The adenovirus genome is a linear double stranded DNA molecule and the current widely accepted model of viral DNA replication proposes linear viral DNA intermediates at all stages of replication. Although the experimental evidence for this mechanism of replication is very strong, circular forms of adenovirus serotype 5 (Ad5) DNA molecules have also been detected in both permissive and non-permissive cell lines. In some experiments the circular structures were detected before the onset of viral DNA replication and thus suggested a possible role for circular forms of Ad5 DNA in the life cycle of the virus after infection. This study was undertaken to better understand the role of circular forms of the adenovirus genome in virus replication. The approach was to subclone the viral junction internally into the linear genome thus creating mutant viruses with embedded terminal sequences and to study the effect of such inserts on DNA structure. A total of five mutant viruses were constructed containing a variety of inserts and viral DNA from infected cells and banded viruses was analyzed by Southern Blot hybridization. The data clearly showed that embedded viral junctions have biological activity in that they generated novel, rear ranged viral DNA molecules, and that embedded single ITR sequences were also biologically active, but to a lesser extent. It was also observed that the copy numbers of the rearranged molecules were variable. It appeared that the embedded viral junction was active in recombination and replication of the viral genome, creating the rearrangements through these two processes. However, the results suggested that circular forms are not obligatory intermediates in DNA replication. The analysis of banded viral DNA in this study suggested that the encapsidation signal from the left end of the linear genome was required in cis for packaging of the viral genome, confirming previous results which identified an encapsidation signal for viral DNA packaging. The banding experiments also showed that truncated viral DNA molecules containing 75% of the viral genome were packaged.

Master of Science (MS)

Thesis