Hypermethylation of the human papillomavirus transgenome in transgenic mice.

We have previously reported an observation that the regulatory sequence of the human papillomavirus (HPV) type 18 genome elicits only basal and unregulated transcriptional activities in transgenic mice [Choo et al., Virology (1992) 188:378-383]. We have now extended the study to other transgenic mice carrying an entire or a subgenomic fragment of HPV-16 and -18. Extremely low levels of HPV transcripts were detected in various organs of the mice only by reverse transcriptase-based polymerase chain reaction. On analysis of the methylation status of the HPV transgenome sequences, hypermethylation was clearly observed, therefore suggesting a possible explanation for low level HPV transgenome transcriptional activity in HPV transgenic mice.

Unregulated and basal transcriptional activities of the regulatory sequence of the type 18 human papillomavirus genome in transgenic mice.

Type 18 human papillomavirus (HPV18) is a genital virus closely associated with cervical carcinoma. To analyze the transcriptional activities of the long control region (LCR) of the HPV18 genome, we have produced 12 transgenic mice harboring the HPV18/LCR sequence fused to a promoterless SV40 T-antigen (TAg) gene. The mice were small in body size, generally very weak, and none lived longer than 110 days. Three mice with the longest life span (58-110 days) developed hyperplastic thymus and/or lymph node and were further analyzed. In these mice, Northern hybridization failed to detect TAg transcripts in any of the 25 organs studied. However, spliced TAg RNA was detected by polymerase chain reaction in the hyperplastic thymus and lymph node and in the normal submaxillary gland, stomach, large intestine, urinary bladder, and the cerebrum, indicating the presence of very low cellular levels of TAg RNA in these organs. When immunostaining was performed on the hyperplastic thymus, TAg protein was detected only in the ductal epithelial cells. Our results appear to indicate that the HPV18/LCR sequence was able to express only unregulated and basal levels of transcriptional activity in transgenic mice. Such a mode of transcription has become a major hindrance in the use of transgenic mouse system for the studies of the biology of the human papillomavirus.

Transgenome transcription and replication in the liver and extrahepatic tissues of a human hepatitis B virus transgenic mouse.

We have produced a transgenic mouse (B32-1) carrying the complete genome of the human hepatitis B virus (HBV). High titers of the viral surface (HBsAg) and the e antigen (HBeAg) were detected in the serum of the mouse. In the liver and 12 of 16 extrahepatic tissues analyzed, Northern blot hybridization indicated the presence of the 2.1-kilobase (kb) and the 3.5-kb major HBV transcripts. A liver cDNA library was constructed from which the liver RNAs from four cDNA clones with splicing were found. Sequencing analysis showed that the splicing occurred between nucleotides 2451 and 487 of the viral genome, resulting in a truncated viral polymerase gene, as in human hepatocytes. Southern blot analysis of total DNA preparations of the tissues revealed the presence of episomal HBV genome, indicating replication of the viral transgenome in these tissues. However, replication was detected only in some but not all of the tissues that transcribed the 3.5-kb RNA. Partial double-stranded as well as full-length and subgenomic-length single-stranded HBV DNA species of discrete sizes were detected which may represent replication intermediates of preferred replication termination sites of the HBV transgenome. Since many molecular characteristics of mouse B32-1 were similar to those found in HBV-infected humans, HBV transgenic mice similar to B32-1 would be useful in further elucidation of other aspects of the replication and transcription mechanisms of HBV in the liver and extrahepatic tissues.

Analysis of the unoccupied site of an integrated human papillomavirus 16 sequence in a cervical carcinoma.

We have previously cloned and analyzed the structure of a type 16 human papillomavirus (HPV16) integration in a primary cervical carcinoma tissue, M50 (Choo et al., J. Virol. 62, 1659-1666, 1988). We found that specific nucleotide sequences within the HPV16 genome influenced the genomic organization of the integrated viral genome. Using the viral-cellular junctions of the M50 DNA as probes, we have now cloned the unoccupied site from a human genomic library. Mapping analysis showed that a deletion of about 1.1 kilobase pairs (kb) had occurred at the integration site of M50. Sequencing of the integration junctions of the unoccupied site and comparison with the viral sequence has revealed short regions of sequence homology between the viral and the cellular genomes at both junctions. Our results are consistent with a mechanism of integration of the HPV16 sequences in the M50 carcinoma involving illegitimate recombination events using short patches of homologous sequences between the two heterologous genomes for anchorage and as guides for crossover. Preferred topoisomerase I cleavage sites and alternating purine and pyrimidine bases, which favor the formation of Z-DNA, could also be identified at the integration regions, supporting a proposed role for the topoisomerase I enzyme in the illegitimate recombination in the viral integration process.

Production of transgenic mice carrying the human hepatitis B virus or the human papillomavirus DNA sequences in Taiwan: analysis of physical structure and hereditary mode.

