Dissertation for Scientific Degree of Dr.biol. (in Molecular Biology)
Tatyana Voronkova
Generation of polyomavirus-derived virus-like particles as a carrier for gene transfer
Universitv of Latvia, Riga.
SUMMARY
Polvomaviruses are non-enveloped, double-stranded DNA viruses. Their capsids are composed ofthree proteins: VP1, VP2, VP3 vvhere VP1 represents the major capsid protein. As the DNA replication of polyomaviruses takes place in the nucleus the capsid, namely the VP1, possesses a nuclear localization signal. The VP1 of different polyomaviruses expressed in heterologous systems fonns virus-like particles (VLPs) without need for the minor proteins VP2 and VP3. Those VP1-derived VLPs are very similar to the native virions in morphological, immunological and functional respect. Purified VP1 VLPs bind to human B- and T-cells, as well as to monkey kidney cells. Therefore, VP1-derived virus-like particles have become an interesting vehicle for gene transfer experiments.
Furthermore, virus-like particles are usually highly antigenic and immunogenic due to the multiple presentation of their epitopes in a native conformation. The presentation of foreign epitopes on the surface of so-called chimeric VLPs was demonstrated to improve the antibody response against them. Thus, new candidate vaccines have been developed on the basis of VLPs.
The aim of our study vvas to establish the hamster polyomavirus (HaPV) capsid as a new carrier for foreign nucleic acids in gene therapy and for foreign epitopes as the basis for vaccine development. The first goal of the study was the analysis of the synthesis in E. coli cells and self-assembly ability of the HaPV major capsid protein VP 1 in different environment conditions. The second goal vvas to predict and experimentally map B-cell epitopes in the VP1. This epitope mapping should be the basis for the selection of potential sites for foreign sequence insertion or deletion ofunwanted epitopes (for the use in the gene transfer).
The VP1 ofHaPV has expressed in E.coli cells at a high level. Two entire HaPV VP1-coding sequences, starting with the authentic and a second upstream ATG were cloned and expressed to high level. VP1-derived VLPs and capsomers could be demonstrated already in the cytoplasm of E.coli cells. Large amounts of VLPs were obtained by purification using Sepharose CL4B column chromatography. The capsid-like assemblies can be stabilized at low ionic strength by the addition of calcium. Hovvever, the presence of Ca2+ did not othenvise appear to influence basic VLP morphology when examined in the electron microscope. The purified VP1 protein assembled spontaneously into VLPs with a structure reasembling that of the native HaPV capsid. These particles has been dissociated and subsequently reassociated in appropriate conditions. During this procedure fbreign plasmid DNA could be encapsidated m vitro. The complexation of the plasmid DNA with the VLPs resulted in its resistance against the action of DNase I. Thus exogenous DNA can be efficiently packaged into VP1 VLPs.
For epitope mapping a VP1 expression library was generated in E. coli cells. This library is based on the generation of gene fusions of the coat protein gene of bacteriophage fr and PCR-amplified segments of the VP1-encoding sequence. The immunoreactive regions were determined by reacting of antibodies vvith nested sets of deletions fragments. By the means of that library several distinct epitopes vvere mapped in the C-terminal region of HaPV-VP1. The mapped epitope regions are potential sites for the insertion of foreign epitopes or other ligands, as receptor binding regions. The present work is decribed in 3 papers and presented on 5 intemational conferences.
List of original papers
1. Hassen Siray, M. Ozel, B. Jandrig, T. Voronkova. W. Jia, R. Zocher, W. Arnold, S. Scherneck, D. H. Kruger, R. Ulrich (1998), Capsid protein-encoding genes of hamster polyomavirus and properties ofthe viral capsid. Virus Genes 18:1, 39-47
2 Hassen Siray, C. Frommel, T. Voronkova. S. Hahn, W. Arnold, J. Schneider-Mergener, S. Scherneck, R. Ulrich. The C-terminal region of hamster polyomavirus VP1 bears an immunodominant, crossreactive B-cell epitope. FEBS Letters, in press
3. T. Voronkova. A. Kazaks, B. Jandrig, H. Siray, W. Arnold, D. H. Kruger, S. Scherneck, R. Ulrich. The C-terminal region of the hamster polyomavirus major capsid protein VP1 carries several distinct epitopes. Intervirology, submitted