Viral-cellular Junctions Research Articles

Viral-Cellular DNA Junctions as Molecular Markers for Assessing Intra-Tumor Heterogeneity in Cervical Cancer and for the Detection of Circulating Tumor DNA.

The development of cervical cancer is frequently accompanied by the integration of human papillomaviruses (HPV) DNA into the host genome. Viral-cellular junction sequences, which arise in consequence, are highly tumor specific. By using these fragments as markers for tumor cell origin, we examined cervical cancer clonality in the context of intra-tumor heterogeneity. Moreover, we assessed the potential of these fragments as molecular tumor markers and analyzed their suitability for the detection of circulating tumor DNA in sera of cervical cancer patients. For intra-tumor heterogeneity analyses tumors of 8 patients with up to 5 integration sites per tumor were included. Tumor islands were micro-dissected from cryosections of several tissue blocks representing different regions of the tumor. Each micro-dissected tumor area served as template for a single junction-specific PCR. For the detection of circulating tumor-DNA (ctDNA) junction-specific PCR-assays were applied to sera of 21 patients. Samples were collected preoperatively and during the course of disease. In 7 of 8 tumors the integration site(s) were shown to be homogenously distributed throughout different tumor regions. Only one tumor displayed intra-tumor heterogeneity. In 5 of 21 analyzed preoperative serum samples we specifically detected junction fragments. Junction-based detection of ctDNA was significantly associated with reduced recurrence-free survival. Our study provides evidence that HPV-DNA integration is as an early step in cervical carcinogenesis. Clonality with respect to HPV integration opens new perspectives for the application of viral-cellular junction sites as molecular biomarkers in a clinical setting such as disease monitoring.

HPV16 integration probably contributes to cervical oncogenesis through interrupting tumor suppressor genes and inducing chromosome instability.

BackgroundThe integration of human papilloma virus (HPV) into host genome is one of the critical steps that lead to the progression of precancerous lesion into cancer. However, the mechanisms and consequences of such integration events are poorly understood. This study aims to explore those questions by studying high risk HPV16 integration in women with cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (SCC).MethodsSpecifically, HPV integration status of 13 HPV16-infected patients were investigated by ligation-mediated PCR (DIPS-PCR) followed by DNA sequencing.ResultsIn total, 8 HPV16 integration sites were identified inside or around genes associated with cancer development. In particular, the well-studied tumor suppressor genes SCAI was found to be integrated by HPV16, which would likely disrupt its expression and therefore facilitate the migration of tumor. On top of that, we observed several cases of chromosome translocation events coincide with HPV integration, which suggests the existence of chromosome instability. Additionally, short overlapping sequences were observed between viral derived and host derived fragments in viral-cellular junctions, indicating that integration was mediated by micro homology-mediated DNA repair pathway.ConclusionsOverall, our study suggests a model in which HPV16 might contribute to oncogenesis not only by disrupting tumor suppressor genes, but also by inducing chromosome instability.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0454-4) contains supplementary material, which is available to authorized users.

Abstract 3173: HPV integration and methylation patterns in cervical intraepithelial neoplasia and invasive cervical cancer via HPV capture and high-throughput sequencing

