Small tumor virus genomes are integrated near nuclear matrix attachment regions in transformed cells

Abstract

More than 15% of human cancers have a viral etiology. In benign lesions induced by the small DNA tumor viruses, viral genomes are typically maintained extrachromosomally. Malignant progression is often associated with viral integration into host cell chromatin. To study the role of viral integration in tumorigenesis, we analyzed the positions of integrated viral genomes in tumors and tumor cell lines induced by the small oncogenic viruses, including the high-risk human papillomaviruses, hepatitis B virus, simian virus 40, and human T-cell leukemia virus type 1. We show that viral integrations in tumor cells lie near cellular sequences identified as nuclear matrix attachment regions (MARs), while integrations in nonneoplastic cells show no significant correlation with these regions. In mammalian cells, the nuclear matrix functions in gene expression and DNA replication. MARs play varied but poorly understood roles in eukaryotic gene expression. Our results suggest that integrated tumor virus genomes are subject to MAR-mediated transcriptional regulation, providing insight into mechanisms of viral carcinogenesis. Furthermore, the viral oncoproteins serve as invaluable tools for the study of mechanisms controlling cellular growth. Similarly, our demonstration that integrated viral genomes may be subject to MAR-mediated transcriptional effects should facilitate elucidation of fundamental mechanisms regulating eukaryotic gene expression.

FIG. 1

Southern hybridization of cellular sequences flanking integrated HPV18 in cervical carcinoma. Alu-PCR products were hybridized with an HPV18-specific oligonucleotide probe. Lanes 1 and 2, HPV18(+) cervical squamous-cell carcinoma; lane 3, HPV18(+) cervical adenocarcinoma; lane 4, HPV16(+) cervical squamous-cell carcinoma; lane 5, normal human genomic DNA; lane 6, Alu-HPV18 hybrid plasmid (2.3 kb); and lane 7, negative control. Tumors 1 and 2 harbored multiple HPV18 integrations.

FIG. 2

MAP of the genomic region surrounding an HPV18 integration in a cervical carcinoma. The HSR, defined using a mathematical algorithm that weights sequence motifs associated with cellular MARs, demonstrates a significant potential for attachment to the nuclear matrix. D_up is the distance from the IP_up to the HSR. In this instance, the position of the downstream viral IP is unknown. The inset shows the full 30-kb analyzed interval, which contains a single HSR. In our study, MAP and HSR boundaries were defined using the MAR prediction algorithm's default detection and clipping parameters. The MAP of the genomic region illustrated here is shown with unclipped peaks for clarity.

FIG. 3

Mean distance to an HSR, d_up, for high-risk HPV and HBV integrations in tumor cells and the distribution of means in Monte Carlo simulations. The distribution was computed under the assumption that viral integration occurs randomly with respect to HSRs. P gives the fraction of simulated mean distances less than or equal to the observed mean; here, P < 0.0002.

FIG. 4

Integrated tumor virus genomes in transformed cells and cell lines are adjacent to MARs. Viral integrations in nonneoplastic cells, and in tumor cells in which the integration disrupted genes involved in cellular growth control, show no association with these regions. MAR-mediated effects on the expression of integrated viral oncogenes might confer a proliferative advantage to a cell and its progeny, promoting tumor development.