| Summary: | Minute Virus of Mice (MVM) is a member of the Parvovirinae genus of the
Parvoviridae family of viruses. This family of small, single-stranded DNA viruses infect
a wide range of eukaryotic hosts ranging from insects to humans. Due to their small size
and limited coding capacity parvoviruses may serve as a suitable tool with which to
examine elements of the host cell DNA replication machinery. Previous studies on
MVM have localized a region of approximately 200 nucleotides inboard of the right viral
genomic termini, known as the Internal Replication Sequence (IRS), which contributes in
cis to viral replication competence.
A comprehensive library of linker scanning mutations across the IRS of MVM
was constructed and assayed in the context of a minigenomic system for replication
competence in an effort to identify short sequence elements contributing to viral
replication. Three elements required for efficient replication were observed. Elements of
the library were also examined for interactions with host cell nuclear factors, and such
interactions were localized to four sites with evidence being obtained to suggest positive
interactions between the sites. These sites were found to be directly adjacent to two of
the elements required for efficient replication and overlapping the third, suggesting a
possible correlation between these functions. Simultaneous deletion of two of these
binding sites was observed to abrogate function of the IRS, further supporting a
functional relationship between factor binding and origin activity. The sequence element found to both bind, host factors and be required for
replication competence was employed as 'bait' in a yeast one-hybrid genetic screen of a
murine cDNA library in an attempt to clone interacting factor(s). A clone recovered from
this, while not considered likely to be directly relevant, leads to postulation of a known
origin binding protein RIP60 as a prospective candidate for action at the IRS origin.
Preliminary studies conducted to determine whether RIP60 binds at the viral origin were
conducted but failed to provide evidence of RIP60 association with the viral genome.
A model is proposed whereby a leading-strand only origin of DNA replication
within the region of the IRS affords a mechanism for the viral rearrangement of its 5'
termini from an extended to a hairpin form during replication. Studies employing the
viral IRS and right-hand terminus as an origin driving the replication of attached
unrelated vector sequences are presented in support of this model.
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