Background: Although subclinical persistent infections with the human polyomaviruses are ubiquitous worldwide, they are known to vary in relation to geographical location, diseases present and may associate with different human tumours, especially in immunocompromised patients. The current study hypothesised that there may be co-operativity between HPV and polyomaviruses, particularly in HIV positive women, that could influence the rate of progression to invasive cervical carcinoma. Patients and Methods: Novel PCR methods were developed for the detection of SV40, MCV, JCV and BKV polyomavirus DNA. These were used to test DNAs extracted from 220 cervical smears and 77 invasive cervical carcinomas (ICCs) from HIV positive and negative Kenyan women of known HPV status. An expression plasmid was constructed containing JCV Large T (LT) antigen and this, in addition to empty vector control, used to stably transfect HPV16 E6/E7 immortalised human keratinocytes. Expression of LT was analysed in transfected cell lines by PCR, immunocytology and Western blotting. These cells were then used to test for changes in Cell contact growth inhibition; Growth rate and Epithelial to Mesenchymal Transition (EMT). Screening of full transcriptome microarrays was carried out on vector and LT transfected cells and their sensitivity to the drug mefloquine tested by comparison of growth rates and live/dead cell assays. Results: PCR accurately detected ~18 copies of SV40, MCV, JCV and BKV DNA in addition to simultaneous detection of JCV and BKV. None of the clinical samples tested were positive for SV40, MCV, or BKV DNA. However, JCV DNA was detected in 24/297 (8%) of cervical specimens. Comparison of the incidence of JCV in cervical smears and ICCs showed a ~3-fold increase in samples from HIV positive women with ICC (P=0.025) whereas no significant difference was found between smears and ICCs from HIV negative women (P=0.553). Analysis of the consequences of ectopic expression of JCV LT in E6/E7 immortalised human keratinocytes showed no difference in either growth rates or contact inhibition and changes in the EMT marker vimentin were found to be related to cellular clonality. Microarray analysis showed LT related alterations in gene expression which could have bearing on its carcinogenic potential in addition to changes related to clonality. JCV LT expressing monoclonal cell were the most sensitive to mefloquine treatment. Conclusion: The simultaneous JCV/BKV detection method, described herein, is unique and has been evaluated by the WHO for this purpose. The results indicate the prevalence JCV and BKV with respect to the African geographical location and suggest that JCV may combine with high-risk HPV in a sub-set of HIV positive women to influence the rate of progression to invasive cervical carcinoma. In vitro JCV LT was found not to be an overt oncogene in the cell system used although cell cloning procedures clearly affected the assays. LT induced changes in total gene expression were consistent with neoplastic progression although a high proportion of genes with unknown function were dsyregulated with respect to clonality. The anti JCV drug mefloquine showed some selectivity for LT expressing cells and further investigation of this indication is warranted.

Capsid of mouse polyomavirus (MPyV) is composed from three structural proteins: major structural protein VP1 and minor structural proteins VP2 and VP3. Posttranslational modifications may affect functions of proteins. This work deals with acetylation of MPyV structural proteins and its impact on the viral replication cycle. First part of the thesis is focused on acetylation of VP1. We showed that the VP1 protein is acetylated in viral particles and that interaction of VP1 with minor proteins supports VP1 acetylation. Further, we showed that cytoplasmatic deacetylase, histone deacetylase 6 (HDAC6), is important for virus infectivity. Overexpression of HDAC6 decreased MPyV infectivity, also decreased infectivity was exhibited by virus isolated from HDAC6 knock out cells. In addition, VP1 protein of virus from HDAC6 knock out cells was more acetylated in comparison with virus from parental cell line. These data suggest that VP1 is substrate for HDAC6. Second part of the thesis is focused on the characterization of N-terminal acetylation of VP3 minor structural protein. It has been previously shown that VP3 protein is N-terminally acetylated and MyPV with mutated (unacetylated) form of VP3 protein is non-infectious. The main aim of this part is to prove the hypothesis that N-terminal acetylation is...

