Biomedical subjects

F O Wettstein

Publications and source records attributed to F O Wettstein.

At least 19 recordsLinked to original sources

PubMed1992

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.

Animals↗

PubMed1992

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.

3T3 Cells↗

PubMed1991

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.

Blotting, Northern↗

PubMed1991

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.

Animals↗

PubMed1990

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.

Animals↗

PubMed1990

The E7 proteins of the nononcogenic human papillomavirus type 6b (HPV-6b) and of the oncogenic HPV-16 differ in retinoblastoma protein binding and other properties.

The E7 early viral protein of the oncogenic human papillomavirus type 16 (HPV-16) has been strongly implicated in the maintenance of the malignant phenotype in cervical cancers and cancer-derived cell lines. HPV-16 E7 is a nuclear phosphoprotein that can cooperate with ras to transform baby rat kidney cells, transactivates the adenovirus E2 promoter, and binds to the retinoblastoma (RB) protein. The E7 phosphoprotein of the nononcogenic HPV-6b, which is generally associated with benign genital warts, is similar to the HPV-16 E7 in amino acid sequence but differs dramatically in migration in sodium dodecyl sulfate-polyacrylamide gels, sedimentation in nondenaturing glycerol gradients, and the ability to bind the RB protein. Our results indicate that the RB protein preferentially binds the phosphorylated form of HPV-6b E7, which comprises a minor fraction of the total E7 expressed in transiently transfected COS-7 cells. These characteristics may help to explain the difference in the oncogenic potential of the oncogenic and nononcogenic types of genital papillomaviruses.

Amino Acid Sequence↗

PubMed1989

Genetic analysis of CRPV pathogenesis: the L1 open reading frame is dispensable for cellular transformation but is required for papilloma formation.

Genetic studies to elucidate the role of papillomaviruses in the development and progression of tumors have been severely hampered because the viruses cannot be grown in tissue culture and therefore mutants are not available. We have employed recombinant DNA for papilloma induction to identify essential sequences involved in papillomavirus pathogenesis. Here, we demonstrated that deleting most of the open reading frame (ORF) L2 did not affect the potential of viral cottontail rabbit papillomavirus (CRPV) DNA to induce papillomas. The extrachromosomally maintained DNA in the papillomas was not rearranged and the major transcripts of 1.3 and 2.0 kb encoded E7 and E6, respectively. A recombinant DNA containing a larger deletion lacking the 3' terminal half of ORF L2 and all of ORF L1 (pdlBc/l) did not induce papillomas. The results indicate that sequences in the late region not required for transformation of NIH 3T3 cells by bovine papillomavirus type-1 essential in CRPV for induction of papillomas.

Animals↗

PubMed1989

Oncogenic and nononcogenic human genital papillomaviruses generate the E7 mRNA by different mechanisms.

A new promoter located within E6 was mapped in human papillomavirus type 6b (HPV6b)- and HPV11-containing benign genital condylomata (genital warts). The RNA transcribed from this promoter represented the major RNA species colinear with open reading frames E6 and E7 and can encode the E7 protein. No equivalent promoter was active in HPV16-containing cancers and cancer-derived cell lines. In those, the major transcripts contained one of two different introns within E6 and the RNAs could encode two different E6 proteins and E7.

Amino Acid Sequence↗

PubMed1988

A study of pituitary thyrotropin, its subunits, and messenger ribonucleic acids in nonthyroidal illness.

We studied serum TSH, pituitary TSH and alpha and beta-subunits of TSH and their messenger ribonucleic acids (mRNAs) in three models of nonthyroidal illness (NTI) in the rat, ie diabetes mellitus (1 wk after 65 micrograms streptozotocin/g BW IP), turpentine oil-injection (8 to 48 hours after after a dose of 5 microliter/g bw SC), and complete fasting for 72 hours. Euthyroid, hypothyroid (two months after thyroidectomy) and hyperthyroid rats (30 micrograms T4/d X 7, SC) were also studied for comparison. Pituitary TSH, alpha and beta subunits and serum TSH, T4, and T3 were measured by RIA. Pituitary mRNAs coding for common delta and TSH-beta subunits were determined by cytoplasmic dot hybridization technique using specific [32P]-cDNA probes. In all NTI models there were significant decreases in serum levels of TSH, T4, and T3, but no significant changes were observed in the pituitary content of TSH, and alpha and TSH-beta subunits. Hypothyroid rats had an increase in serum TSH, pituitary TSH, and pituitary TSH-beta subunit and a decrease in pituitary alpha subunit. On the other hand, hyperthyroid rats showed a decrease in serum TSH, pituitary TSH, and pituitary TSH-beta subunit, while there was no change in the alpha subunit. A significant reduction in the pituitary TSH-beta mRNA levels was observed in all NTI models and hyperthyroidism, while TSH-beta mRNA was increased in thyroidectomized rats. alpha-mRNA was increased only in the pituitary of hypothyroid rats; there was no appreciable change in the pituitary alpha-mRNA in the various other pituitary groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Identification and characterization of the CRPV E7 protein expressed in COS-7 cells.