Abstract Persistent infection with high-risk human papillomaviruses (HPV) types is essential for the development of cervical cancer and its immediate precursor, cervical intraepithelial neoplasia 3 (CIN3). However, HPV infection alone is not sufficient to promote cervical carcinogenesis, thus it is hypothesized that the progression to invasive disease is dependent on viral and host factors. For example, it is well established that HPV integration into the host genome is a signature of invasive cervical cancer (ICC). Also, HPV genomes have CpG sites scattered throughout their genes that, with advancing disease, are increasingly methylated by the host cell's DNA methyltransferases. This methylation may alter the expression patterns of viral genes that are relevant to transformation. The goal of the current study was to investigate the landscape of viral-host integration and associated methylation in CIN and ICC. DNA from the lesions of patients diagnosed with either CIN or ICC was captured utilizing HPV and target gene enrichment and next-generation sequencing. DNA libraries were prepared using SureSelectXT Methyl-Seq Target Enrichment Kit (Agilent Technologies) and hybridized with a custom SureSelectXT Library (Agilent Technologies) designed to target 63 HPV types and xGen Lockdown Probes (Integrated DNA Technologies) designed to target 6 cancer-relevant genes. Library samples were pooled and sequenced using the Illumina HiSeq 2000. Bisulfate treated sequence reads obtained from Illumina Hi-Seq 2000 were analyzed by MethylCoder that generates per-base resolution of methylation data. In MethylCoder, the Genomic short-read nucleotide alignment program (GSNAP) was used for aligning short reads into reference genomes (HPV and hg19). For each CpG site, the methylation rate was calculated as the percentage of unconverted cytosines in each sample. In support of previous work, we found that multiple HPV types exhibited integration into various host genes in ICC. Also, HPV integration occurs in different patterns within the same gene. For example, in one tumor, HPV16 exhibited three distinct integrations into the PVT1 oncogene. Global methylation patterns were found to be unique among HPV types and samples, yet preliminary analysis suggests that the percent methylation in HPV may track with that of the host genome. Further, the overall methylation pattern of the virus was significantly different in CIN versus ICC. Here we provide a discussion of specific nonrandom integration sites across multiple ICC samples and representative HPV methylation profiles from CIN and ICC samples. Our data provide novel insights into the HPV CpG methylation sites and gene targets at viral-cellular junctions of different HPV types from CIN through ICC, thus potentially identifying new biomarkers for use in the screening and diagnosis of cervical cancer. Citation Format: Marissa Iden, Samantha Fye, Yi-Wen Huang, Pengyuan Liu, Janet S. Rader. HPV integration and methylation patterns in cervical intraepithelial neoplasia and invasive cervical cancer via HPV capture and high-throughput sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3173. doi:10.1158/1538-7445.AM2014-3173

Multiplex Identification of Human Papillomavirus 16 DNA Integration Sites in Cervical Carcinomas

Cervical cancer is caused by high-risk human papillomaviruses (HPV), in more than half of the worldwide cases by HPV16. Viral DNA integration into the host genome is a frequent mutation in cervical carcinogenesis. Because integration occurs into different genomic locations, it creates unique viral-cellular DNA junctions in every single case. This singularity complicates the precise identification of HPV integration sites enormously. We report here the development of a novel multiplex strategy for sequence determination of HPV16 DNA integration sites. It includes DNA fragmentation and adapter tagging, PCR enrichment of the HPV16 early region, Illumina next-generation sequencing, data processing, and validation of candidate integration sites by junction-PCR. This strategy was performed with 51 cervical cancer samples (47 primary tumors and 4 cell lines). Altogether 75 HPV16 integration sites (3′-junctions) were identified and assigned to the individual samples. By comparing the DNA junctions with the presence of viral oncogene fusion transcripts, 44 tumors could be classified into four groups: Tumors with one transcriptionally active HPV16 integrate (n = 12), tumors with transcribed and silent DNA junctions (n = 8), tumors carrying episomal HPV16 DNA (n = 10), and tumors with one to six DNA junctions, but without fusion transcripts (n = 14). The 3′-breakpoints of integrated HPV16 DNA show a statistically significant (p<0.05) preferential distribution within the early region segment upstream of the major splice acceptor underscoring the importance of deregulated viral oncogene expression for carcinogenesis. Half of the mapped HPV16 integration sites target cellular genes pointing to a direct influence of HPV integration on host genes (insertional mutagenesis). In summary, the multiplex strategy for HPV16 integration site determination worked very efficiently. It will open new avenues for comprehensive mapping of HPV integration sites and for the possible use of HPV integration sites as individualized biomarkers after cancer treatment of patients for the early diagnosis of residual and recurrent disease.

