents in harvested goods. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first multi-year study investigating mycotoxin contamination in maize kernels as well as in crop residues. The results indicate a high need to identify cropping factors influencing the infection of maize by Fusarium species to establish recommendations for growers.

Comparing steroid estrogen, and nonylphenol content across a range of European sewage plants with different treatment and management practices.

The effluent of 17 sewage treatment works (STW) across Norway, Sweden, Finland, The Netherlands, Belgium, Germany, France and Switzerland was studied for the presence of estradiol (E2), estrone (E1), ethinylestradiol (EE2) and nonylphenol (NP). Treatment processes included primary and chemical treatment only, submerged aerated filter, oxidation ditch, activated sludge (AS) and combined trickling filter with activated sludge. The effluent strength ranged between 87 and 846 L/PE (population equivalent), the total hydraulic retention time (HRT) ranged between 4 and 120 h, sludge retention time (SRT) between 3 and 30 d, and water temperature ranged from 12 to 21 degrees C. The highest estrogen values were detected in the effluent of the STW which only used primary treatment (13 ng/L E2 and 35 ng/L E1) and on one occasion in one of the STW using the AS system (6.5 ng/L E2, 50.5 ng/L E1, but on three other occasions the concentrations in this STW were at least a factor of 6 lower). For the 16 STW employing secondary treatment E2 was only detected in the effluent of six works during the study period (average 0.7-5.7 ng/L). E1 was detected in the effluent of 13 of the same STW. The median value for E1 for the 16 STW with secondary treatment was 3.0 ng/L. EE2 was only detected in two STW (1.1, <0.8-2.8 ng/L). NP could be detected in the effluent of all 14 STW where this measurement was attempted, with a median of 0.31 microg/L and values ranging from 0.05 to 1.31 microg/L. A comparison of removal performance for E1 was carried out following prediction of the probable influent concentration. A weak but significant (alpha<5%) correlation between E1 removal and HRT or SRT was observed.

Long-term survival estimates of cast gold inlays and onlays with their analysis of failures.

The aim of the present study was to assess the clinical behaviour of cast gold restorations with respect to define a gold control against current and future ceramic and composite restorations. The study sample included 50 patients with 303 cast gold restorations. All restorations were cemented with a non-adhesive technique. A total of 303 restorations were evaluated clinically and radiographically using modified United States Public Health Service criteria. Restorations recorded as having an A- or a B-rating were defined as successful. Of the 303 restorations, 42 were judged as failures, which resulted in a failure rate of 13.8% for a mean observation time (+/- s.d.) of 18.7 ( +/- 9.5) years. The estimated Kaplan-Meier survival rates (s.e.) were 96.1% (+/- 1.1%) at 10 years, 87.0% (+/- 2.2%) at 20 years and 73.5% (+/- 5.4%) at 30 years. In total, biological reasons were counted 25 times in comparison to 17 technical reasons for those 42 failed cast gold restorations, with 17 secondary caries (40%) as the most common biological reason and with 13 retention losses (31%) as the most common technical reason. The endodontically treated tooth was exclusively identified as a risk factor. The restoration type (inlay versus onlay) did not influence the survival rate.

Recombinant Listeria monocytogenes vaccination eliminates papillomavirus-induced tumors and prevents papilloma formation from viral DNA.

Listeria monocytogenes is a gram-positive, facultative intracellular bacterium that enters the cytoplasm of infected cells and spreads directly into neighboring cells without encountering the extracellular environment. Cytoplasmic L. monocytogenes efficiently presents secreted proteins to the major histocompatibility complex class I pathway which can stimulate protective T-cell-mediated immune responses. We have used a cottontail rabbit papillomavirus (CRPV) rabbit model to test the ability of recombinant L. monocytogenes strains secreting the viral E1 protein (E1-rLm) to protect outbred rabbits against CRPV- and CRPV DNA-induced tumors. CRPV infection of outbred rabbits serves as a model for oncogenic papillomaviruses since CRPV-induced papillomas progress with high frequency to malignant carcinoma. Rabbits were vaccinated with wild-type L. monocytogenes or E1-rLm and then challenged with CRPV or viral DNA. In contrast to 0% papilloma regression in control animals, 77% of E1-rLm-vaccinated rabbits generated protective immunity that controlled and induced complete regression of tumors induced by CRPV. Latent viral DNA was not detected at 71% of the papilloma regression sites examined 4.5 months postregression. E1-rLm responder rabbits were completely resistant to papilloma formation from viral DNA. In contrast to controls, peripheral blood mononuclear cells from E1-rLm responder rabbits were able to proliferate in response to in vitro E1 stimulation. These results indicate that E1-rLm immunization generated a systemic anti-CRPV E1 cell-mediated immune response which protected outbred rabbits from tumors induced by CRPV or CRPV DNA challenge.

