FIG 5.
Recombinant CMV mutant lacking expression of pUL31-YFP exhibits an MOI-dependent replication defect. (A) UL31 was disrupted in CMV TB40/E (TBwt) by replacing 1,200 bp of the UL31 ORF with the galK gene (UL31del) or insertion of galK into the first ATG of UL31 (UL31in). The locations of the first two ATG sequences are indicated. (B) MRC-5 fibroblasts were infected using TBGFP, UL31del, or UL31in virus at an MOI of 3. Viral titers from culture supernatants at 72 and 96 hpi were quantified using a TCID50 assay. Data are from three biological replicates and two technical replicates, with error bars representing standard deviations from the means. (C) Whole-cell lysates were collected at 96 hpi and analyzed using Western blotting and the indicated antibodies. (D) A stop codon, TAG, was introduced into the second ATG of AD169 UL31YFP, resulting in UL31stop virus. (E) Fibroblasts were infected with UL31YFP or UL31stop virus at an MOI of 3. Total RNA was collected at 96 hpi and analyzed using qRT-PCR with primers to UL32 (pp150) and GAPDH. Data include three biological replicates and two technical replicates, with error bars representing standard deviations from the means. Whole-cell lysates were analyzed by Western blotting. (F) Fibroblasts were infected with UL31YFP or UL31stop virus at an MOI of 0.05 IU per cell. Whole-cell DNA was isolated at the indicated times, and relative viral genomes were determined by qPCR using primers for UL123 and GAPDH. Viral titers were determined from cell-free virus at 120 hpi. (G) Titers were also determined following infection at an MOI of 3. Data include three biological replicates and two technical replicates, with error bars representing standard deviations from the means.