In order to produce suitable animal model systems for the analysis of possible synergistic interaction between hepatitis B virus (HBV) or the human papillomavirus (HPV) with other viral or chemical co-factors, we have produced transgenic mice carrying HBV, HPV16 or HPV18. Transfer of 3343 micro-injected one-cell (C57/BL6 X CBA) F1 embryos into pseudo-pregnant ICR recipients produced 181 pups. Seventeen of 151 of the pups analysed were found to be transgenics. Southern blot and slot blot analysis showed that the copy number of the transgenome in the transgenic mice ranged from 1-50 copies per cell. All the transgenomes analysed appeared to be in tandem repeats without detectable sequence rearrangements. The transgenome sequences were also stable on transmission to the F3 generation. On breeding, six of the ten transgenic mice analysed showed the Mendelian mode of inheritance with integration at single or multiple loci. Of the remaining four mice, three were mosaics and one was sterile. Analysis of the gene expression of the transgenic mice is currently in progress.

Presence of catenated human papillomavirus type 16 episomes in a cervical carcinoma cell line.

Human papillomavirus (HPV) is frequently associated with cervical carcinoma and derived cell lines. In primary tissues of the carcinoma, the viral genome may be present in episomal or integrated configuration. In cell lines, however, only integrated HPV sequences have been reported. In this article, we describe the presence of episomal type 16 HPV (HPV16), demonstrated by electron microscopy and two-dimensional agarose gel electrophoresis, in a cervical carcinoma cell line, CC7T/VGH, established in 1980 in Taiwan. In CC7T/VGH, the HPV16 sequences are transcriptionally active, and at least three major HPV16 RNA species were detected in Northern blots. Results from restriction enzyme and S1 nuclease analysis suggest a composition of oligomeric HPV16 molecules in dimeric repeats. In addition, the HPV16 oligomers exist as catenated molecules of interlocking rings instead of concatemers. A monomeric copy of the HPV16 episome was cloned from a Hirt supernatant of CC7T/VGH by using a plasmid vector. Mapping and partial sequencing studies revealed an internal deletion of 163 base pairs within the L1 open reading frame. However, insertion of an A.C nucleotide pair at the deletion junction restored the otherwise frame-shifted L1 open reading frame. Two base transitions were also found within the E7 and the E1 open reading frames. Our findings suggest the need for closer examination for HPV episomal catenation in other cervical carcinoma cell lines as well as in primary carcinoma tissues of the uterine cervix and the anogenital tract. With CC7T/VGH, a way is now available for studies of many important aspects of the biology of HPV such as replication and gene expression of the extrachromosomal viral genome.

Analysis of the structure and expression of the c-myc oncogene in cervical tumor and in cervical tumor-derived cell lines.

The structure of the c-myc oncogene in 17 cervical tumors and patient-matched nontumor tissues from Chinese patients residing in Taiwan was analysed. In contrast to recent reports on Mexican patients, none of the samples showed rearrangements and sequence amplification in the c-myc gene. The discrepancy may be explained by different carcinogenesis mechanisms being in operation in different geographic regions. Although no structural alterations in the c-myc gene were found in seven cervical carcinoma cell lines analysed, Northern blot analysis indicated different levels of c-myc gene expression which may be related to the presence of human papillomavirus (HPV) sequence in the cell and suggests a possible c-myc-hpv interaction in some stages of the transformation process.

Sequence duplication and internal deletion in the integrated human papillomavirus type 16 genome cloned from a cervical carcinoma.

Integrated human papillomavirus type 16 (HPV16) sequences were cloned from a cervical carcinoma and analyzed by restriction mapping and nucleotide sequencing. The viral integration sites were mapped within the E1 and E2 open reading frames (ORFs). The E4 and E5 ORFs were entirely deleted. An internal deletion of 376 base pairs (bp) was found disrupting the L1 and L2 ORFs. Sequencing analysis showed that an AGATGT/ACATCT inverted repeat marked the deletion junction with two flanking direct repeats 14 and 8 bp in length. A 1,330-bp sequence duplication containing the long control region (LCR) and the E6 and E7 ORFs was also found. The duplication junction was formed by two 24-bp direct repeats with 79% (19 of 24) homology located within the LCR and the E2 ORF of the prototype viral genome, respectively. This observation leads us to propose that the initial viral integration involved an HPV16 dimer in which the direct repeats in tandem units recombined, resulting in reiteration of only a portion of the original duplication. A guanosine insertion between nucleotides 1137 and 1138 created a continuous E1 ORF which was previously shown to be disrupted. Results from this study indicate that sequence reiteration and internal deletion in the integrated, and possibly in the episomal, HPV16 genome are influenced by specific nucleotide sequences in the viral genome. Moreover, reiteration of the LCR/E6/E7 sequences further supports the hypothesis that the E6/E7 ORFs may code for oncogenic proteins and that regulatory signals in the LCR may play a role in cellular transformation.