Recombinant Listeria monocytogenes as a live vaccine vehicle and a probe for studying cell-mediated immunity.

The ability of Listeria monocytogenes (L. monocytogenes) to enter the cytosol of host cells allows secreted proteins to efficiently enter the endogenous antigen-processing pathway leading to presentation by MHC class I molecules. L. monocytogenes has recently been exploited as a live vaccine vehicle for the induction of immunological memory against heterologous antigens. We have established a genetic system for site-specific integration of antigen expression cassettes into the Listeria genome which allows regulated expression and secretion of heterologous proteins. The ability of recombinant strains to stimulate long-term immunological memory and CD8+ T-cell-mediated protective immunity was investigated using the lymphocytic choriomeningitis virus (LCMV) murine infection model. Vaccination of mice with recombinant Listeria strains expressing LCMV antigens induced LCMV-specific CD8+ T cells which protected mice against LCMV challenge. We have also used a cottontail rabbit papillomavirus model to test the ability of recombinant Listeria strains to stimulate protective antitumor immunity in domestic rabbits. These studies have demonstrated the protective efficacy of recombinant L. monocytogenes vaccines and have established an experimental system for systematic analysis of cytotoxic T-cell induction by an intracellular bacterium.

Regression of papillomas induced by cottontail rabbit papillomavirus is associated with infiltration of CD8+ cells and persistence of viral DNA after regression.

Cottontail rabbit papillomavirus (CRPV) is a highly oncogenic papillomavirus and has been successfully used as a model to develop protective vaccines against papillomaviruses. Papillomas induced by the virus may spontaneously regress, suggesting that CRPV can also serve as a model to develop therapeutic vaccines. As a first step toward this goal, we have analyzed immunologic and viral aspects associated with papilloma regression and have identified several features unique to regression. Immunohistochemical staining of biopsies from growing and regressing papillomas and from sites after complete regression showed infiltration of CD8+ cells into the basal and suprabasal layers of the epidermis only during active regression. In situ hybridizations with mRNA-specific probes were strongly positive for E6 and E7 mRNAs during regression, but no late mRNA was present. Viral DNA was detected by in situ hybridization during regression but not after regression. However, analysis by PCR revealed persistence of viral DNA for several months at the majority of regression sites. The results suggest that stimulation of a strong CD8+ response to virus-infected cells is important for an effective therapeutic vaccine and that special attention should be given to the suppression of latent infection.

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.

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.

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.

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.

Comparison of the properties of the E6 and E7 genes of low- and high-risk cutaneous papillomaviruses reveals strongly transforming and high Rb-binding activity for the E7 protein of the low-risk human papillomavirus type 1.

A comparative analysis of different properties of the E6 and E7 proteins of high-risk and low-risk cutaneous papillomaviruses was performed. The corresponding genomic regions of human papillomavirus types 1 and 8 (HPV1 and HPV8) and of the cottontail rabbit papillomavirus (CRPV) were cloned into the eucaryotic expression vector pZipNeo-SV(X)-1 and into vectors for in vitro transcription and translation. With the help of these vectors, the individual proteins were investigated for their ability to transform C127 and NIH 3T3 rodent fibroblasts, bind the Rb protein in vitro, transactivate the adenovirus E2 promoter, and cooperate in the immortalization of primary human keratinocytes. Expression vectors for HPV16 E6 and E7 were used as a positive control. A highly transformed phenotype could be observed with rodent cell lines expressing HPV8 E6, HPV16 E6 and E7, and, surprisingly, HPV1 E7. In contrast, no transformation was detected with CRPV long E6 and HPV8 E7, whereas cells expressing HPV1 E6 and CRPV short E6 exhibited a weakly transformed phenotype. Although neither CRPV E6 nor CRPV E7 caused morphological transformation of C127 cells, CRPV E6 was able to induce anchorage-independent growth in both rodent cell lines, whereas CRPV E7 led to high cloning efficiencies only in NIH 3T3 cells. The in vitro Rb-binding affinities relative to that of HPV 16 E7 were 66% for HPV1 E7, 34% for HPV8 E7, and 11% for CRPV E7. In spite of its high Rb-binding affinity, HPV1 E7 did not trans activate the adenovirus E2 promoter, whereas HPV8 E7 and CRPV E7 showed low activities. Complementation studies in primary human keratinocytes revealed a weak immortalizing potential for HPV8 E7 and indicated a low degree of cooperativity between CRPV E7 and CRPV or HPV16 E6.

