Mouse polyomavirus is a type species of Polyomaviridae family and serves as model for studying viral infection of human pathogenic polyomaviruses. Minor proteins of viral capsid have been found to be necessary for effective initiation of infection. In order to study their role in the early steps of infection we utilized the novel Cre-LoxP system for production of the viral mutant lacking both minor proteins. Virus produced this way was compared with virus produced by standard method and we found that both systems facilitate production of mutant virus with the comparable quality and quantity. The mutant virus contained reduced amount of viral DNA and formed virions with impaired stability. For further studies of intracellular virion trafficking we prepared virions with genomes modified by thymidine analogues 5- bromo-2'-deoxyuridine (BrdU) and 5-Ethynyl-2'-deoxyuridine (EdU) and optimized the methods for analogue detection. The viral genome become accessible for detection 4 hours post infection. For ultramicroscopic analysis of translocation of virus to the nucleus we used freeze substitution. All this methods will be utilized for detailed study of distinct steps in viral infection. Key words: Mouse polyomavirus, minor proteins,...

Polyomaviruses are small non-enveloped DNA viruses infecting birds and mammals, including human. Their capsid consists of the major capsid protein, VP1, and two minor capsid proteins, VP2 and VP3. The VP2 and VP3 proteins are supposed to have an important function in the transport of viral genome into the cell nucleus, which is a key step to facilitate viral replication. VP2 and VP3 proteins of mouse polyomavirus and SV40 have an ability to bind and disrupt cellular membranes. This feature is believed to be involved in the transport of viral genome into the nucleus. Plasmids carrying genes of the minor capsid proteins of Merkel cell polyomavirus were prepared in order to produce and visualize these proteins in mammalian cells. These proteins are known to have very unusual sequences compared to other human polyomaviruses or related mouse polyomavirus. When produced alone, the minor capsid proteins of Merkel cell polyomavirus did not significantly interact with cellular membranes, unlike the minor proteins of the mouse polyomavirus. The second goal of this work was to prepare mouse polyomavirus mutants with deletion in hydrophobic domains of VP2 and VP3 proteins. These domains are likely responsible for the mentioned membrane interactions. Prepared mutants were non-infectious. The loss of infectivity was not...

Various methods for extraction and analysis of high value minor proteins (lactoferrin, lactoperoxidase and immunoglobulins) directly from raw milk were explored. Extraction, purification and analysis of high-value minor proteins directly from milk without pre-treatment are major challenges for dairy industry, largely due to the complexity of milk and the presence of colloidal solids (casein micelles and milk fat globules). To overcome some of these challenges, this work focused on three main objectives: 1) characterization of cryogel monolith chromatography for purification of lactoferrin (LF) and lactoperoxidase (LP) directly from raw milk in single step, 2) identification and characterization of Protein A Mimetic affinity ligands for purification of immunoglobulins (Igs) from milk and 3) development and validation of a surface plasmon resonance method for simultaneous quantification of five whey proteins in multiple samples. Results portrayed the possibility of 40–50 column volumes of various milk samples (whole milk, skim milk and acid whey) to pass through a 5 mL cryogel monolith chromatography column at 525 cm hr⁻¹ without exceeding its pressure limits if the processing temperature is maintained around 35–37°C. Ideally, this should be the milk secretion temperature. The dynamic binding capacity obtained for the cryogel matrix (2.1 mg mL⁻¹) was similar to that of the binding capacity (2.01 mg mL⁻¹) at equilibrium with 0.1 mg mL⁻¹ of lactoferrin in the feed samples. Lactoferrin and lactoperoxidase was selectively bound to the cryogel column with trivial leakage in flowthrough fractions. Lactoferrin was recovered from elution fractions with a yield of 85% and a purity of 90%. These results, together with the ease of manufacture, low cost and versatile surface chemistry of cryogels suggest that they may be a good alternative to packed-bed chromatography for direct capture of proteins from milk, provided that the binding capacity can be increased. A Protein A Mimetic (PAM) hexapeptide (HWRGWV) peptide ligand that binds to the Fc portion of antibody molecules was explored for affinity purification of immunoglobulins from milk. The peptide has the ability to purify IgG from various milk and whey samples with a purity of greater than 85% in single step. More than 90% bound IgG was recovered with 0.2 M acetate buffer at pH 4.0 and total column regeneration was successfully achieved by 2.0 M guanidine-HCl. At 9.0 mg mL⁻¹ of IgG feed concentration, an equilibrium binding capacity of 21.7 mg mL⁻¹ and dynamic binding capacity of approximately 12.0 mg mL⁻¹ of resin was obtained. Recoveries and yields of IgG were significantly influenced by the feed IgG concentration. PAM hexamer ligand also contributed a significant amount of cross-reactivity with casein, glycomacropeptides and β-lactoglobulin proteins, however majority of these proteins were recovered in the regeneration step, except β-lactoglobulin, which co-eluted with IgG. Higher IgG concentration in feed vastly reduced the amount of cross-reactivity whilst increasing the recoveries and purities in the final product. PAM affinity ligands also showed interactions towards other classes of bovine immunoglobulins. These findings established the possibility of using PAM hexamer peptide as an alternative to conventional Protein A/G affinity chromatography for the isolation of Igs from milk in single step process. A surface plasmon resonance (SPR) method was developed for simultaneous, quantitative determination of commercially important whey proteins in raw and processed milk samples, whey fractions and various milk-derived products, with six samples per assay. Immobilized antibody stability and reproducibility of analyses were studied over time for 25 independent runs (n=300), giving a relative standard deviation (RSD) of <4%. Immobilized antibodies showed negligible non-specific interactions (<2–4 SPR response units (RU)) and no cross-reactivity towards other milk components (<1 RU). Regeneration of immobilised antibodies with glycine at pH 1.75 was determined to be optimal for maintaining the SPR response between samples. This method compared and validated well with reversed phase high performance liquid chromatography (RP-HPLC) and standard enzyme-linked immunosorbent assays (ELISA).

