In recent years it has become common practice to use viruses in the development of new methodologies for improving strategies useful to human health. The knowledge of the genetics and biology of viral-host interactions, as well as the establishment of the structural configuration of the viral particles, has proved pertinent in validating the system adopted. One of the best studied viruses is the oncogenic murine virus polyoma (Py). Two of its characteristics render it a favourite object of study: the shortness of; its DNA genome (5296 bp) that encodes only three early and three late proteins and the advantage of having mouse, genetically the best analysed mammal, as its natural host. ! the three early genes code for multifunctional proteins that control the interaction with the host cells and regulate transcription and replication of the virus. The three late proteins form the capsid in which the viral genome is packed. VP1 is the major of these three proteins in size and in relative amounts (85%) in the mature capsid. Its' role was considered, until very recently, as mainly structural. Its three dimensional structure has been determined, as well as that of the mature capsid. VP1 forms pentamers in-vivo and can polymerise to form ``infectious'' pseudocapsid structures, as has been shown by one of the participating groups. Mutations in VPl have been selected which display tissue specific modified host range of growth and degree of tumorigenicity in-vivo (point mutation) or with altered host range in-vivo and in-vitro (amino acid deletions). The deletion of these latter mutants (selected by one of the participating groups) involves the D-E loop of the protein. This loop is exposed in the outer face of the viral capsid and is highly antigenic. The genetic analysis of these mutants strongly suggests an active involvement of VPl in the formation of transcriptional-replicative complexes involved in virus expression and growth.
The aim of this proposal is to produce an efficient vector for gene transfer and for immunological purposes taking advantage of the genetic knowledge of polyomavirus-; host interaction. In fact, it is a quite unique condition to be able to study in a well genetically defined animal model a system of gene transfer that can be easily adapted to other mammals. The modifications we propose in order to achieve new cell specificity or immunological properties are based on principles that basically are conserved in all higher organisms. The specific questions that the five research groups pose concern which aminoacid, modifications of the VPl molecule, compatible with its structural constraints, can be introduced to: 1) alter the viral in-vivo host specificity of absorption and growth; 2)determine which modifications are useful for pseudocapsid-cell specificity, 3) optimise the pseudocapsid formation as a stable carrier of heterologous DNA and other biologically relevant molecules into mammalian cells; 4) modify pseudocapsids antigenic, properties so as to induce in-vivo the production of antibodies against epitopes of health interest, testing the possibility for using capsids and pseudocapsids for immunological therapeutic purposes. In order to develop these studies it is essential to acquire knowledge of VPl structural properties that will allow the introduction of modifications useful for the alteration of the in-vivo viral growth properties, the' optimization of its potentiality to form pseudocapsids with different properties such as, the size, the tissue specificity and organ routing, and the alteration of its antigenic properties.
To achieve the proposed objectives a more detailed knowledge of the interaction of VP1 with host-cell proteins is needed. Therefore, we programme additional research on this subject using methodologies that can give further information on specific protein-protein interactions involving VP1.
Funding SchemeCSC - Cost-sharing contracts
128 44 Praha 2
141 86 Huddinge