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Functional characterization of the non-coding control region of human polyomaviruses

Ajuh, Elvis Tasih. Functional characterization of the non-coding control region of human polyomaviruses. 2017, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_12386

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Abstract

With the advent of advanced molecular biology techniques, 13 human polyomaviruses (HPyVs) have been identified from different human body compartments. Generally, infection with HPyVs is harmless in immunocompetent people. However, some of these viruses are known to cause severe morbidities in the immunosuppressed. Five HPyVs, BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), Merkel cell polyomavirus (MCPyV), Trichodysplasia spinolosa polyomavirus (TSPyV) and HPyV7 are known to cause diseases. Although other HPyVs have been detected in diseased tissues or cancers, evidence of their involvement is lacking. Furthermore, less is known about the cell tropism, replication in cell culture, and gene regulation of HPyVs. The non-coding control region (NCCR) of HPyVs functions as a bi-directional promoter/enhancer system, coordinating the respective steps of the viral replication cycle. Furthermore, it also determines viral persistence, host cell specificity, replication and virulence. Efficient replication of BKPyV and JCPyV have been demonstrated in cell cultures, but less is known with regards to the novel HPyVs. NCCR activity which can be used as an indicator of HPyV cell tropism and replication in cell culture is lacking for the newly discovered HPyVs. Despite similarities in the genome organization of HPyVs, the NCCRs of the HPyVs are different with respect to large T antigen binding sites, transcriptional factor binding sites (TFBS) and length. We hypothesized that the HPyV-NCCRs will display different activity in the same or different host cells. A bi-directional reporter vector recapitulating the HPyV genome with a red fluorescence protein (dsRed2) and a green fluorescence protein (EGFP) as markers of EVGR and LVGR, respectively, was designed. The reporter was used to analyze the NCCR activity of the 13 HPyVs in different cell lines originating from kidney, skin, lungs, cervix, brain and colon cancers. Our result demonstrated that the bi-directional HPyV-NCCR activity substantially differ in the same and different host cells. Indicating that the HPyV-NCCRs differentially sense and interpret the host cells’ transcription factors and different host cells’ transcription factors modulate the basal HPyV-NCCRs expression. As previously reported for BKPyV and JCPyV, rearranged (rr)-NCCR variants of newly discovered HPyVs associated with diseases showed higher EVGR expression compared to their respective archetype, indicating the NCCR a major pathogenicity determinant. Analyzing the HPyV-NCCRs’ activity in cell lines expressing T antigens (Tags) displayed activation of EVGR expression, suggesting that the bi-directional reporter recapitulates an essential NCCR response reported for the viral replication cycle. Furthermore, it indicates that cell lines expressing Tags of respective HPyVs can be used for the propagation of respective HPyVs. This data serves as a basis for understanding host and viral factors that may permit the identification of suitable cell culture systems and secondary host cell tropism for HPyVs. However, actual viral replication studies are needed to confirm replication of HPyV genomes in some of the cell lines tested. Furthermore, the role of the LTag-binding motif with regards to the regulation of EVGR and LVGR has been well-characterized in the monkey polyomavirus, simian virus 40, although very little is known with respect to the role of the LTag-binding motif for HPyVs. Given that the LTag recognition sites influence EVGR and LVGR expression and the LTag-binding sites organization of SV40 is similar to that of some HPyVs, such as BKPyV, we hypothesized that the role of the LTag-binding motif in the regulation of EVGR and LVGR expression in BKPyV is potentially similar to that of SV40. The EVGR and LVGR expression of BKPyV archetype (ww)-NCCR was compared with those of BKPyV ww-NCCR defective in one or more LTag-binding motifs in HEK293, HEK293T and HEK293TT cell lines, expressing none, small and large amounts of SV40 LTag, respectively. Our results indicated that abundant LTag decreased EVGR-expression by probably interacting with LTag binding site A, suggesting abundant LTag expression may down regulate EVGR expression, similar to SV40’s-EVGR autoregulation. Contrarily, the LVGR expression is proportionally increased in the presence of increasing amounts of LTag expression (HEK293T and HEK293TT cells), by LTag potentially interacting with binding sites A and D. These results may open avenues for designing new therapeutic strategies targeting this reduction of EVGR expression in the presence of abundant LTag expression. Actual viral replication studies are needed to verify whether these mutants are replicative competent or not.
Concluding, a basic approach was used to elucidate factors that may allow the replication of HPyVs in cell cultures, that will greatly enhance our understanding of the basic biology of these viruses. Furthermore, putative host cell tropism of HPyVs was suggested. The effect of LTag-binding sites with respect to BKPyV’s EVGR and LVGR expression, which could be extrapolated to other HPyVs was shown.
Advisors:Tanner, Marcel and Hirsch, Hans H.
Faculties and Departments:03 Faculty of Medicine > Departement Public Health > Sozial- und Präventivmedizin > Malaria Vaccines (Tanner)
09 Associated Institutions > Swiss Tropical and Public Health Institute (Swiss TPH) > Department of Epidemiology and Public Health (EPH) > Health Interventions > Malaria Vaccines (Tanner)
UniBasel Contributors:Tanner, Marcel and Hirsch, Hans H.
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:12386
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:1 Online-Ressource (220 Seiten)
Language:English
Identification Number:
Last Modified:22 Jan 2018 15:53
Deposited On:15 Jan 2018 17:26

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