FIG 2.

Comparison of ICP27t2 and ICP27 in transfected cells. (A) Expression of ICP27t2 from pT2-27. Vero cells were transfected with pT2-27, harboring ICP27t2, or pBH27, harboring ICP27. At 2 days posttransfection, total protein extracts were prepared and analyzed by immunoblotting using MAb H1119. (B) Stimulation of gC expression from a transfected plasmid. Vero cells were transfected with vector DNA only or with pgCΔpro minus or plus pBH27 or pT2-27. Total proteins were harvested after 2 days, and gC levels were analyzed by immunoblotting. (C) Regulation of gC mRNA. Vero cells were transfected as shown, and total cell RNA was prepared 2 days posttransfection and subjected to Northern analysis with a gC gene-specific probe. The band labeled “u” corresponds to unspliced gC mRNA, while the band labeled “s” corresponds to the spliced form. (D) Analysis of effects on ICP0 and ICP4 localization. Vero cells growing on coverslips were transfected with an ICP0-carrying or ICP4-carrying plasmid, minus or plus plasmids harboring ICP27 (pC27) or ICP27t2 (pT2-27). The cells were fixed 1 day later and analyzed by immunofluorescence using MAb specific for ICP0 or ICP4. (E). Complementation of HSV-1 ICP27 null mutant d27-1. Vero cells were transfected in triplicate with pUC19 DNA as a control, or with pBH27 or pT2-27 DNA. One day posttransfection, the cells were infected with ICP27 mutant d27-1 and incubated for a further day. Viral titers were determined by plaque assay on V27 cells. Error bars denote standard errors of the means.