Transfection. PacI-digested cosmids Tn23, Tn26, Tn44, Tn45, Tn46, Tn47, Tn51, and Tol11 (10 μm each) (31) were mixed with 2 μg of Sal-digested pON303ΔAcc (or pON303) (16, 32). To construct RC303Acc or Towne/Tol11 1–1, 10 μg of the pON303ΔAcc- or pON303-containing cosmid mixtures were used to transfect 106 ihfie1.3 cells by the calcium phosphate precipitation technique as described (31).
DNA Blot Hybridization and Immunoblot Analyses. Total infected cell DNA was isolated (33) from infected HF or ihfie1.3 cells; 1 μg was digested with EcoRI, ClaI, or BamHI (New England Biolabs) using the manufacturer’s recommended conditions and was transferred to Hybond-N+ (Amersham) before hybridization with [32P]dCTP (Amersham) random-primed radiolabeled pON303 or pON2307 (an exon 4-specific probe) (34). Immunoblot analysis was performed according to published protocols using human CMV-seropositive antiserum, murine monoclonal antibodies CH443 (specific for exon 4; unpublished results) and CH160 (specific for exon 2; ref. 35), renamed 1203 (Goodwin Institute, Ft. Lauderdale, FL), or rabbit polyclonal antibody raised against the glutathione S-transferase fusion protein carrying aa 232– 400 of IE1491aa from exon 4. Horseradish peroxidase-conjugated goat anti-human IgG, goat anti-rabbit IgG, and goat anti-mouse IgG (Vector Laboratories) were used as secondary antibodies and blots were developed using the Enhanced Chemiluminescence System (Amersham) according to the manufacturer’s protocol.
Construction of RC303ΔAcc. Recombinant viruses were constructed from seven Towne strain cosmids (Tn23, Tn26, Tn44, Tn45, Tn46, Tn47, and Tn51) and one Toledo strain cosmid (Tol11) (31) that formed an overlapping set except for a 15-bp gap between Tn51 and Tol11 (170,506–170,520) as depicted in Fig. 1. This gap was bridged with either pON303ΔAcc (16) or pON303 (32), resulting in the production of either ie1-deficient RC303ΔAcc or wild-type (wt) Towne/ Tol11 1.1 (Fig. 1). We used established transfection conditions for generation of recombinant CMV (31). SalI-linearized plasmid was added in a molar amount equivalent to an individual of PacI-linearized cosmids and cotransfection of this mixture was carried out on ihfie1.3 cells to complement IE1491aa in trans. When the genome structure of the resulting recombinants was analyzed, two of five independent pools arising from the pON303ΔAcc-containing set carried a uniform population of the expected mutant, and two pools made with pON303 contained the expected Towne/Toledo wt chimera (31). Blot hybridization of EcoRI-digested DNA from mutant RC303ΔAcc and wt Towne/Tol11 1.1 transfection pools and from plaque pure RC303ΔAcc produced the expected patterns (Fig. 2). Hybridization with pON303 probe revealed a 10-kbp EcoRI fragment in the wt virus, which was reduced to 8.6 kbp in the mutant (Fig. 2A). An ie1 exon 4 probe (pON2307) hybridized with the 10-kbp fragment in Towne/ Tol11 1.1 but failed to hybridize with any fragment in the mutant. A 3.9-kbp ClaI fragment containing ie1 was reduced to 2.5 kbp as a result of the deletion (Fig. 2B). After evaluation of BamHI digests (data not shown) and EcoRI and ClaI digests, we concluded that the two independent RC303ΔAcc isolates and two independent Towne/Tol11 1.1 isolates exhibited no differences outside of the 1.4-kbp deletion that had been engineered into the mutant virus.