Preferential sites for the integration and disruption of human papillomavirus 16 in cervical lesions

BackgroundPersistent infection with high-risk human papillomavirus (HPV) is necessary to cause cervical cancer, and integrating viral DNA into the host genome may contribute to the process of carcinogenesis. The underlying mechanisms are still unclear. ObjectiveIn this study, we aimed to investigate the distribution of HPV 16 integration in the host genome and disrupted sites in the viral genome. Study designThe physical status of HPV 16 genomes in 46 cervical precancerous and cancerous lesions was determined via ligation-mediated chain reaction (DIPS) using 15 previously published primer sets and 12 newly designed primer sets. ResultsA total of 60 viral-cellular junctions were identified in 31 of 46 specimens, and over 80% of the integration sites in the human genome were located in regions of repetitive elements. The proportion of LSIL-, HSIL-, and SCC-containing integration sites near cancer-relevant genes was 10%, 18.8%, and 33.3%, respectively. The frequency of viral gene disruption was significantly higher (P<0.05) in the L2 gene than in other regions of the viral genome. ConclusionThere are sites of preferential HPV 16 integration. The integration sites tend to be located in repetitive regions of the host genome, and some sites are found near cancer-relevant genes. In addition, the HPV 16 genome is more likely to be disrupted in the L2 gene locus.

Determination of Integrated HPV58 Sequences in Cervical Lesions

Integration of high-risk HPV in host genome is an important event in cervical cancer development. This study was aimed to analyze the integration of HPV58, a high-risk type that is prevalent in cervical lesions from southeastern Asia. Detection of integrated papillomavirus sequences by ligation-mediated polymerase chain reaction followed by DNA sequencing revealed 8 integrations in 5 samples, and virus integration was found present in 2 samples with early lesion. Sequence analysis of the viral-cellular junctions showed that E1 disruption in virus genome was an early and common event during HPV58 infection. In 6 integrations, DNA fragments of HPV58 genome integrated into the repetitive element sequences of host genome.

Identification of hepatitis B virus integration sites in hepatocellular carcinoma tissues from patients with chronic hepatitis B

Hepatitis B virus (HBV) integration into the host genome is frequently detected in HBV positive hepatocellular carcinoma (HCC) in China. The aim of this study is to carry out a large-scale screening for the HBV integrations sites in HCC samples from Chinese patients. Cellular DNA was extracted from 40 HBV-related HCC by proteinase K digestion/phenol extraction method. One primer specific to HBV sequence and another primer directed to human Alu repeat were used to amplify the virus/cellular DNA junction. To avoid undesirable amplifications between Alu sequences, primers were constructed with dUTPs and destroyed by uracil DNA glycosylase treatment after 15 initial cycles of amplification. Only desirable fragments were then further amplified with specific primers to the known region and to a tag sequence introduced in the Alu-Specific primer. The PCR product was purified and subject to direct sequencing by ABI 3700 Auto sequencer. NCBI (national center for biotechnology information) BLAST and MapViewer search were used for identification of HBV location on human genomes. In 40 HBsAg positive HCC samples, 34 (85%) were showed to have at least one copy of HBV fragment in host genome, indicating HBV-Alu-PCR is a rapid way for identification of new cellular DNA sequences adjacent to HBV. Analysis from the 68 isolated viral-cellular junctions, X gene was found to be interrupted at any length, not specifically at DR1 and DR2 regions. Three-prime-deleted X gene was observed in 65 (96%) cases. HBV preferred to integrate into the intron and the up-stream regulatory region of the cellular genes. In no case HBV inserted into the exon. Our results also demonstrated that the cellular genes targeted by HBV are usually key regulators of cell proliferation and cell death. Three genes, myeloid/lymphoid or mixed-lineage leukemia 4, G protein alpha transducing activity polypeptide 1 and fibronectin, were found to be recurrently targeted by HBV. HBV-Alu-PCR is a powerful tool for the study of HBV integration sites. Truncated X is a major form existed in the HBV integrants. HBV integration is not distributed evenly throughout the host genome and viral insertional mutagenesis may play an important role in the development of HCC.

