Viral-cellular Fusion Transcripts Research Articles

Differences in Stability of Viral and Viral-Cellular Fusion Transcripts in HPV-Induced Cervical Cancers.

HPV-DNA integration results in dysregulation of viral oncogene expression. Because viral-cellular fusion transcripts inherently lack the viral AU-rich elements of the 3’UTR, they are considered to be more stable than episome-derived transcripts. The aim of this study is to provide formal proof for this assumption by comparing the stability of viral early transcripts derived from episomal and integrated HPV16 DNA, respectively. Full-length cDNA of three fusion transcripts comprising viral and cellular sequences in sense orientation were amplified and cloned into the adeno-viral-vector pAd/CMV/V5-DEST. The most abundant HPV16 oncogene transcript E6*I-E7-E1vE4-E5 with and without 3’UTR, served as reference and control, respectively. Human primary keratinocytes were transduced using high titer virus stocks. qRT-PCR was performed to determine mRNA stability in relation to GAPDH in the presence of actinomycin-D. In four independent transduction experiments, all three viral-cellular fusion transcripts were significantly more stable compared to the episome-derived reference. Among the three viral-cellular fusion transcripts the most stable transcript was devoid of the instability core motif “AUUUA”. Unexpectedly, there was no significant difference in the stability between the episome-derived transcripts either with or without 3’UTR, indicating that the AU-rich elements of the 3’UTR are not contributing to RNA stability. Instead, the three “AUUUA” motifs located in the untranslated region between the viral E4 and E5 genes may be responsible for the instability. This is the first report showing that authentic viral-cellular fusion transcripts are more stable than episome-derived transcripts. The longer half-life of the fusion transcripts may result in increased levels of viral oncoproteins and thereby drive the carcinogenic process.

Transcriptionally active regions are the preferred targets for chromosomal HPV integration in cervical carcinogenesis.

Integration of human papillomavirus (HPV) into the host genome is regarded as a determining event in cervical carcinogenesis. However, the exact mechanism for integration, and the role of integration in stimulating cancer progression, is not fully characterized. Although integration sites are reported to appear randomly distributed over all chromosomes, fragile sites, translocation break points and transcriptionally active regions have all been suggested as being preferred sites for integration. In addition, more recent studies have reported integration events occurring within or surrounding essential cancer-related genes, raising the question whether these may reflect key events in the molecular genesis of HPV induced carcinomas. In a search for possible common denominators of the integration sites, we utilized the chromosomal coordinates of 121 viral-cellular fusion transcripts, and examined for statistical overrepresentation of integration sites with various features of ENCODE chromatin information data, using the Genomic HyperBrowser. We find that integration sites coincide with DNA that is transcriptionally active in mucosal epithelium, as judged by the relationship of integration sites to DNase hypersensitivity and H3K4me3 methylation data. Finding an association between integration and transcription is highly informative with regard to the spatio-temporal characteristics of the integration process. These results suggest that integration is an early event in carcinogenesis, more than a late product of chromosomal instability. If the viral integrations were more likely to occur in destabilized regions of the DNA, a completely random distribution of the integration sites would be expected. As a by-product of integration in actively transcribing DNA, a tendency of integration in or close to genes is likely to be observed. This increases the possibility of viral signals to modulate the expression of these genes, potentially contributing to the progression towards cancer.

Non-Random Integration of the HPV Genome in Cervical Cancer

HPV DNA integration into the host genome is a characteristic but not an exclusive step during cervical carcinogenesis. It is still a matter of debate whether viral integration contributes to the transformation process beyond ensuring the constitutive expression of the viral oncogenes. There is mounting evidence for a non-random distribution of integration loci and the direct involvement of cellular cancer-related genes. In this study we addressed this topic by extending the existing data set by an additional 47 HPV16 and HPV18 positive cervical carcinoma. We provide supportive evidence for previously defined integration hotspots and have revealed another cluster of integration sites within the cytogenetic band 3q28. Moreover, in the vicinity of these hotspots numerous microRNAs (miRNAs) are located and may be influenced by the integrated HPV DNA. By compiling our data and published reports 9 genes could be identified which were affected by HPV integration at least twice in independent tumors. In some tumors the viral-cellular fusion transcripts were even identical with respect to the viral donor and cellular acceptor sites used. However, the exact integration sites are likely to differ since none of the integration sites analysed thus far have shown more than a few nucleotides of homology between viral and host sequences. Therefore, DNA recombination involving large stretches of homology at the integration site can be ruled out. It is however intriguing that by sequence alignment several regions of the HPV16 genome were found to have highly homologous stretches of up to 50 nucleotides to the aforementioned genes and the integration hotspots. One common region of homologies with cellular sequences is between the viral gene E5 and L2 (nucleotides positions 4100 to 4240). We speculate that this and other regions of homology are involved in the integration process. Our observations suggest that targeted disruption, possibly also of critical cellular genes, by HPV integration remains an issue to be fully resolved.

