Detection of SV40 DNA Sequences in Infected and Transfected B Cells 64

Acknowledgements I would like to give thanks to all who contributed to this work by way of varied assistance, advice and even warm smiles. First of all, I would like to thank my supervisor, Prof. Mauro Tognon, for giving me the opportunity to undertake the experiments that resulted to this thesis, and for his encouragements that helped me to achieve my aims. My gratitude and regards to Dr. Elisa Mazzoni, an inspirator, a mentor, a kind and good friend who enlightened me much in my study. I thank my colleagues in Prof. Tognon’s laboratory, Dr. Cecilia Pancaldi, Dr. Stefania Maniero, Dr. Marco Manfirini, Dr. Silvia Bosi, Dr. John Rotondo, Dr. Ilaria Bononi. My special thanks to Prof. Fernanda Martini, for giving me free assess to her laboratory whenever I had the need; to Dr. Lara Rizotto of Hematology and Pathophysiology section, St Anna’s Hospital, for her unconditional support throughout the research period. Thank you all—your kindness and constant willingness to help created a fantastic work environment and made my time in the laboratory thoroughly interesting. I have not forgotten the contribution of Prof. Eldibert Van Driesche, Prof. Stefano Margez and Greet Devuyst of Vriji University Brussels, Belgium, in shaping my academic ambition. I am sure they will be as glad as I am today in seeing this study program come through. I am indebted to my friends and to the Alaribe/Nnadozie family for their patience and support during my studies. My sincere regards to my uncle, Monsignor Dr. Innocent Alaribe for being the mastermind of this long journey; my husband Dr Gabriel C. Nnadozie and my son for their support, love and care; my cousin Calistus Agbakwuru and family for their advice and help. ii Table of Contents Page Acknowledgements ii Introduction 1.1 The discovery of simian virus 40 (SV40) 1 1.2 Polyomavirus infection in the natural host 2 1.3 SV40 infection in humans 4 1.4 SV40 structure and genome 8 1.4.1 SV40 late genome region 9 1.4.2 SV40 early genome region 15 1.4.3 SV40 regulatory region and micro RNA 20 1.5 Life cycle of SV40 in host cells 23 1.6 The immune response to SV40 29 1.7 SV40 association with human cancers 31 1.8 Association of SV40 with lymphomagenesis/non-Hodgkin’s lymphoma (NHL) 35 2. Objective of the experimental thesis 39 3. Material and Methods 41 3.1 PBMCs and isolation of B/T cells from buffy coats 41 3.2 Flow cytometric analysis, infection and transfection of human purified B and T cells 42 3.3 Cell lines 42 3.4 Alamar blue assay for cell viability and proliferation 43 3.5 Population doubling time 44 3.6.1 DNA purification (FOR EXPERIMENT i) 44 iii 3.6.2 DNA and RNA extraction (FOR EXPERIMENT ii and iii 45 3.7.1 PCR (EXPERIMENT i) 45 3.7.2 PCR (FOR EXPERIMENT ii and iii) 46 3.8 RT-PCR analysis 46 3.9 Ultrastructural studies 47 3.10 Indirect immunoflourescence 47 3.11 Cytopathic effects of infected and T and B cells with determined titers 48 3.12 Plaque assay 48 3. 13 Statistical significance 49 4 Results Experiment I Simian virus 40 (SV40) sequences in blood specimen o healthy individual from Aviano (Abstract) 50 4.1 SV40 DNA sequences in 60 subjects of Aviano 50 Experiment II Simian virus 40 infection of human T lymphocytes (abstract) 52 4.2 Viability of the SV40 infected T lymphocyte cells 52 4.3 SV40 sequences analysis in infected T cells by single and nested PCR 53 4.4 SV40 Tag oncoprotein and capsid Vps expression in T lymphocyte 55 4.5 SV40 Effective production of progeny by infected T cells 55 4.6 Structure distortion in SV40 infected T cells 56 Experiment III Transformation of human B cells by SV40, a small DNA tumour virus (Abstract) 58 iv 4.7 FACs analysis, infection and transfection 59 4.8 Cell viability and proliferation capacity 60 4.9 Growth behavior of infected and transfected cells 63 4.10 Detection of SV40 DNA sequences in infected and transfected B cells 64 4.11 Expression of SV40 Tag and VP1 in human transfected and infected B cells 65 4.12 Structure distortion in transfected and infected B cells 67 4.13 Effective production of progeny in infected and transfected B cells with CV-1 permissive monolayer cells 69 4.14 Plaque assay viral titer 69 DISCUSSION 72 CONCLUSION 82 ABBREVIATIONS 84 REFERENCES 86 PUBLICATIONS 99 List of Figures Figure 1.1 Simian virus 40 Genome 9 Figure 1.2 S40 minichromosomes revealing the structural capsid proteins 10 Figure 1.3 Model for SV40 entry and penetration of the ER membrane 11 Figure 1.4 Proposed mechanism by which SV40 regulates its life cycle 14 Figure 1.5 A schematic model representing functional domains of SV40 Tag 16 Figure 1.6 Domains of SV40 small tag 17 Figure 1.7 Cell transformation mediated by small tag