Characterization of Adeno-Associated Virus Genomes Isolated from Human Tissues

Infection with wild-type adeno-associated virus (AAV) is common in humans, but very little is known about the in vivo biology of AAV. On a molecular level, it has been shown in cultured cells that AAV integrates in a site-specific manner on human chromosome 19, but this has never been demonstrated directly in infected human tissues. To that end, we tested 175 tissue samples for the presence of AAV DNA, and when present, examined the specific form of the viral DNA. AAV was detected in 7 of 101 tonsil-adenoid samples and in 2 of 74 other tissue samples (spleen and lung). In these nine samples, we were unable to detect AAV integration in the AAVS1 locus using a sensitive PCR assay designed to amplify specific viral-cellular DNA junctions. Additionally, we used a second complementary assay, linear amplification-mediated-PCR (LAM-PCR) to widen our search for integration events. Analysis of individual LAM-PCR products revealed that the AAV genomes were arranged predominantly in a head-to-tail array, with deletions and extensive rearrangements in the inverted terminal repeat sequences. A single AAV-cellular junction was identified from a tonsil sample and it mapped to a highly repetitive satellite DNA element on chromosome 1. Given these data, we entertained the possibility that instead of integrated forms, AAV genomes were present as extrachromosomal forms. We used a novel amplification assay (linear rolling-circle amplification) to show that the majority of wild-type AAV DNA existed as circular double-stranded episomes in our tissues. Thus, following naturally acquired infection, AAV DNA can persist mainly as circular episomes in human tissues. These findings are consistent with the circular episomal forms of recombinant AAV vectors that have been isolated and characterized from in vivo transduced tissues.

Mapping and analysis of HPV16 integration sites in a head and neck cancer cell line.

Human papillomavirus (HPV) is a circular double-stranded DNA virus implicated in at least 90% of cervical and anogenital cancers and has been observed in approximately 20% of squamous cell carcinomas of the head and neck (SCCHN). Transcription of the viral oncogenes E6 and E7 is regulated by expression of the E2 protein. Disruption of the E2 gene sequence due to viral integration results in upregulation of E6 and E7, which promote tumorigenesis by abrogating p53 and pRb functions. HPV integration sites in cervical and anogenital cancers have been mapped primarily to chromosomal fragile sites and in some cases have been shown to integrate within tumor suppressor genes or other cancer-related genes. To study viral integration sites in SCCHN, we examined an HPV16-infected SCCHN cell line cultured from a tongue-base tumor. HPV fluorescence in situ hybridization (FISH) revealed multiple integrated viral DNA copies in blocks throughout the genome. Sequential FISH and spectral karyotyping identified integration sites on chromosomes 3, 6, 9q, 13q and t(1;8)(q;?). Restriction site-polymerase chain reaction (RS-PCR) was performed to identify the viral-cellular junctions. Sequence analyses confirmed integration sites at 9q31.1 and 6p21 and revealed a novel junction at 16p12.3. Subsequent chromosome breakage studies suggested that the observed viral-cellular integration sites may have occurred within common fragile sites. Additional studies using RT-PCR for E6--E7 viral transcripts showed oncoprotein expression from episomal and integrated viral sequences. Our results suggest that viral integration of HPV in SCCHN appears to occur nonrandomly through targeting specific chromosomal sequences prone to breakage.

Detection of integrated papillomavirus sequences by ligation‐mediated PCR (DIPS‐PCR) and molecular characterization in cervical cancer cells

Human papillomavirus (HPV) genomes usually persist as episomal molecules in HPV associated preneoplastic lesions whereas they are frequently integrated into the host cell genome in HPV-related cancers cells. This suggests that malignant conversion of HPV-infected epithelia is linked to recombination of cellular and viral sequences. Due to technical limitations, precise sequence information on viral–cellular junctions were obtained only for few cell lines and primary lesions. In order to facilitate the molecular analysis of genomic HPV integration, we established a ligation-mediated PCR assay for the detection of integrated papillomavirus sequences (DIPS-PCR). DIPS-PCR was initially used to amplify genomic viral–cellular junctions from HPV-associated cervical cancer cell lines (C4-I, C4-II, SW756, and HeLa) and HPV-immortalized keratinocyte lines (HPKIA, HPKII). In addition to junctions already reported in public data bases, various new fusion fragments were identified. Subsequently, 22 different viral–cellular junctions were amplified from 17 cervical carcinomas and 1 vulval intraepithelial neoplasia (VIN III). Sequence analysis of each junction revealed that the viral E1 open reading frame (ORF) was fused to cellular sequences in 20 of 22 (91%) cases. Chromosomal integration loci mapped to chromosomes 1 (2n), 2 (3n), 7 (2n), 8 (3n), 10 (1n), 14 (5n), 16 (1n), 17 (2n), and mitochondrial DNA (1n), suggesting random distribution of chromosomal integration sites. Precise sequence information obtained by DIPS-PCR was further used to monitor the monoclonal origin of 4 cervical cancers, 1 case of recurrent premalignant lesions and 1 lymph node metastasis. Therefore, DIPS-PCR might allow efficient therapy control and prediction of relapse in patients with HPV-associated anogenital cancers. © 2001 Wiley-Liss, Inc.