350. Vector Design-Dependent Modulation of Viral-Cellular Chimeric Fusion Transcripts Derived from Self-Inactivating (SIN) Lentiviral Vectors

350. Vector Design-Dependent Modulation of Viral-Cellular Chimeric Fusion Transcripts Derived from Self-Inactivating (SIN) Lentiviral Vectors

Loss of gene function as a consequence of human papillomavirus DNA integration

Integration of the human papillomavirus (HPV) genome into the host chromatin is a characteristic step in cervical carcinogenesis. Integration ensures constitutive expression of the viral oncogenes E6 and E7 which drive carcinogenesis. However, integration has also an impact on host DNA. There is increasing evidence that integration not only occurs in fragile sites and translocation breakpoints but also in transcriptionally active regions. Indeed, a substantial number of integration sites actually disrupt host genes and may thereby affect gene expression. No doubt, even subtle changes in gene expression may influence the cell phenotype but small fold changes are difficult to quantify reliably in biopsy material. We have, therefore, addressed the question whether a complete loss of gene function that is insertional mutagenesis in combination with deletion or epigenetic modification of the second allele is also a phenomenon pertinent to cervical cancer. Out of the ten preselected squamous cell carcinomas analyzed, all viral integration sites were located within the intron sequences of known genes, giving rise to viral-cellular fusion transcripts of sense orientation. Moreover, for two tumors, we provide evidence for complete functional loss of the gene affected by HPV integration. Of particular note is that one of the genes involved is the recently described novel tumor suppressor gene castor zinc finger 1. Although our study provides no functional proof that any of the genes affected by HPV integration are causally involved in the transformation process, an exhaustive systematic look at the role of insertional mutagenesis in cervical cancer appears to be warranted.

Risk assessment in skin gene therapy: viral–cellular fusion transcripts generated by proviral transcriptional read-through in keratinocytes transduced with self-inactivating lentiviral vectors

Cutaneous gene therapy can be envisioned through the use of keratinocyte stem cell clones in which retroviral genotoxic risks can be pre-assessed. While transactivation of cellular genes by the retroviral long terminal repeat enhancer has been proven in experimental and clinical settings, the formation of chimeric viral-cellular transcripts originated by the inefficient termination (read-through) of retroviral transcripts remains to be studied in depth. We now demonstrate the widespread presence of viral-cellular fusion transcripts derived from integrated proviruses in keratinocytes transduced with self-inactivating (SIN) retroviral vectors. We have detected high molecular weight RNAs in northern blot analysis of retroviral vector expression in individual cell clones. Characterization of some of these transcripts revealed that they originate from genes located at the proviral integration sites. One class of transcripts corresponds to fusions of the viral vectors with intronic sequences, terminating at cryptic polyadenylation sites located in introns. A second class comprises fusion transcripts with coding sequences of genes at the integration sites. These are generated through splicing from a cryptic, not previously described donor site in the lentiviral vectors to exons of cellular genes, and have the potential to encode unintended open reading frames, although they are downregulated by cellular mechanisms. Our data contribute to a better understanding of the impact of SIN lentiviral vector integration on cellular gene transcription, and will be helpful in improving the design of this type of vectors.

