Parvoviruses are unique among all known organisms in having DNA genomes which are both single-stranded and linear. The long term goal of this research program is to understand, at the molecular level, how these viruses inveigle their host cell into replicating and packaging such apparently alien molecules. The autonomously replicating parvoviruses are incapable of inducing resting cells to enter S-phase and thus replicate exclusively in dividing cells. This constraint appears to underlie the ability of vectors derived from them to induce persistent immunity to antigens they are constructed to encode. The model we will explore is the autonomous parvovirus Minute Virus of Mice (MVM), which, although a murine virus, can be replicated by human proteins both in vivo and in vitro, in the presence of its own replication initiator polypeptide NS1. We will exploit a newly-developed reverse genetic system to explore structural aspects of the viral hairpin telomeres, particularly that at the left-end of the genome, in an attempt to learn why the virus requires this telomere to be in a single sequence orientation. We will use degenerate oligonucleotide selection approaches, combined with an in vitro nicking reaction reconstituted with recombinant viral and cellular proteins, in order to determine the consensus binding and nicking sites for the viral initiator protein NS1. This information will be used to explore further the mechanism of concatemer resolution in a reconstituted in vitro system. The occupancy of the many NS1 binding sites throughout the genome will be explored in vivo in order to establish the extent to which the virus assembles """"""""pseudochromatin"""""""" during infection, in which NS1 substitutes for the normal role of cellular histones. Model genome substrates will be used to determine whether these NS1 interactions potentiate viral DNA replication and/or packaging of progeny single strands. Novel recombinant genomes and viral assembly mutants will be used to explore the nature of the packaging substrate, the mechanism of strand selection, and to develop an in vitro packaging system. The knowledge gained from these studies can be applied directly to the development of safer and more efficient vaccine vectors for potential use in animals and humans. ? ? ?
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