Changes in RNA expression pattern during the malignant progression of cottontail rabbit papillomavirus-induced tumors in rabbits.

Cottontail rabbit papillomavirus induces strictly epithelial tumors in both cottontail and domestic rabbits. A high proportion of the initial benign papillomas progress within 8 to 14 months to invasive carcinomas. With the help of mRNA-specific riboprobes for E6, E7, E1, E2, L1 and L2, we investigated by in situ hybridization the RNA expression pattern of cottontail rabbit papillomavirus in tissue sections of biopsies from different stages of tumor development. Common features of all lesions were high levels of E6 and E7 mRNAs and low levels of E1 and E2 mRNAs. In agreement with earlier reports, there was no evidence for a major mRNA class equivalent to the prominent E1-E4 RNA of human papillomavirus types 6/11 and 16. In cottontail rabbit papillomas, high levels of E6 and E7 mRNAs were present in the upper differentiated epithelial layers. These layers also contained most of the E1 and E2 mRNAs and the viral DNA. In contrast, papillomas of domestic rabbits revealed the opposite differentiation-dependent expression pattern for the E6 and E7 mRNAs; there were strong signals in the basal layers, and these declined with increased differentiation. Transcripts encoding the L1 mRNA were detected only in a few isolated cells of the granular layer. There was no difference between the amounts of E6, E7, E1, and E2 mRNAs present in highly dysplastic tissue and those present in adjacent normal papillomatous epithelium within a progressing papilloma. However, late transcripts and viral DNA detectable only in the upper layers of the papilloma were present throughout the thickness of the dysplastic tissue, indicating a newly acquired permissiveness of the dysplastic cells for viral DNA replication and late transcription. Carcinomas in general had the same expression patterns for E6, E7, and E1 but were dissimilar in the levels of expression of E2 and late transcripts.

T-cell response to cottontail rabbit papillomavirus structural proteins in infected rabbits.

Cottontail rabbit papillomavirus (CRPV)-induced papillomas progress at a high frequency to carcinomas and thus can serve as a model for high-cancer-risk human papillomavirus infection. Previously, we have shown that antibodies to nonstructural and structural proteins are detected in only a fraction of papilloma-bearing animals. However, the antibody response to structural proteins drastically increases as papillomas progress to carcinoma (Y.-L. Lin, L. A. Borenstein, R. Selvakumar, R. Ahmed, and F. O. Wettstein, J. Virol. 67:382-389, 1993). Here we have monitored the cellular immune response to viral proteins during the course of infection and particularly during progression from papilloma to carcinoma. This was done by measuring the in vitro proliferation response of peripheral blood mononuclear cells (PBMCs) to CRPV structural proteins L1 and L2. The proliferating cells were identified as T cells by selective removal of B or T cells. In general, the T-cell response was low for rabbits at the papilloma stage and none responded to L2. Lymphocytes from animals with carcinomas more frequently and more strongly responded to L1, and more than half also responded to L2. In addition to stimulation of PBMCs, L1- and L2-specific proliferation could also be demonstrated with lymph node and spleen cells. Overall, our data show that progression of papilloma to carcinoma is associated with an increased T-cell response to CRPV structural proteins in addition to an increased humoral response. This greater immune reactivity, however, was not associated with a selectively increased expression of structural proteins, since RNA isolated from papillomas and carcinomas contained similar relative levels of late and early RNA as shown by dot blot analysis. Thus, the heightened immune reactivity seen in carcinoma-bearing rabbits most likely reflects greater stimulation of the immune system owing to dissemination of the tumor. These findings suggest that increased immune responses to papillomavirus proteins may be prognostic of progression to carcinoma and particularly of the development of metastases.

Cottontail rabbit papillomavirus L1 protein-based vaccines: protection is achieved only with a full-length, nondenatured product.

