Studying a poxvirus gene capture model through recombination and reactivation

Introduction: Vaccinia poxvirus (VACV) is a double stranded DNA virus that replicates in the cytoplasm of infected cells. Some VACV genes resemble homologs of host genes and appear to have been captured from the cell; however, since poxviruses are confined to the cytoplasm, researchers are unclear as to how these viruses acquire this homology (1). If a cellular mRNA was accidentally reverse transcribed into cDNA, which could occur during retrovirus co-infection, a poxvirus might be able to incorporate this sequence into its own genome through rare non-homologous (homology-independent) recombination. Methods: We modeled this process using two different recombination systems and substituted a DNA encoding mycophenolic acid (MPA), a selectable marker, for the hypothetical non-homologous host cDNA. We prepared DNA constructs containing this marker along with 20 base pairs homologous to the 5’ and 3’ flanking regions of the VACV-encoded NotI restriction site. A construct without this flanking homology was also prepared. The “passive” recombination system used a helper poxvirus to reactivate VACV DNA; in contrast, VACV infected BSC40 cells were transfected with the construct in the “active” recombination system. Results: The “passive” recombination system generated 105 PFU/mL of reactivated VACV; however, no recombinants containing the selectable marker were detected. The “active recombination” method generated 106 PFU/mL of total VACV and approximately 10 PFU/mL of recombinant virus for both homology containing and non-homology containing constructs. Discussions: We were unable to determine the recombination frequency of the “passive system” because recombinant virus was not detected. Based on virus titers determined from plaque assays, we approximated the recombination frequency of the “active system” to be ≤ 10-5. We are currently cloning and sequencing viruses resulting from non-homologous recombination to determine where the MPA marker is located. Preliminary analysis of these types of clones (data not shown in this paper) suggests that the transfected DNAs are being incorporated into a diversity of sites, some located near the boundary of the VACV genome where the right terminal inverted repeat begins. In summary, our findings suggest that the recombination frequencies in both methods are very low and better methods of selection are needed to observe these rare events. Future studies of recombinant clones are needed to gain a better understanding of this non- homologous gene capture process.

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