Detection of integrated papillomavirus sequences by ligation-mediated PCR (DIPS-PCR) and molecular characterization in cervical cancer cells

Human papillomavirus (HPV) genomes usually persist as episomal molecules in HPV associated preneoplastic lesions whereas they are frequently integrated into the host cell genome in HPV-related cancers cells. This suggests that malignant conversion of HPV-infected epithelia is linked to recombination of cellular and viral sequences. Due to technical limitations, precise sequence information on viral-cellular junctions were obtained only for few cell lines and primary lesions. In order to facilitate the molecular analysis of genomic HPV integration, we established a ligation-mediated PCR assay for the detection of integrated papillomavirus sequences (DIPS-PCR). DIPS-PCR was initially used to amplify genomic viral-cellular junctions from HPV-associated cervical cancer cell lines (C4-I, C4-II, SW756, and HeLa) and HPV-immortalized keratinocyte lines (HPKIA, HPKII). In addition to junctions already reported in public data bases, various new fusion fragments were identified. Subsequently, 22 different viral-cellular junctions were amplified from 17 cervical carcinomas and 1 vulval intraepithelial neoplasia (VIN III). Sequence analysis of each junction revealed that the viral E1 open reading frame (ORF) was fused to cellular sequences in 20 of 22 (91%) cases. Chromosomal integration loci mapped to chromosomes 1 (2n), 2 (3n), 7 (2n), 8 (3n), 10 (1n), 14 (5n), 16 (1n), 17 (2n), and mitochondrial DNA (1n), suggesting random distribution of chromosomal integration sites. Precise sequence information obtained by DIPS-PCR was further used to monitor the monoclonal origin of 4 cervical cancers, 1 case of recurrent premalignant lesions and 1 lymph node metastasis. Therefore, DIPS-PCR might allow efficient therapy control and prediction of relapse in patients with HPV-associated anogenital cancers.

Analysis of integrated hepatitis B virus DNA and flanking cellular sequence by inverse polymerase chain reaction

Although hepatitis B virus (HBV) DNA has been detected in the human hepatoma cell line, HAGS 2.1, viral and cellular junction sequences have not been investigated fully. To facilitate the analysis of HBV DNA integration sites in HAGS 2.1 cells, a combination of conventional polymerase chain reaction (PCR) and inverse PCR (IPCR) was carried out to identify the junction between the viral and the cellular gene. The HBV integrant and its cellular counterpart sequence were cloned and analyzed. The sequencing data indicated that the breakpoints on the HBV integrant are at nucleotide 2111 of the C gene and nucleotide 1558 of the X gene. The length of the integrated HBV DNA in HAGS 2.1 was approximately 2.6 kb, which includes partial C, P, and X genes and an intact S gene. The cellular sequence flanking the integrated HBV gene was very similar to a human satellite III repetitive sequence with 43 and 56 of GGAAT repeats on the left- and right-hand side, respectively. Although the findings on the viral–cellular junction in HAGS 2.1 cells cannot explain the liver tumorigenesis, the current study shows that by choosing the nearest restriction site, which can be determined by conventional PCR rather than using a unique site within the integrated viral sequence to do IPCR, gives a higher successful rate for cloning and the subsequent analysis of the viral–cellular junctions.

