Fig 8.

Analysis of viral NLS and rescue mutants. (A) Multistep growth kinetics of HCMV wild type, the revertant TB-UL48mut-bC2_REV, and respective virus mutants were performed using HFFs infected at an MOI of 0.01 PFU. Each virus was controlled in triplicate, and each growth kinetic was repeated three times. The virus yield of each supernatant was determined from day 0 every 3 days until day 18 postinfection. Growth curves include the mean of all determined virus yields, including the standard deviation. (B) Intracellular distribution of pUL48 was controlled for the revertant and mutant viruses in comparison to the wild type in indirect immunofluorescence experiments of infected HFFs at 3 dpi. pUL48 was detected by indirect immunofluorescence in wild-type, TB-UL48mut-bC2_REV, and TB-UL48NLSrepl36 virus-infected cells with our polyclonal anti-pUL48 serum and by EGFP fluorescence in cells infected with TB-EGFP-UL48 and TB-UL48NLS-rsc viruses. In addition, the tegument protein pp28 was detected with an anti-pp28 MAb. Nuclei were stained with DAPI.