FIG. 6.

Effect of M-F UTR on F protein translation and incorporation into infectious virions. (A) Western blot analysis of viral F protein. VerodogSLAMtag cells were infected with the 5804PeH, 58utrMF-NP, 58utrMF-NPΔF₁₀₆, and 58ΔF₁₀₆ viruses at an MOI of 0.01 and overlaid with 0.5% methylcellulose. Cell lysates were extracted at 72 h postinfection and subjected to Western blot analysis. The membranes were probed with rabbit antipeptide sera specific to the CDV P, F, and H proteins. (B) Normalized F and H protein expression levels. Band intensities of the P, F, and H protein bands were determined by densitometry (optical density [OD]) from nonsaturated exposures, using Kodak Molecular Imaging software. The F and H protein expression levels were normalized by calculating the F/P and H/P ratios for each virus. The graph represents the results from three independent experiments. Error bars indicate the standard deviations. Statistical analysis was performed using a one-way analysis of variance with Tukey's multiple comparison test and the asterisk (*) indicates statistically different (P < 0.005) samples. (C) Western blot analysis of F protein incorporation in infectious virions. VerodogSLAMtag cells were infected with the 5804PeH, 58utrMF-NP, 58utrMF-NPΔF₁₀₆, and 58ΔF₁₀₆ viruses, supernatants were collected, and virions were purified by ultracentrifugation through a 20%/60% discontinuous sucrose gradient. Purified virus titers were determined, and 10⁵ TCID₅₀ were loaded in each well. Membranes were probed with rabbit antipeptide sera specific to the CDV P and F proteins. The top panel displays the relative F protein incorporation, expressed as a ratio between the OD values of the P and F protein bands of each virus. The middle panel indicates the relative infectivity, as a ratio between the OD of the P proteins of the respective viruses and that of the parental 5804PeH virus.