351. Clonal Analysis Allows Characterization and Risk Assessment of Viral-Cellular Fusion Transcripts Generated by Transcriptional Termination Read-Through in Keratinocytes Transduced with Self-Inactivating Retroviral Vectors

351. Clonal Analysis Allows Characterization and Risk Assessment of Viral-Cellular Fusion Transcripts Generated by Transcriptional Termination Read-Through in Keratinocytes Transduced with Self-Inactivating Retroviral Vectors

The Majority of Viral-Cellular Fusion Transcripts in Cervical Carcinomas Cotranscribe Cellular Sequences of Known or Predicted Genes

Integration of human papillomavirus (HPV) DNA into the host genome is a frequent event in cervical carcinogenesis and is reported to occur at randomly selected chromosomal sites. However, as the databases are being up-dated continuously, the knowledge based on sequenced viral integration sites also expands. In this study, viral-cellular fusion transcripts of a preselected group of 74 cervical carcinoma or cervical intraepithelial neoplasia grade 3 (CIN3) biopsies harboring integrated HPV16, HPV18, HPV31, HPV33, or HPV45 DNA were amplified by 3'-rapid amplification of cDNA ends PCR and sequenced. Consistent with previous reports, integration sites were found to be distributed throughout the genome. However, 23% (17 of 74) of the integration sites were located within the cytogenetic bands 4q13.3, 8q24.21, 13q22.1, and 17q21, in clusters ranging from 86 to 900 kb. Of note is that clusters 8q24.21 and 13q22.1 are within 1.5 Mbp of an adjacent fragile site whereas clusters 4q13.3 and 17q21 are >15 Mbp distant to any known fragile sites. It is tempting to speculate that as yet unknown fragile sites may be identified on the basis of HPV integration hotspots. No correlation between HPV type and specific integration loci was found. Of 74 fusion transcripts, 28 contained cellular sequences, which were homologous to known genes, and 40 samples contained sequences of predicted genes. In 33 fusion transcripts, both viral and cellular sequences were in sense orientation, indicating that the gene itself or upstream sequences were affected by integration. These data suggest that the influence of HPV integration on host gene expression may not be a rare effect and should encourage more detailed analyses.

Integration of HPV-16 and HPV-18 DNA in vulvar intraepithelial neoplasia

Objective. Vulvar intraepithelial neoplasia (VIN) is a premalignant disease of the lower genital tract. The increased occurrence of high-risk human papillomavirus (HPV) infection seems to be associated with the increasing frequency of VIN. Integration of HPV DNA into host chromosome has been hypothesized to be a critical step in the carcinogenesis of cervical neoplasia resulting in altered expression of two viral transforming genes E6 and E7. Method. We analyzed HPV-16 and HPV-18 DNA, and integrated transcripts of HPV-16 and HPV-18 genomes in 30 VIN cases with 53 lesions using a PCR-based protocol for the amplification of papillomavirus oncogene transcripts (APOT). Result. 24 of 30 VIN lesions (80%) harbored HPV-16 (in 23 cases) and HPV-18 DNA. Integration of HPV-16 and HPV-18 genome was observed in eight (38.1%) of 21 HPV-16/18 positive VIN III cases. All eight VIN were multifocal and had multicentric disease (CIN/VAIN) including one case that progressed to vulvar carcinoma. Five of eight lesions were found to have E7 specific viral–cellular fusion transcripts only, two of eight showed E7–E4 viral–cellular fusion transcripts, and one of eight had both episomally derived and E7–E4 viral–cellular fusion transcripts. In 10 (83.3%) of 12 multifocal VIN III patients, all specimens derived from the same patient harbored the same HPV type and HPV transcript pattern suggesting monoclonality. Conclusion. HPV-16 is the most prevalent type among VIN II/III. HPV-16 and HPV-18 DNA integration into host cell genome seems to be related to the progression stage of vulvar dysplasia and, therefore, may be necessary for development of HPV-associated invasive vulvar carcinoma.

Hypothetical Genes Are Frequent Targets of HPV DNA Integration in Cervical Carcinomas

