Analysis of the internal replication sequence of minute virus of mice

• Main Author: Brunstein, John
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/8603

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.