Analysis of the UL77 protein expression in cells infected with the recombinant virus with or without insertion of a stop codon downstream of the ATG start codon for UL76 ORF.

(a) Quantity of viral major immediate early (IE1/2), early p52 (UL44), and UL77 gene transcription with the recombinant viruses. RNAs were analyzed with MIE, UL44, and UL77 specific primers and probes by real-time RT-PCR at 2, and 3 d. p.i. as described in the Materials and Methods. The assay was performed in triplicate, and the standard error of the mean was determined. HCMV RNAs were normalized to G6PD RNA, and each value was relative to the level of the RflagUL76revertantflagUL77 RNA at 3 d. p.i. (b) Western blot analysis of immediate-early pIE72 (UL123), pIE86 (UL122), early p52 (UL44), and flag-pUL77 proteins at the indicated times after infection with RUL76stopflagUL77 or RUL76revertantflagUL77 at an MOI of 1. Lanes: 1 and 2, 1 d.p.i.; 3 and 4, 2 d.p.i.; 5 and 6, 3 d. p.i.; 7 and 8, 4 d p.i.; 1, 3, 5, and 7, RUL76stopflagUL77; 2, 4, 6, and 8, RUL76revertantflagUL77. (c) HFF cells stably expressing EGFP-UL76 fusion protein. The EFGP-UL76 fusion protein in HFF cells was detected by Western blot analysis using the polyclonal antibody against EGFP. (d) Western blot analysis for the detection of the immediate-early pIE72 (UL123), pIE86 (UL122), early p52 (UL44), and flag-pUL77 proteins in HFF cells stably expressing the EGFP (Lanes: 1, 3, 5, and 7) or EGFP-UL76 fusion protein (Lanes: 2, 4, 6, and 8) at the indicated times after infection with recombinant virus RUL76stopflagUL77 at an MOI of 1. Lanes: 1 and 2, 1 d.p.i.; 3 and 4, 2 d.p.i.; 5 and 6, 3 d. p.i.; 7 and 8, 4 d p.i.