Identification of the role of VP19C NES in HSV-1 life cycle. (A) RFLP analysis of BAC recombinant variants. Parental p17-37BAC, pVP19C NESm/Kan/17-37BAC and pVP19C NESm/17-37BAC were digested with Bam HI. The sizes of the molecular markers are shown on the left. (B) VP19C NESm/17-37BAC recombinant virus reconstitution. pVP19C NESm/17-37BAC DNA electroporated into Vero cells caused cytopathic effects (CPE) (middle panel) with green fluorescence (right panel), while no CPE were observed in mock-transfected Vero cells (left panel). (C) The sequence of the VP19C NESm recombinant virus at the VP19C loci is shown. (D) The growth properties of wild-type HSV-1, parental 17-37BAC and VP19C NESm/17-37BAC were compared. Vero cells were infected with wild-type HSV-1, parental 17-37BAC or VP19C NESm/17-37BAC virus at an MOI of 1, and virus was harvested at the indicated time points and titrated on a Vero monolayer. The data plotted show the mean of three independent experiments. (E) Individual plaque area assays of wild-type, parental and recombinant VP19C NESm/17-37BAC virus are shown. The diameter of 80 plaques was measured for each virus, and the means ± standard deviations of the diameters were calculated and are shown. (F) Immunofluorescence assay for detecting the subcellular localization of VP19C in wild-type HSV-1 and VP19C NESm/17-37BAC virus-infected cells. Vero cells were infected with wild-type HSV-1 or VP19C NESm/17-37BAC virus at an MOI of 3 for 12 h and then probed with antibody against VP19C. (G) Western blot analysis of virion lysates was performed. Vero cells were infected with wild-type HSV-1, parental 17-37BAC or the VP19C NESm/17-37BAC virus at an MOI of 3. Expression was determined in cell lysates by Western blot analysis with antibodies against VP19C, VP22 and VP23. (H) The relative expression of virion proteins in the lysates was analyzed using Quantity One Imaging Software (Bio-Rad).