Autonome Parvoviren

Oncosuppressive properties and a low pathogenicity in adult animals make the two rodent parvoviruses MVMp and H1 promising vectors for cancer gene therapy. Recombinant vectors were developed from genomes of these viruses in which a part of the capsid-protein-coding sequence was replaced by a transgene. These recombinant viruses can replicate their genome and express their transgene, but they are unable to produce progeny viruses. Such vectors are produced by cotransfection of the recombinant virus genome and a helper plasmid which provides the capsid proteins. During this procedure, RCVs (Replication Competent Viruses) can be generated, probably by homologous recombination. As a consequence, RCVs can express their capsid proteins and produce progeny viruses. In the present work, second-generation H1- and MVMp- based recombinant virus genomes were developed in order to minimise RCV levels in the recombinant virus preparations. By constructing chimeras and pseudotypes between MVMp and H1 viruses, virus preparations with high titres were obtained which did not show any detectable RCVs in plaque-assays. This corresponds to a 100-17000fold decrease of RCVs for the chimeras and pseudotypes. In this work we also characterised the host-cell-tropism of the chimeras and the pseudotypes. The chimeric vectors and Pseudotypes allowed us to show that the restriction for viral replication of the rat H1 genome in murine A9 cells is not due to the H1 capsids, but can be ascribed to a 1800 bp long DNA region of the H1 genome, located in the NS coding sequence. To our knowledge this is the first report that convincingly shows that the tropism of a rodent parvovirus is determined by the viral genome. Since a recombinant MVMp genome packaged with either H1 or MVMp capsids led to viral DNA replication in A9 cells, virus entry is not responsible for the lack of recombinant H1 vector replication in these cells. After transfection of A9 cells with H1 viral DNA, replication and viral protein expression took place, whereas after H1 infection it did not. Thus we can conclude that the cause for the restricted H1 virus replication has to be found after virus entry but before the beginning of viral gene transcription. It is usually accepted that viral gene transcription needs the conversion of single-stranded Summary 134 DNA into double-stranded monomeric replicative form. Most probably this conversion reaction is disordered in A9 cells for the H1 genome. A defective double-stranded conversion might be due to an interaction of VP proteins with the viral single stranded DNA sequence. Another possibility is an interaction of a cellular protein with the viral single stranded DNA which then leads to an inhibition of the viral DNA conversion. It might be also possible that a cellular factor which is needed for the conversion reaction can not bind to the H1 genome in A9 cells. Interestingly, in most of the tested murine cell lines the tropism was determined by the MVMp genome, while for all human cell lines the tropism was determined by the virus capsid. The second part of this work focused on the effect that H1 virus infection on cellular gene expression. For this purpose, the cDNA array technique was used. It was previously shown that parvovirus H1 infection leads to the activation of caspase 3 and apoptotic cell death of the human monocytic U937 cell, in a way similar to the apoptotic cell death induced by TNFα. Moreover, the c-myc gene, overexpressed in these cells, is down-regulated during H1 infection. In agreement with these data, we were able to show -by comparing the gene expression profiles between buffer treated and H1 infected synchronised U937 cells- genes coding for caspases 2, 4 and 8 were upregulated after H1 infection. In addition, different genes associated with the TNFα complex were found to be up-regulated, as well as the mad4 gene, which is able to inhibit the transcriptional activity of c-myc by binding its main partner max. Moreover, 2 genes involved in oestrogen-synthesis were found to be up-regulated after infection. Oestrogen was shown to have some protective effect against TNFα induced apoptosis in U937 cells. Induction of these genes might reflect a defence mechanism of the cell. Also, a gene encoding for a subunit of the 26s proteosome and different genes involved in differentiation processes or in apoptosis were up-regulated in infected cells. Few genes were shown to be down-regulated after infection, such as the adenylate cyclase and cAMP dependent transcription factor 1. Altogether, our data give a better understanding of the molecular pathways which are used by the parvovirus and enlighten the diversity of cellular responses to a H1 virus infection in U937 cells.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Ludwig-Maximilians-Universität

Year

2002

Author dc:creator

• Wrzesinski, Claudia

Repository record source_url

https://edoc.ub.uni-muenchen.de/1718/

OAI identifier oai:identifier

oai:edoc.ub.uni-muenchen.de:1718