Construction of replication-defective (E1−) Ad vectors expressing gE or gI. The full-length gE and gI genes were subcloned into the Ad shuttle plasmid pCA3, which contains the left end of the Ad type 5 genome. In pCA3gE and pCA3gI, the gE and gI genes, respectively, were flanked by the HCMV immediate-early promoter and the SV40 poly(A) sequence, so that transcription was in the leftward direction, opposite the direction of E1 transcription. Recombinant Ads expressing either gE [Ad(E1−)gE] or gI [Ad(E1−)gI]) were obtained by cotransfecting 293 cells with pBHG10 and either pCA3gE or pCA3gI by the calcium phosphate technique. mu, map units.