Objective: Integration of HPV DNA into the host genome is a frequent event in cervical carcinogenesis. Viral integration sites seem to be randomly distributed among all chromosomes with a clear preference for genomic fragile sites. To date about 200 HPV DNA integration sites have been mapped. However, this number is still too small to fully comprehend the different mechanisms by which HPV DNA integration contributes to carcinogenesis. Methods: 55 cervical carcinoma samples of FIGO stage IA to IIIB positive for either HPV16 (n=34) or HPV18 (n=21) were analysed. Viral-cellular fusion transcripts were detected using the APOT assay (amplification of papillomavirus oncogene transcripts) introduced by Klaes and colleagues (Klaes et al., 1999). All putative viral-cellular fusion transcripts were analysed by sequencing. The genomic localisation of the integration sites were determined by database query of all available public databases. Results: The detection rate of viral-cellular fusion transcripts from HPV16 positive tumours was significantly lower (p=0.012, two-sided t-test) than that of HPV18 positive tumours (53% versus 81%, respectively). A BLASTN comparison of the fusion transcripts with the NCBI Database showed homology in 16 out of 35 cases, with 10 fusion transcripts containing known genes and 6 transcripts containing parts of Expressed Sequence Tags (EST). 16 integration sites were mapped within hypothetical genes listed in the UCSC Genome Browser Database. Conclusion: One striking finding of this study is the high proportion of viral integration sites within in silico predicted genes (46%). In contrast, only 29% of integration sites were located within known genes. This not only suggests that virus integration occurs within transcriptionally active chromosomal sites, but also that the APOT assay may be a useful tool for the detection of hypothetical genes which have not been analysed in the context of carcinogenesis thus far.

DNA aneuploidy and integration of human papillomavirus type 16 e6/e7 oncogenes in intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix uteri.

Increasingly deregulated expression of the E6-E7 oncogenes of high-risk human papillomaviruses (HR-HPVs) has been identified as the major transforming factor in the pathogenesis of cervical dysplasia and derived cancers. The expression of these genes in epithelial stem cells first results in chromosomal instability and induces chromosomal aneuploidy. It is speculated that this subsequently favors integration of HR-HPV genomes into cellular chromosomes. This in turn leads to expression of viral cellular fusion transcripts and further enhanced expression of the E6-E7 oncoproteins. Chromosomal instability and aneuploidization thus seems to precede and favor integration of HR-HPV genomes. To prove this sequential concept, we analyzed here the sequence of events of DNA aneuploidization and integration in a series of HPV-16-positive cervical dysplastic lesions and carcinomas. Eighty-five punch biopsies of HPV-16-positive cervical lesions (20 CIN1/2, 50 CIN3, and 15 CxCa) were analyzed for DNA ploidy by DNA flow cytometry and for integration of HPV E6/E7 oncogenes using the amplification of papillomavirus oncogene transcripts assay, a reverse transcription-PCR method to detect integrate-derived human papillomavirus oncogene transcripts. DNA aneuploidy and viral genome integration were both associated with increasing dysplasia (P < 0.001, chi(2) test for trend). In addition, DNA aneuploidy was associated with increased viral integration (P < 0.01, Fisher's exact test). Nineteen of 20 (95%) lesions with integrated viral genomes had aneuploid cell lines; however, only 19 of 32 (59%) lesions with aneuploid cell lines had integrated viral genomes. These data support the hypothesis that aneuploidization precedes integration of HR-HPV genomes in the progression of cervical dysplasia. Accordingly, deregulated viral oncogene expression appears to result first in chromosomal instability and aneuploidization and is subsequently followed by integration of HR-HPV genomes in the affected cell clones.

Involvement of intact HPV16 E6/E7 gene expression in head and neck cancers with unaltered p53 status and perturbed pRb cell cycle control.

We have identified parameters which define a causal role of HPV16 in head and neck cancer. Twenty-eight tumours which were typed positive for HPV16 DNA, were comprehensively analysed for expression of the viral oncogenes E6 and E7, the status of the p53 gene, and the protein status of pRb and p16(INK4a). In a subset of cases, we have searched for integrated viral DNA, and have determined the genomic status of the E6 gene. Expression of E6/E7 was found in 12 tumours most of which were derived from the oropharynx, whereas p53 mutations were present in 13 tumours from various sites. The tumours either carried p53 mutations but did not express E6/E7, or they did express E6/E7 but were p53-wild-type. Coexistence of E6/E7 expression with a mutated p53 was found in only one case. Strikingly, in most p53-mutated tumours without E6/E7 expression, we found the E6 gene to be disrupted. E6/E7 expression was associated with reduced pRb and overexpressed p16(INK4a). Viral-cellular fusion transcripts were found in two cases. Our data demonstrate that HPV16 DNA-positivity in head and neck cancers is not indicative of a causal role. A causal role of HPV16 in head and neck cancer is defined by: E6/E7 expression, viral integration with an intact E6 gene, and perturbation of pRb cell cycle control. Mostly, the p53 gene is wild-type.