The polyomavirus enhancer consists of functionally redundant DNA sub-elements. One such sub-element, element 2, comprises a region with contiguous binding sites, or motifs, for at least three nuclear factors, designated as PEA1, PEA2, and PEA3. Although little is known of PEA2, PEA1 is presumed to be a murine homolog of human transcription activator protein 1 (AP-1), and PEA3 has recently been shown to be encoded by a member of the Ets family of oncogenes. The contributions of each factor to enhancer function are not understood. A cell-free system was devised to assay the individual abilities of the DNA motifs recognized by PEA1, PEA2, and PEA3 to confer transcriptional activation upon a minimal promoter. The motifs were cloned and tested as monomers, as multiple tandem copies, and in paired combinations. The results of these in vitro studies indicate that the PEA1 motif behaves as a low affinity AP-1 binding site; that PEA1 and PEA3, but not PEA2, activate transcription; and that both the PEA1 and PEA3 motifs act synergistically. Band shift titration experiments demonstrated that neither PEA1 nor PEA3 bound to their DNA motifs co-operatively, indicating that synergistic activation of transcription by these factors is not due to cooperative binding. Finally, additional in vitro transcription experiments suggest that PEA1 and PEA3 may co-operate with each other to stimulate transcription. A current model proposes that the minimal sub-units of enhancer structure are small (8-10 base pair) DNA motifs, called enhansons, that act synergistically. I propose that the motifs for PEA1 and PEA3, but not PEA2, are enhansons of the polyomavirus enhancer. / Thesis / Master of Science (MS)

The aim of this thesis is to investigate the targeting potential of mouse polyomavirus (MPyV) based virus-like particles (VLPs) as vectors for directed cell delivery of therapeutic or diagnostic compounds. Major capsid protein VP1 of MPyV is able to selfassemble into the noninfectious VLPs. Our main goal is to retarget these VLPs from its native receptor to the prostatic cancer cells by changing the receptor binding site in the surface-exposed loop of VP1. We introduced a peptide ligand CTITSKRTC, which binds prostate-specific membrane antigen (PSMA), by insertion or substitution into BC loop of VP1. These modifications did not change the stability of the particles and genetic substitution prevented the native receptor binding. PSMA-specific binding of modified VLPs was tested by pull-down assay and surface plasmon resonance. In order to further utilize these VLPs, we tested several approaches for preparation of VLPs as vehicles for compounds delivery into eukaryotic cells. Although the method for encapsidation of the DNA into the VLPs in cellular nuclear extracts, which mimic the in vivo conditions, did not enabled us to produce pseudocapsids, we successfully optimized procedure for dissassembly and reassembly of purified particles. This method will be use for encapsidation of molecules into the...

Aim of this diploma thesis was to prepare two protein antigens and two monoclonal antibodies, all based on VP2 minor protein of human polyomaviruses BK virus and Merkel Cell Polyomavirus. One monoclonal antibody was being prepared against unique part of VP2 protein (N-terminal epitope, not present in VP3 protein). A cell line producing such monoclonal antibody has never been established before due to low immunogenicity of the epitope. Our approach was successful in terms of mouse immunization, however, serious problems with hybridoma line stability appeared later during the preparation process. Preparation of antibody targeted to the sequence of VP2 protein of Merkel Cell Polyomavirus was another aim of this thesis. Mouse immunization and hybridoma fusion were performed successfully. After four rounds of cloning in order to purify an established clone, nine clones were cultivated in larger scale. This cultivation probably led to diminished antibody specificity and loss of production ability in most of the hybridoma cells. One more cloning should give rise to an established clone with sufficient production. Two preparations of protein antigens were performed in two expression systems. DNA encoding C-terminally truncated protein VP2 of BK virus fused with His-tag was cloned into a vector suitable for...

Exosomes are extracellular vesicles of endosomal origin. It was thought, that exosomes are used by cells only as carriers for cellular waste, but it was found out, that exosomes serve in the cellular communication and have a role in viral infections. Exosomes are exploited by viruses for example for the transport of viral protein or viral RNA/DNA. One of the viruses, where the mechanism of exploitation is unknown (if any exists) is murine polyomavirus. Murine polyomavirus belongs to the family Polyomaviridae, to which other human viruses belong for example, JC virus or virus of Merkel cell carcinoma. Murine polyomavirus codes for small, large and middle T antigen and three capsid proteins. Middle T antigen is known to bind to cellular membranes. Exosomes are membrane derived structures, so we investigated a possible transfer of middle T antigen. To this goal the successful isolation of exosomes and their characterization was necessary. Exosomes were isolated by ultracentrifugation and further purified by the density gradient OptiPrep. Exosomes were characterized by electron microscopy, NanoSight and by protein exosomal markers. These markers are for example Alix and flotillin-1. The cells were transfected in order to produce middle T antigen. It was shown, that exosomes isolated from these cells...