Papillomas induced by the cottontail rabbit papillomavirus (CRPV) progress at a high frequency to carcinomas. In this regard, CRPV and its tumors can serve as an animal model for highly oncogenic human papillomaviruses. We have previously shown that immunization with major structural protein L1 elicits neutralizing antibodies and protects rabbits from papilloma development (Y.-L. Lin, L.A. Borenstein, R. Selvakumar, R. Ahmed, and F.O. Wettstein, Virology 187:612-619, 1992). In this study, we demonstrated that vaccination with the TrpE-L1 fusion protein not only protected rabbits from papilloma development but also prevented latent infection. This was indicated by the failure to amplify CRPV sequences by polymerase chain reaction in biopsies from infection sites of immunized animals. Furthermore, we showed that TrpE-L1 immunization protected rabbits from papilloma formation induced by virus but not from that induced by viral DNA. To explore the possibility of developing vaccines based on L1 subfragments, we mapped the linear L1 epitopes recognized by TrpE-L1-immunized rabbits and by virus-infected rabbits resistant to superinfection. Sera from papilloma-bearing rabbits reacted with one major epitope located at the carboxy-terminal end of L1, between amino acids (aa) 480 and 505. A second epitope, and in some animals a third one, was located in the amino-terminal region, between aa 78 and 101, as well as between aa 37 and 62. Sera from TrpE-L1-immunized animals recognized only one major epitope, located between aa 6 and 37. Immunization of rabbits with L1 subfragment fusion proteins led to seroconversion, but no neutralizing antibodies were produced and the animals were not protected against papilloma formation. The data indicate that a successful papillomavirus vaccine must be based on immunization with full-length native L1 and that further simplification to smaller peptides containing major linear epitopes is not feasible.

Progression from papilloma to carcinoma is accompanied by changes in antibody response to papillomavirus proteins.

Cottontail rabbit papillomavirus induces benign tumors, papillomas, in rabbits which progress at a high frequency to malignant tumors, carcinomas. Cottontail rabbit papillomavirus therefore provides an experimental model for oncogenic human papillomaviruses. The nature of the antigens recognized by the host has not been identified at any stage of tumor development. Here, we characterized the humoral immune response to viral antigens in cottontail and domestic rabbits at the papilloma stage, in domestic rabbits at the carcinoma stage, and in animals in which papillomas had regressed. Antibodies to linear epitopes were identified by Western blotting (immunoblotting) with bacterial fusion proteins, and evidence for recognition of conformational epitopes was obtained by immunoprecipitation. An immune response to the early proteins E1, E2, E6, and E7 was detected only in a fraction of the animals, and all animals were negative for E4 and E5. The response to E6 and E7 peaked around 7 months and then decreased, while that to E1 and E2 remained level after an initial raise. The antibody response to structural proteins was low at the papilloma stage, and antibodies to L1 recognized predominantly conformational epitopes. As papillomas progressed to carcinomas, there was a drastic increase in the response to L1 and L2, suggesting a change in interaction between virus-infected host cells and the host's immune system.

Effective vaccination against papilloma development by immunization with L1 or L2 structural protein of cottontail rabbit papillomavirus.

Immunization of rabbits with either L1, the major structural protein, or L2, a minor structural protein of cottontail rabbit papillomavirus (CRPV), protected against challenge with the virus. Neutralizing antibodies were elicited by both the L1 and L2 trpE fusion proteins. Neutralization with anti-L1 serum, however, was more efficient than with anti-L2 serum. In contrast, when tested on Western blots the immune response to L2 was stronger than to L1. Rabbits were also protected against CRPV infection by immunization with L1 expressing recombinant vaccinia virus. Sera from two of three rabbits immunized with recombinant vaccinia virus were negative on Western blots but all three were positive in ELISA's with nondenatured fusion protein or in immunoprecipitations. The results suggest that both the viral structural proteins, L1 and L2, merit consideration in the development of a vaccine against papillomavirus.

Identification of three transforming proteins encoded by cottontail rabbit papillomavirus.

Cottontail rabbit papillomavirus (CRPV) provides an animal model for human papillomaviruses associated with a high risk of cancer development. So far, nothing is known about the transforming functions of CRPV genes because of the lack of an assay system. We have recently developed two systems to assay for CRPV transforming functions. One is based on the finding that transformation of NIH 3T3 cells by CRPV is considerably increased by deleting sequences in open reading frame L2. The second one is based on the use of a cottontail rabbit skin epithelial cell line, sf1Ep (C. Meyers and F. O. Wettstein, Virology 181:637-646, 1991). Mutations were introduced which abolished expression of the full-length E6 protein (LE6), the short E6 protein (SE6) initiated at the second ATG of E6, the E7 protein, or the E5 protein. Mutations affecting LE6 or E7, but not SE6, reduced transformation of NIH 3T3 and sf1Ep cells. Transformed NIH 3T3 cell lines with mutations in LE6 and E7 did not grow in soft agar, while those with mutations in SE6 and E5 grew with a reduced efficiency. The cell lines with mutations in LE6, SE6, or E7 still did induce tumors in nude mice. These mutations, however, abolished the ability to induce papillomas in rabbits. When expressed individually with a retroviral vector, LE6, SE6, or E7, but not E5, conferred anchorage-independent growth. The level of viral protein expression in these cell lines was generally low, and a comparison of the abundance of virus-specific mRNA showed that cell lines contained 20 to 50 times less mRNA than a cottontail rabbit papilloma. These data demonstrate that CRPV encodes at least three transforming proteins.

