The encapsidation of polyomavirus is not defined by a sequence-specific encapsidation signal

AbstractMouse polyomavirus (MPyV) is considered a potential tool for the application of gene therapy; however, the current knowledge of the encapsulation of DNA into virions is vague. We used a series of assays based on the encapsidation of a reporter vector into MPyV pseudovirions to identify putative cis-acting elements that are involved in DNA encapsidation. None of the sequences that were derived from MPyV have been shown to solely enhance the encapsidation of a reporter vector in the assay. The frequency of encapsidation strongly correlated with the total intracellular amount of the vector after transfection. The encapsidation of target DNA into the pseudovirions was shown to be non-specific, and the packaging of non-replicated DNA was observed. We propose that the actual concentration of target DNA at the sites of virion formation is the primary factor that determines its selection for encapsidation

Interactions of heterologous DNA with polyomavirus major structural protein, VP1

Abstract`Empty' polyomavirus pseudocapsids, self-assembled from the major structural protein VP1, bind DNA non-specifically and can deliver it into the nuclei of mammalian cells for expression [Forstová et al. (1995) Hum. Gene Ther. 6, 297–306]. Formation of suitable VP1-DNA complexes appears to be the limiting step in this route of gene delivery. Here, the character of VP1-DNA interactions has been studied in detail. Electron microscopy revealed that VP1 pseudocapsids can create in vitro at least two types of interactions with double-stranded DNA: (i) highly stable complexes, requiring free DNA ends, where the DNA is partially encapsidated; and, (ii) weaker interactions of pseudocapsids with internal parts of the DNA chain

The Compulsory Share and Related Matters

Diplomová práce je zaměřena na problematiku "Povinný díl a otázky s ním spojené" V práci se uceleně zabývám vývojem institutu povinného dílu a s ním souvisejících otázek, tj. nepominutelných dědiců, započtení na povinný díl a institutu vydědění. Zároveň prostřednictvím shrnutí historického vývoje dané výseče dědického práva nastiňuji inspirační sílu právní úpravy těchto otázek obsažených v současném občanském zákoníku předchozími občanskými zákoníky a zároveň tak dospívám k určité vývojové linii institutu povinného dílu. Tyto instituty dědického práva jsou tradičními a dle mého názoru velmi důležitými, s nimiž se v praxi často setkáváme, a proto považuji za důležité nahlížet na ně komplexně, což je z mého úhlu pohledu možné pouze v případě, že je čtenáři osvětlena nejen jejich současná právní úprava, ale také jejich historický vývoj.ObhájenoThe thesis is focused on the issues of The Compulsory Share and Related Matters in Civil law. I concentrate on the evolution of the institute of compulsory share and its relating matters i.e. forced heirs, collation with respect to a compulsory share and disinheritance. Alongside through by summarization of historical progress of inheritance law I outline the inspiration for legal adjustment of these matters covered in current Czech Civil Code by the previous Civil codes and show the development line of the compulsory share institute. Institutes of the inheritance law are traditional and by my opinion very important ones, with whom we come across in practice often, and because of that I find it crucial to look at them in its complexity, which is possible only if the reader understands not only related current legislation but also its historical development

Interaction of the Mouse Polyomavirus Capsid Proteins with Importins Is Required for Efficient Import of Viral DNA into the Cell Nucleus

The mechanism used by mouse polyomavirus (MPyV) to overcome the crowded cytosol to reach the nucleus has not been fully elucidated. Here, we investigated the involvement of importin α/β1 mediated transport in the delivery of MPyV genomes into the nucleus. Interactions of the virus with importin β1 were studied by co-immunoprecipitation and proximity ligation assay. For infectivity and nucleus delivery assays, the virus and its capsid proteins mutated in the nuclear localization signals (NLSs) were prepared and produced. We found that at early times post infection, virions bound importin β1 in a time dependent manner with a peak of interactions at 6 h post infection. Mutation analysis revealed that only when the NLSs of both VP1 and VP2/3 were disrupted, virus did not bind efficiently to importin β1 and its infectivity remarkably decreased (by 80%). Nuclear targeting of capsid proteins was improved when VP1 and VP2 were co-expressed. VP1 and VP2 were effectively delivered into the nucleus, even when one of the NLS, either VP1 or VP2, was disrupted. Altogether, our results showed that MPyV virions can use VP1 and/or VP2/VP3 NLSs in concert or individually to bind importins to deliver their genomes into the cell nucleus