Mouse polyomavirus (MPyV) capsid is composed of the main capsid protein VP1 and minor capsid proteins VP2 and VP3. Minor proteins are not essential capsid assembly, but they are key for efficient viral infection. The first part of this thesis studies the modifications of VP2 and VP3, the deamidation of Asn at 253 of VP2 (137 of VP3) and N-terminal acetylation of Ala of VP3, which could be the cause of double bands for VP2 and VP3 on SDS-PAGE. Mutated genomes of MPyV N253D (Asn to Asp) and N253E (Asn to Glu) simulating deamidation and A117V (Ala to Val) with reduced acetylation were prepared previously. We prepared three isolations of the mutant viruses and we confirmed that the deamidation is the cause of the double bands. Mutant viruses were compared to the wild type in terms of efficiency of infection, but the role of deamidation could not be proven. Virus A117V is noninfectious either due to lowered acetylation or the substitution of amino acid at this position. This thesis also studies the role of -tubulin acetylation in the infection of MPyV. The role of -tubulin acetylation in viral infection is being investigated to find new antiviral strategies. Acetylation rises after MPyV infection, but this is not due to a change in mRNA expression of tubulin acetylating (TAT1) or deacetylating enzyme...

Merkel cell polyomavirus (MCPyV) is a recently discovered human virus, having it's genome often integrated in a genome of Merkel carcinoma cells. Although this type of carcinoma is not so usual, it is very aggressive and it's incidence has been rising in last few years. It is not surprising that this virus is nowadays in the centre of scientific interest, as well as other pathogens and mechanisms affecting human life. Because the virus was discovered not so long ago, its research has been at the whole beginning. This diploma thesisaims to contribute to the study of this virus from the molecular-virology point of view. A neutralizing monoclonal antibody, type IgG2a, targeted against the main capsid protein of MCPyV, VP1, and recognizing its conformational epitote was prepared. This antibody was then used for a pilot study of VP1 VLPs MCPyV movement in mammalian cells. Results showed that the studied virus, at least particularly, utilizes caveolin-1-carrying vesicles for its movement in cells (colocalisation of VP1 VLPs and caveolin-1 was observedColocalisation with EEA1 marker of early endosomes, LamP2 marker of endolysosomal compartments or with BiP marker of endoplasmic reticulum was sporadic but significant. These preliminary results suggest that MCPyV might utilise an endocytic pathway leading...