Characterization of viral-cellular fusion transcripts in a large series of HPV16 and 18 positive anogenital lesions.

Persistent high risk type human papillomavirus (HR-HPVs) infections induce dysplasia or cancer of the anogenital tract, most notably of the uterine cervix. The viral genome usually persists and replicates as an episomal molecule in early dysplasia, whereas in advanced dysplasia or cervical cancer HPV genomes are frequently integrated into the chromosomal DNA of the host cell. Previous studies suggested that modification of critical cellular sequences by integration of HPV genomes might significantly contribute to the neoplastic transformation of anogenital epithelia (insertional mutagenesis). This prompted us to characterize the integration loci of high risk HPV genomes in a large set of genital lesions. We amplified E6/E7 oncogene transcripts derived from integrated HPV16 and HPV18 genomes and characterized in detail the co-transcribed cellular sequences of 64 primary genital lesions and five cervical cancer cell lines. Database analyses of the cellular parts of these fusion transcripts revealed 51 different integration loci, including 26 transcribed genes (14 known genes, 12 EST sequences with unknown gene function). Seventeen sequences showed similarity to repetitive elements, and 26 sequences did not show any database match other than genomic sequence. Chromosomal integration loci were distributed over almost all human chromosomes. Although we found HPV sequences integrated into cancer related genes and close to fragile sites, no preferential site or integration motif could be identified. These data demonstrate that target directed insertional mutagenesis might occur in few HPV-induced anogenital lesions, however, it is rather the exception than the rule.

APM-1, a novel human gene, identified by aberrant co-transcription with papillomavirus oncogenes in a cervical carcinoma cell line, encodes a BTB/POZ-zinc finger protein with growth inhibitory activity.

Integration of human papillomavirus (HPV) DNA into the host cell genome is an important step in cervical carcinogenesis. In tumour cells with integrated HPV DNA, transcription of viral oncogenes E6 and E7 continues into the flanking cellular sequences thereby producing viral-cellular fusion transcripts. Analysis of cellular sequences flanking the integrated HPV68 DNA in the cervical carcinoma cell line ME180 revealed homozygosity of the mutant allele in ME180 cells. We speculated that this could indicate the existence of a cellular tumour suppressor gene in the integration region. We report here the identification of a novel human gene, named APM-1, which is co-transcribed with the HPV68 E6 and E7 genes and is present in the 3'-cellular part of the ME180 viral-cellular fusion transcripts. The APM-1 gene encodes a protein with a BTB/POZ domain and four zinc fingers, and is located at chromosome 18q21. APM-1 transcripts are detected in normal cervical keratinocytes, but not in the majority of cervical carcinoma cell lines analysed. The APM-1 gene caused a reduction of clonal cell growth in vitro of HeLa and CaSki tumour cells. These characteristics make APM-1, the first novel human gene identified in a HPV integration region, a likely candidate for the postulated tumour suppressor gene.

Enzymatic Properties of Overexpressed HBV-Mevalonate Kinase Fusion Proteins and Mevalonate Kinase Proteins in the Human Hepatoma Cell Line PLC/PRF/5

We have previously reported a case of integration of HBV sequences in the 5′-noncoding region of the gene for mevalonate kinase (Mk) (EC.2.7.1.36) in the human hepatoma cell line PLC/PRF/5, resulting in overexpression of viral-cellular fusion transcripts and enhanced intracellular levels of the enzyme. Here, we present an evaluation of the functionalities of Mk and HBV/Mk fusion proteins derived from viral-cellular fusion- and Mk-transcripts, some of which lack 156 bp in the Mk coding region as a result of a differential splicing process. cDNA clones with a full-length Mk-ORF produce proteins which can metabolize mevalonate to its monophosphorylated form. Our results suggest that the enhanced and inappropriate expression of Mk may lead to increased metabolism of mevalonate and phosphorylation of hitherto unknown cellular proteins. This consequence of HBV-DNA insertion could thus be related to the activation of proteins that may be relevant in oncogenesis.