Down‐regulation of integrated Epstein‐Barr virus nuclear antigen 1 and 2 genes in a burkitt lymphoma cell line after lymphoblastoid cells somatic cell fusion with autologous EBV‐immortalized

Epstein Barr virus (EBV) latent gene expression was analyzed in somatic cell hybrids between an EBV-positive Burkitt lymphoma (BL) cell line (BL 60) and an autologous EBV-immortalized lymphoblastoid cell line (LCL, IARC 277). The EBV genomes carried by the parental cell lines differ in sequence and in their physical state. The BL 60 EBV genome is integrated into the host cell genome whereas the LCL IARC 277 carries exclusively episomal EBV molecules. The hybrid cells contain both EBV genomes and display the differentiation phenotype of the parental LCL with regard to growth characteristics and cell-surface antigen expression in vitro and in vivo. While the EBNA 1 and EBNA 2 gene expression of the LCL-derived EBV is maintained in these hybrid cells, the BL-60-derived EBNA 1 and EBNA 2 genes are transcriptionally down-regulated. Mapping of the genomic region surrounding the latent Cp promoter of the BL-60-derived EBV revealed an extensive deletion upstream of the Cp promoter including the enhancer element in the ori P region, the origin of latent viral replication (ori P), the coding sequences for the EBV latent membrane protein (LMP) and the EBV terminal protein (TP), and suggested that one viral-cellular junction sequence is located near the Cp promoter. Integration of EBV into the host cell genome together with the extensive deletion might be causally related with the altered latent gene expression pattern after introduction of a lymphoblastoid host-cell background by somatic cell fusion. Down-regulation of the BL-60-derived EBNA genes could be due to loss of regulatory sequences in the BL-derived EBV necessary for EBNA 1 and EBNA 2 transcription in the lymphoblastoid hybrid cells, but not in the parental BL cells.

A stable flank of unstable lymphotropic papovavirus integration sites is associated with a cellular S1 nuclease-sensitive sequence

Integration of viral DNA sequences is associated with stable transformation by viruses of the polyomavirus genus. In LPV-HE cells, a hamster embryonal cell line transformed by the African green monkey lymphotropic papovavirus (LPV), viral integration is unstable during tissue culture passage, leading to subclones with rearrangements of the original integration locus. Three of four viral integrations that were molecularly cloned together with flanking cellular sequences are identical in one junction but are different from each other at the other flank. The "stable" flank which is already present in early passage cells contains an S1 nuclease-sensitive sequence located approximately 60 bp outside of the viral-cellular junction. The presence of this site may contribute to the stability of the flanking region.

Human papillomavirus type 16 transcripts expressed from viral-cellular junctions and full-length viral copies in CaSki cells and in a cervical carcinoma

We have mapped using the RNA PCR the viral-cellular junctions of HPV16 viral-cellular cotranscripts expressed in CaSki cells and a cervical carcinoma to nt 3728 and 881, respectively. Both junctions were located within the E1–E2 region. Examination of the cellular sequences of the cotranscripts showed the presence of a polyadenylation signal in each of the transcripts. In CaSki cells and in the cervical carcinoma transcripts derived from the full-length early region including the E2 transcript were also detected. Our results suggest that the utilization of a cellular polyadenylation site could be important in the development of cancer by HPV.

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.

Different type of hepatitis B virus (HBV) DNA integrants that may reflect the integration process

Through analyses of HBV DNA integratns in human cellular DNA, we identified three different integrant types, each of which may reflect the process of primary integrant formation by the viral DNA. The first type, which we call "simple type" consists of integrants found in some hepatocellular carcinomas (HCC's). The structure of the viral genome is simple, and part of it is deleted. The viral cohesive end sequence appears at one of the viral-cellular DNA junctions, and integration has elicited a microdeletion in the target cellular DNA sequence. This structure suggests viral DNA replication intermediates as substrates for integration. Judging from its frequency in HCC, this type may represent the most preferred one, if not all, among the primary integration products. The second type, which we call "complex type" is essentially the same as the first type, except tht the viral genome structure is complex. We considered the possibility that they may have been produced via the same process, using preformed complex viral genomes such as "novel form DNA's" (Rogler and Summers, 1982) as substrates. In cultured fetal hepatocytes, integration of HBV DNA can occur only a few days after infection. Among such integrants, we found a third type integrant, having a simple viral genome, but having a larger cellular DNA deletion. We propose that different forms of viral DNA may be used as substrates in the integration process, and the process is characterized by its eliciting of deletions of different size in the target cellular DNA. The most preferred substrate may be the one producing the simple type integrants, and the most frequently occurring deletion in the target DNA may be the microdeletion.(ABSTRACT TRUNCATED AT 250 WORDS)

A viral-cellular junction fragment from a human papillomavirus type 16-positive tumor is competent in transformation of NIH 3T3 cells.

