Fast screening procedures for random transposon libraries of cloned herpesvirus genomes: Mutational analysis of human cytomegalovirus envelope glycoprotein genes

We have cloned the human cytomegalovirus (HCMV) genome as an infectious bac terial artificial chromosome (BAC) in Escherichia coli. Here, we have subje cted the HCMV BAC to random transposon (Tn) mutagenesis using a Tn1721-deri ved insertion sequence and have provided the conditions for excision of the BAC cassette. We report on a fast and efficient screening procedure for a Tn insertion library. Bacterial clones containing randomly mutated full-len gth HCMV genomes were transferred into 96-well microtiter plates, A PCR scr eening method based on two Tn primers and one primer specific far the desir ed genomic position of the Tn insertion was established. Within three conse cutive rounds of PCR a Tn insertion of interest can be assigned to a specif ic bacterial clone. We applied this method to retrieve mutants of HCMV enve lope glycoprotein genes. To determine the infectivities of the mutant HCMV genomes, the DNA of the identified BACs was transfected into permissive fib roblasts. In contrast to BACs with mutations in the genes coding for gB, gH , gL, and gM, which did not yield infectious virus, BACs with disruptions o f open reading frame UL4 (gp48) or UL74 (gO) were viable, although gO-defic ient viruses showed a severe growth deficit. Thus, gO (UL74), a component o f the glycoprotein complex III, is dispensable for viral growth. We conclud e that our approach of PCR screening for Tn insertions will greatly facilit ate the functional analysis of herpesvirus genomes.