The papillomavirus E7 protein may play an important role in oncogenesis as it is the major viral protein present in human cervical cancer-derived cell lines. Because of the relevance of the cottontail rabbit papillomavirus (CRPV) system for the study of viral-induced malignancies, we characterized its E7 protein expressed by a heterologous strong promoter. An E7-specific antiserum was obtained by immunizing rabbits with a Trp E-E7 fusion protein containing the 88 carboxyl-terminal amino acids of E7. This antiserum specifically precipitated from [35S]cysteine but not from 32PO4-labeled transiently transfected COS-7 cells a 14-kDa protein. The protein was present only in the soluble cytoplasmic fraction and exhibited a heterogeneous sedimentation rate in nondenaturing glycerol gradients.

Animals↗

PubMed1988

The two proteins encoded by the cottontail rabbit papillomavirus E6 open reading frame differ with respect to localization and phosphorylation.

Cottontail rabbit papillomavirus-induced tumors contain two E6-coding transcripts. A major transcript can code for a short E6 protein initiated at AUG codon 2, and a minor one could code for a long E6 initiated at AUG 1. We have identified the two proteins expressed in COS-7 cells (M. Barbosa and F. O. Wettstein, J. Virol. 61:2938-2942, 1987). The properties of the two proteins are distinctly different. The long E6 is predominantly present in the nucleus, in which it appears to be associated with the nuclear matrix. Minor portions of the long E6 are located in equal amounts in both the soluble cytoplasmic and the membrane fractions. The short E6 is a soluble cytoplasmic protein phosphorylated at serine residues.

Animals↗

PubMed1988

E2 of cottontail rabbit papillomavirus is a nuclear phosphoprotein translated from an mRNA encoding multiple open reading frames.

The papillomavirus E2 protein is a transcription trans-activator and as such as a paramount effect on viral functions. We have identified and characterized the cottontail rabbit papillomavirus E2 protein expressed from the late simian virus 40 promoter in COS-7 cells. E2 was shown to be a highly phosphorylated 49-kilodalton protein. Subcellular fractionation and indirect immunofluorescent staining indicated that E2 was located in the nuclei. E2 was expressed from a vector which contained just the open reading frame. ORF E2 and also from vectors which extended farther upstream and also expressed E7 or the short E6 protein. However, the level of E2 was very low in cells transfected with a vector expressing the long E6 protein. Mapping of transcripts with nuclease S1 and exonuclease ExoVII in cells expressing the short E6 and E2 proteins showed that E2 was translated from an RNA which encoded the short E6, E7, and E2 proteins but served as mRNA for only the short E6 and E2 proteins.

Animals↗

PubMed1987

A variant of CRPV DNA preferentially maintained as a plasmid in NIH 3T3 cells and characterization of its transcripts in nude mouse tumors.

Rabbit and human papillomaviruses are strictly epitheliotropic and their DNA replicates extrachromosomally in benign lesions (warts) of their natural host. Their tissue and host specificity is thought to be genetically controlled and may account for the inefficiency in transforming heterologous cells. In our hands, CRPV DNA did not induce foci in NIH 3T3 cells and in cotransfections with a selectable marker only integrated DNA was found. The viral DNA appeared to be transcriptionally inactive since no transcripts could be detected and in cells were not tumorigenic for nude mice. In contrast to these results a spontaneously derived CRPV variant DNA was able to replicate extrachromosomally and a majority of focus-derived cell lines were tumorigenic for nude mice. In nude mouse tumors the variant DNA remained exclusively extrachromosomal and viral transcripts were detected. The sizes of the major transcripts were 2.0 and 1.3 kb and this suggested that E6 and E7 were expressed. The situation in rabbit tissue was different; both variant and wild-type DNA were maintained extrachromosomally. The extrachromosomal maintenance of the variant but not of wild-type DNA in mouse cells suggests that the variant lacks sequences which may play a role in the host and the tissue restriction of CRPV. The deletion in the variant DNA was located in the late region and included most or all of L1 and a carboxy terminal segment of L2. A second deletion eliminated some pBR322 sequences.