A 4.4-kilobase DNA fragment (T4.4) from a human tumor (comprising part of the human papillomavirus type 16 E6 promoter; the E6, E7, and part of the E1 open reading frames; and cellular sequences) was found to be competent to fully transform NIH 3T3 cells. This competency resides in the whole hybrid DNA fragment, since the separate viral or cellular DNA sequences were not active. Abundant E6-E7 transcripts were found in the transformed cells. When the cellular fragments were substituted with polyadenylation sequences from polyomavirus or simian virus 40 DNA, little or no restoration of transforming activity was observed. In experiments in which an exogenous reporting gene, that for chloramphenicol acetyltransferase, was used, the possibility was excluded that the cellular flanking sequences act as a traditional enhancer; yet, when the cellular sequences were placed downstream of a chloramphenicol acetyltransferase expression vector (pSV2 CAT), activity of the reference gene was clearly enhanced. These results indicate that DNA containing human papillomavirus type 16 open reading frames E6 and E7 isolated from the genome of a human tumor has transforming potential, that this potential is realized when the viral DNA is joined to cellular sequences, and that the cellular sequences function in a more complex way than by simply providing polyadenylation signals.

‘Illegitimate’ recombination events in polyoma-transformed rat cells

In the LPT line of polyoma (Py)-transformed rat cells, amplification of the integrated viral DNA and of cell nucleotide sequences flanking the viral integration site, can be induced either spontaneously or by treatment with carcinogens. We show here that the amplified DNA includes interspersed viral and cellular sequences generated by ‘illegitimate’ recombination events. Genomic libraries have been prepared in phage λ vectors from LPT cells treated with the inducing agent mitomycin C and from untreated LPT cells. Four phages, including viral-cell DNA recombinants, have been isolated from these libraries. Sequencing through the recombination sites revealed the following characteristics: (i) The crossover points map at four different positions in the viral DNA and at four different positions in the flanking cell DNA. (ii) There are very short homologous sequences of 1, 2, or 4 bp, at the recombination sites, (iii) Aside from the exchanges between the viral and the cellular DNA, no further rearrangements occurred around the new viral-cellular DNA junctions, (iv) Next to the recombination sites, there are blocks of homopurine-homopyrimidine sequences, which may assume a structure that differs from the Watson-Crick double helix, (v) Clustered homologous sequence blocks of up to 10 bp are present less than 200 bp away from the recombination sites. These homologies are not in register. Based on these results, we propose a model that may account for these recombination events and, more generally, for recombination events that occur during gene amplification in mammalian cells.

Structure of viral DNA in a rat cell line, GY1, transformed by Ad12 HindIII fragment-G

The cell line GY1, established by transformation of a rat cell line 3Y1 with the Ad12 HindIII fragment-G (leftmost 6.8%, nucleotide 1 to 2322), contains more than 100 viral copies per haploid genome. The viral DNAs in this cell line were cloned into a phage vector, λgt WESλB, and recloned into pBR322 with their flanking cellular DNAs. Independently isolated 39 clones were analyzed by restriction enzyme cleavage and Southern blot hybridization experiment and divided into 11 classes. Some of classes contained multiple identical clones, at maximum 16 clones. It may be interpreted that amplification of some of the recombined sequences had occurred after the multiple integrations of transfected DNAs within cells. Using five clones from different classes the sequences of recombination sites were determined. Viral DNAs deleted with varying degrees at both ends were flanked by quite different cellular sequences in different clones and no common sequences were revealed around viral-cellular junctions. Tandemly repeated viral DNAS were found in one of the clones to be integrated in a head to tail manner into cellular DNA. The linkage of these two viral DNAs had occurred at the site where parental viral DNAs shared 2 bp. Palindrome structures could be constructed around viral-cellular and viral-viral junction sites and around the regions of parental viral DNAs corresponding to the junction sites in all of the cases investigated.