Helen Isok-Paas will defend her doctoral thesis titled „Viral-host interactions in the life cycle of human papillomaviruses“ on 23 March 2017 at 9:15.
Senior Research Fellow Ene Ustav,
Professor Mart Ustav
Thomas Melendy, associate professor, Ph.D. Department of Microbiology and Immunology, University at Buffalo, the State University of New York, USA.
Summary: Human papillomaviruses (HPVs) are very widely distributed and almost all people become infected with this virus at some point during their life. Generally, the viral infection does not cause significant illness but generates benign warts on skin (β-HPVs) and condylomas on genitals (α-HPVs). The clinical importance of papillomaviruses is leaning on the fact that HPV genomes (or parts of it) are found in almost all cervical cancer cases. It is believed that the infection with papillomaviruses that belong to high-risk group of α-HPVs (e.g. HPV16, HPV18) is one of the most important step in the development of cervical cancer. When the infection with low-risk α-HPVs (e.g. HPV6, HPV11) is eventually eliminated by the immune system (usually taking up to 2 years), then the infection with high-risk HPVs become easily persistent. The limited treatment opportunities to cure persistent HPV infection makes the early detection of HPV infection highly important.
The successful life cycle of HPVs is mediated through extrachromosomal molecule and the integration to the host cell chromosomes is not essential. However, in cervical cancer cells the parts of HPV genomes are often found integrated in to the host genetic material. The partial integration of HPV genomes disrupts the expression of many viral genes (incl. genes that encode proteins for DNA replication and for capsid formation), therefore, the integration to host chromosomes is considered as the dead end for the virus. On the other hand, as the integration site is not determined, the uncontrollable growth of cancer cells may be the result of viral integration to the chromosome regions that are responsible for the regulation of cell growth. Also, the large-scale genetic changes found in cervical cancer cells, are probably triggered by the initial viral integration event.
Since, cells carry approximately 100 copies of HPV genomes and the integration encompasses only few viral genomes, majority of the genomes remain as extrachromosomal molecules. The co-occurrence of two forms of viral DNA in one cell is a real threat to genomic stability as HPVs integrate in to the host genetic material in a way that DNA replication origin, that cannot be controlled by cellular checkpoint pathways, is intact. The integration event usually disrupts the genes that encode replication proteins, however, the expression of replication proteins from extrachromosomal molecule is still possible. Multiple replication initiations from the integrated DNA replication origin by episome-derived viral replication proteins lead to the amplification of not only integrated parts of viral DNA but also the flanking cellular sequences. Depending on the site of the integration, the flanking cellular sequences may contain oncogenes or other regulatory elements which amplification may lead to uncontrollable growth of cells or to genomic instability. In addition, the unlicensed DNA re-replication characteristic to HPVs often cause the collision of replication forks and the accumulation of aberrant DNA replication products. The co-localization of the key molecules of the cellular DNA damage response (DDR) pathways of homologous recombination and non-homologous-end-joining to HPV DNA replication centers indicate to the generation of double-stranded DNA breaks by unlicensed DNA re-replication. It seems that the activation of cellular DDR pathways has an important role in repairing the double-stranded DNA breaks that occur during viral DNA replication. While most of the double-stranded breaks are repaired properly, the cross-chromosomal translocations of the amplified viral integration sites detected within this thesis may be the result of the failed attempt to repair the DNA damage. The generation of multiple modifications in host cell genomic material may eventually lead to the genomic instability and to the formation of malignancy.
The study of human papillomaviruses in their natural host cells in vitro is quite challenging as keratinocytes have adapted a very complex life cycle in order to form the layered tissue of the epithelia. While, the undifferentiated keratinocytes are responsible for the renewal of the basal cell compartment by continuous cell division, they also undergo terminal differentiation program to form the stratified structure of the epithelia. As papillomaviruses infect the basal layer of undifferentiated keratinocytes and pack their viral particles at the terminally differentiated cell residues, the virus has adjusted with all the stages of the host cell life cycle. The cultivation of keratinocytes in cell culture and mimicking their differentiation program is technically demanding, time-consuming and cost-expensive. In addition, keratinocyte system is not suitable for the high-throughput screening of chemical compounds in order to find drugs that would reduce the viability of papillomaviruses. The need for a simpler system to study the various aspects of HPV life cycle inspired our research group for years to find a cell-line that is easy to culture and where the replication of HPV genomes could be monitored. The development of U2OS cell-based system turned out to be a suitable environment for HPVs as the transient, stable, and amplificational genome replication of various types of HPVs (β-HPV5, β-HPV8, α-HPV6b, α-HPV11, α-HPV16, α-HPV18) was detected. As the human osteosarcoma cell line U2OS are not the native hosts of papillomaviruses, the suitability of this cell line in the study of papillomaviruses was further confirmed by the complete mapping of HPV11 transcripts. Similar analysis was also carried out with HPV5 and HPV18 by the other members in our research group. The similarity of the gene expression of all the three HPV types with the previously characterized results in native host cells or in clinical samples confirmed that U2OS cells are suitable for the research of human papillomaviruses as well as for the preliminary studies of anti-HPV drugs.
Interestingly, while the life cycle of HPVs function through unit-sized (1n) extrachromosomal molecule and ultimately only one genome is packed into viral particles, the analysis of replication intermediates detected, in addition to 1n molecules, also larger than unit-sized HPV-specific molecules. The subsequent analysis of replication intermediates confirmed that the larger molecules are head-to-tail concatemers (further referred as oligomers). Similar DNA replication products have been described also in previously analyzed clinical samples and were also detected from HPV16 and HPV18-positive patient probes collected within this thesis. The further analysis of the formation of the oligomeric forms of HPV molecules suggest that recombination-dependent DNA replication mode might be involved in this process.