Animals↗

PubMed1987

Identification of the major cottontail rabbit papillomavirus late RNA cap site and mapping and quantitation of an E2 and minor E6 coding mRNA in papillomas and carcinomas.

The capsite of the 2.6- and 4.8-kb major late transcripts of cottontail rabbit papillomavirus (CRPV) has been mapped by primer extension. A leader exon of about 300 nucleotides common to both RNAs is located in the untranslated region of the genome upstream of the capsites for early transcripts. In contrast to the early capsites which are all preceded by TATA boxes, no such sequence is present 30 nucleotides upstream of the late capsite. These data indicate that the switch from early to late transcription involves recognition of a new promoter and suppression of transcription termination at the early polyadenylation site. We have also identified a minor exon with a coding potential for a putative E2 transactivating protein. Quantitation by S1 mapping of the E2 coding exon and a minor exon coding for a full-sized E6 protein unique in size to the highly oncogenic CRPV did not reveal differences in the level of transcription between papillomas and carcinomas.

Animals↗

PubMed1987

The major human papillomavirus protein in cervical cancers is a cytoplasmic phosphoprotein.

In a previous study, the most abundant viral transcript in a human papillomavirus type 16-associated cervical cancer and in a cancer-derived cell line was characterized, and its translation product, the E7 protein, was identified (D. Smotkin and F. O. Wettstein, Proc. Natl. Acad. Sci. USA 68:4680-4684, 1986). Here we show that the E7 protein had a half life of about 1 h and was located in the soluble cytoplasmic fraction. The protein was phosphorylated at serine residues and exhibited a high heterogeneous sedimentation rate in nondenaturing glycerol gradients, suggesting an oligomer formation or association with cellular protein.

Cell Line↗

PubMed1987

Transcription of the cottontail rabbit papillomavirus early region and identification of two E6 polypeptides in COS-7 cells.

Cottontail rabbit papillomavirus (CRPV) early proteins are present at very low levels in virus-induced tumors and cannot be detected by immunological methods. Furthermore, cells in culture are not readily transformed by the virus. To overcome these difficulties in identifying and characterizing the putative transforming protein(s) coded by the E6 open reading frame, the early cottontail rabbit papillomavirus region was expressed under the control of the late simian virus 40 promoter. Mapping of the transcripts in transiently transfected COS-7 cells indicated that transcription was initiated in the late region of simian virus 40. Two E6-coded polypeptides were identified, representing translation products initiated at the first and second AUG codons.

Animals↗

PubMed1986

Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein.

Human papillomavirus type 16 DNA and RNA were characterized in the cervical cancer-derived CaSki cell line, which contains only integrated DNA, and in a cervical cancer, which contains predominantly plasmid DNA. In both, a major RNA can code for the early open reading frame E7 and a minor one can code for E6. The cervical cancer, but not the CaSki cell line, contains a minor RNA that can code for an intact E2 protein, and this may relate to the continued presence of plasmid DNA. The RNA mapping data suggest that the poly(A)+ RNA is transcribed from a minor fraction of the several hundred gene copies present, and in the cervical cancer these genomes appear to be integrated. The E7 protein has been identified in CaSki cells and the prevalence of its mRNA suggests a possible function in progression to, or long-term maintenance of, the malignant state.

Cell Line↗

PubMed1986

Human papillomavirus deoxyribonucleic acid in adenocarcinoma and adenosquamous carcinoma of the uterine cervix.

This report describes the detection of human papillomavirus type 16 or 18 deoxyribonucleic acid (DNA) in nine of 15 invasive tumors of the cervix, including three squamous carcinomas, four adenosquamous carcinomas, one glassy cell carcinoma, and one adenocarcinoma. The viral DNA was identified by Southern blotting and DNA hybridization. Human papillomaviruses may play an etiologic role in the development of at least some adenocarcinomas and adenosquamous carcinomas as well as most squamous tumors of the cervix.

Adenocarcinoma↗