DEFINED LARGE-SCALE ALTERATION OF THE HUMAN CYTOMEGALOVIRUS GENOME CONSTRUCTED BY COTRANSFECTION OF OVERLAPPING COSMIDS

We have constructed defined human cytomegalovirus (CMV) mutants by cot ransfecting overlapping cosmid clones spanning the 230-kbp genome, Usi ng this strategy, we have introduced a 13-kbp region of DNA from a vir ulent strain of CMV into a defined position within the avirulent CMV(T owne) genome. Although more than 80% of the genome of these recombinan t viruses was derived from Towne DNA, their plaque morphology more clo sely resembled that of Toledo. To date, CMV is the largest virus and r equires the greatest number of cosmids to be regenerated via overlappi ng cosmid cotransfection.