downstream expression 1 9 v Figure 1.8 Schematic representation of the interaction of SV40 Tag with tumour suppressor proteins-pRB and p53 25 Figure 1.9 Possible outcomes of SV40 infection in different cells 27 Figure 1.10 Schematic representation of the three most frequently cited cell transformation stages/phases their characteristics 28 Figure 3.1 Structure of the pSV3neo plasmid with intact SV40 early region 42 Figure 3.2 Positive control cell lines 43 Figure 4.1 Viability graph for SV40 infected human T cells in culture 53 Figure 4.2 Single and nested PCR of SV40 infected T cells up to 80 d.p.i 54 Figure 4.3 Immunoflourescence staining of T cell Tag (43a) and VP1 (43b) 55 Figure 4.4 TEM results of T cells 57 Figure 4.5 FACs results of % population of normal B cells isolated from PBMCs of 6 healthy donors buffy coat 59 Figure 4.6 Trypan blue analysis of % survival of infected transfected and normal 60 human B cells from 5-100 d.p.i 61 Figure 4.7 % alamar blue reduction of B cells with different incubation periods and at different initial cell densities 62 Figure 4.8 Population doubling time of cells 63 Figure 4.9 PCR and RT-PCR analysis of the infected and transfected B cells 64 Figure 4.10 Immunoflourescence detection of SV40 Tag in infected and transfected B cells 66 Figure 4.11 Detection of VP1 in infected and transfected human B cells 67 Figure 4.12 Transmission electron microscope for ultrastructural observation in B infected, transfected and normal cells 68 Figure 4.13 C.P.E observed in SV40 infected B cells with CV-1 monolayer cells 70 Figure 4.14 Viral titer of SV40 infected cells using plaque assay 71 vi List of Tables Table 1.1 Members of the polyomaviridae family 4 Table 1.2 Detection of SV40 DNA sequences in human lymphoploriferative 37 disorders/NHL Table 4.1 Numbers detected SV40 DNA sequences from the 60 examined subjects 51 Table 4.2 Oligonucleotides used as primers in PCR and RT-PCR analysis 51 Table 4.3 Viral titers showed by SV40 776 strain released by infected human T 55 lymphocytes in CV-1(45-54-2cv-1) cells Table 4.4 List of Antibodies used in FACs and immunoflourescence analysis 60 Table 4.5 Viral titers determined in B cells infected and transfected cells with CV-1 monolayer cells using end point dilution assay 70 vii Introduction S imian virus 40 (SV40) is a small DNA tumor virus, linked with specific human cancers such as malignant pleural mesothelioma (MPM), brain and bone tumors, leukemia, lymphoma diseases. Its DNA sequences is also detected in healthy blood donors. While the debate on SV40 association, rate of prevalence and detection of its DNA sequences in different human cancers including non- Hodgkin lymphomas (NHL) are at increase, indebt studies on the interaction between lymphocytes and SV40 has not yet been fully described. The highlighted issue calls for further imperative investigation. Hence, this study investigated the interaction of SV40 virus in healthy blood donors using three different experiments. Experiment I: ―Simian virus 40 sequences of healthy individuals from Aviano Cancer center (Italy).‖ The experiment aimed at detecting SV40 DNA sequences in healthy blood donors. Experiment II: ― SV40 infection of human T lymphocyte.‖ It aimed at investigating the rate of susceptibility of purified T lymphocytes to SV40 infection. While the third experiment-―Transformation of human B cells by SV40, a small DNA tumour virus‖ investigated the interaction of SV40 with purified B lymphocytes to observe the capacity of SV40 to infect, transform, and immortalize B cells. These three studies/experiments indeed, indicated the potential role of SV40 in Lymphomagenesis, its relationship and effects with human lymphocytes. Detailed information of these experiments is depicted in results session, each starting with a short abstract. 1.1: The discovery of the simian virus 40, SV40 SV40 belongs to the Polyomaviridae, a family of small DNA viruses that comprises the human polyomaviruses BK virus (BKV) and JC (JCV). BKV and JCV infect 70-90% of the adult population (1,2,3,4). The family name is derived from the first recognised member of the 1 Polyomaviridae, the murine polyomavirus (MuPyV), originally discovered in 1953 by Ludwik Gross as a source of salivary gland tumours in mice. MuPyV was initially designated 'polyomavirus' from the Greek 'poly' meaning many and 'oma' meaning tumours, due to its ability to induce a variety of solid tumours in mice (5,6). SV40 discovery was linked to the development of the anti-polio vaccines in the 1950s and 1960s as poliovirus was grown in primary kidney cells of rhesus and cynomolgus macaques, which were often naturally infected with SV40 (7,8,9).

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