Interaction of polyomaviruses with proteasomal system of host cells Abstract: Viral family Polyomaviridae includes besides model organisms - mouse polyomavirus and SV40 virus, also human pathogens, for example, BK virus. Polyomaviruses are small non- enveloped viruses with double-stranded DNA. Understanding of their life cycle is important for their use in gene therapy and immunotherapy as well as for prevention and treatment of complications caused by these viruses. This thesis is focused on early phases of MPyV and SV40 infection studying, mainly on delivery of viral genome to nucleus and role of proteasomal system in this stage of infection. It was found out that inhibition of proteasomes by specific inhibitor leads to increase of early non-structural protein LT expression, which was chosen as marker for viral entry to the nucleus and successful viral expression. Relative localization of proteasomes and VP1 protein of MPyV and SV40 was monitored and it showed 10% colocalization of mentioned structures. Further, it was found out that proteasomal inhibitor MG-132 negatively influences the replication of both viral and cellular DNA. Next aim of this diploma thesis was to prepare antigen - unique part of VP2 protein of BKV - for producing antibody. Expression vector with inserted fragment of unique part of...

Murine polyomavirus (MPyV) is a member of the Polyomaviridae family. Its capsid is composed of the major capsid protein, VP1, and the minor proteins, VP2 and VP3. The minor capsid proteins probably assure delivery of the viral genome through the endoplasmic reticulum membrane to the nucleus during early phase of infection. However, precise mechanism is not known. Expression plasmids encoding mutated VP2 or VP3 fused with EGFP have been constructed to study the interaction of VP2 and VP3 with membranes. The mutated proteins have deletions in the predicted hydrophobic domains. In this thesis, cell localisation of mutated proteins was followed. The study revealed that the hydrophobic domain 2 is the most important for association of VP2 and VP3 with membranes, while domains 1 and 3 are rather expendable. Further, nature of VP2 and VP3 isoforms has been studied. Isoforms with different electrophoretic mobility were separated on SDS-PAGE. Consequent mass spectrometry analysis showed that they differ in deamidation of asparagine, present at both minor proteins (position 253 of VP2 and 137 of VP3). Previously, acetylation of VP3 N-terminal alanine has been identified. To elucidate the function of these modifications, mutated viruses were constructed with substitution of these amino acids. Pilot...

Mouse polyomavirus (MPyV) is a model virus of the Polyomaviridae family. Polyomaviruses are small non-enveloped DNA viruses. They cause severe problems to immunocompromised patients. Their oncogenic potential is known in animals and humans. Trafficking of MPyV within the cell is not clear yet. The virus enters via smooth monopinocytic vesicles and continues to early and late endosomes. From there, the virus is transported to the ER by unknown mechanism. It bypasses Golgi aparatus (GA). One possible pathway is from late endosomes to trans-Golgi network (TGN) facilitated by Rab9 GTPase and then in COPI vesicles to the ER. In this thesis, the effect of inhibitors of retrograde transport (Brefeldin A, Golgicide A) on MPyV infection was evaluated. Brefeldin A is not completely specific; it has effect on whole endosomal system. Golgicide A causes specific disruption of transport via TGN and GA. Both inhibitors suppressed infection of MPyV. Confocal microscopy revealed colocalization of some MPyV virions with markers of TGN and COPI vesicles. MPyV didn't colocalize with cis-Golgi marker. Unfortunately, the effect of overexpression of Rab9 dominant negative mutant couldn't been evaluated due to its high cytotoxicity. However, overexpression of wild type Rab9 slightly increased infectivity. The results...

Murine polyomavirus is an important member of Polyomaviridae family offering potential applications in gene therapy and immunotherapy. Viral mutant analysis is crucial for study of the virus, however, commonly used methods of its production are laborious and give low yields. This thesis involves development of the new experimental system that can produce intact viral genome from recombinant plasmid in vivo using Cre/loxP-mediated recombination. One loxP site is unavoidably introduced into newly generated viral genome during recombination. Two variants of production plasmids generating wild type viral genome with incorporation of loxP between the poly(A) signal sites of early and late genes or into the intronic region of early genes were prepared. LoxP insertion between the poly(A) signal sites has a dramatic effect on viral gene expression and leads to complete loss of virus infectivity. Conversely, the infectious virus was obtained from the viral genome containing loxP site in the early intronic region. To ensure expression of Cre recombinase I also prepared stably transfected cell lines which can simplify the virus production. This thesis shows that newly designed system gives satisfactory yield of the virus, solves restrictions connected with commonly used methods and can be used for low infectious viral...