Human papillomavirus type 16 immortalized cervical keratinocytes contain transcripts encoding E6, E7, and E2 initiated at the P97 promoter and express high levels of E7.

Human cervical keratinocytes represent the specific host for the genital human papillomaviruses (HPV). Transfection of these cells with the DNA of a number of the oncogenic HPVs including type 16 was recently shown to result in their immortalization but not in malignant transformation. In this report we show that viral transcripts for E6 and E7 in these cells were as abundant as in cancer derived cell lines. However, in contrast to cancer derived cell lines, immortalized cervical keratinocytes contained RNA with the potential to encode a full-length E2 protein. In addition, the levels of the E7 oncoprotein were at least as high as in cancer derived cell lines, suggesting that E2 interruption, observed in cancer derived cell lines, is not causally related to the high level of E7 expression and, therefore, deregulation of the P97 promoter may not be a prerequisite for HPV-16 associated cancer development. Furthermore, we show that E6, E7, and E2 encoding transcripts all originate from the viral promoter, P97. Unlike in cancer derived cell lines, all transcripts terminated at the early poly(A) site.

The late region differentially regulates the in vitro transformation by cottontail rabbit papillomavirus DNA in different cell types.

Papilloma induced by the cottontail rabbit papillomavirus (CRPV) progress at a high frequency to cancers. This property makes the CRPV system unique to study the role of papillomaviruses in cancer development. In contrast to bovine papillomavirus type 1, no convenient in vitro transformation system for CRPV has been available. Here, we describe two in vitro systems that we have developed. Transformation of NIH 3T3 cells is greatly facilitated by deletions in the CRPV late region. Specifically, a 300-bp segment located in the 5' half of open reading frame L2 inhibits transformation of NIH 3T3 cells by CRPV DNA. In a second system involving the natural host cell of CRPV, rabbit skin epithelial cells, transformation is a two-stage phenomenon leading first to cells with an increased growth potential which subsequently become morphologically transformed. Transformation of rabbit skin epithelial cells does not require deletions in the L2 ORF. However, late region sequences located in the 3' half of ORF L1 are needed, as they are for papilloma induction in rabbits.

The E7 protein of human papillomavirus 8 is a nonphosphorylated protein of 17 kDa and can be generated by two different mechanisms.

The E7 protein of human papillomavirus (HPV) 8 shows no in vitro transforming activity, in contrast to E7 of the genital HPVs, but seems to be involved in the control of viral DNA replication. To determine whether functional differences between the E7 proteins of HPV16 and HPV8 were reflected in differences in their biochemical properties, we characterized the E7 protein of HPV8 expressed from the late simian virus 40 promoter in COS 7 cells. An E7-specific antiserum was obtained by immunizing rabbits with a beta-gal-E7 fusion protein containing all of the E7 polypeptide. This antiserum specifically precipitated from [35S]cysteine but not from 32PO4-labeled transiently transfected COS 7 cells a protein with a low mobility of 17 kDa in SDS-PAGE. The high sedimentation rate in nondenaturing glycerol gradients pointed to an interaction with other proteins or to the existence of E7 oligomers. An association with the retinoblastoma protein could, however, be excluded. The 5' ends of HPV8 transcripts derived from the E6-E7 region in G418 selected rodent cells, which were mapped by nuclease S1 digestion, are located upstream of ORFs E6 and E7, respectively. No evidence for an E6*I-like mRNA was found. In COS 7 cells transfected with a plasmid expressing the E6-E7 region of HPV8 under the control of the late SV40 promoter, however, the E7 protein was translated from a polycistronic mRNA potentially encoding E6 and E7. These data indicate that the E7 protein of HPV8 may be expressed in two ways: (i) through translation of an E7-specific mRNA, as with HPV6 and HPV11, or (ii) through internal initiation of translation at the ATG of E7.