FIG. 1.
Generation of plasmids and virus mutants. (A) Schematic map of the PrV genome, which consists of a unique long (UL) region and a unique short (US) region, which is bracketed by inverted-repeat sequences (IR and TR). The positions of BamHI restriction fragments and of genes relevant for this study are indicated. (B) Enlarged map of a plasmid-cloned DNA fragment of PrV-Ka (pUC-B1K) including relevant restriction sites. Transcriptional organization of genes UL50 to UL47 (pointed rectangles) is also shown. Deletion mutants PrV-ΔUL48 and PrV-ΔUL48/49 contain the tetracycline resistance gene (encoding TetR), since they were generated by mutagenesis of an infectious clone of PrV-Ka with plasmid pUC-B1KNNT or pUCB1KXNT in E. coli. (C) A UL48-GST fusion protein was expressed from pGex-UL48, whereas pcDNA-UL48 was used for in vitro transcription from the bacteriophage T7 and SP6 promoters (PT7, PSP6) and subsequent in vitro translation. Plasmid pIRES-UL48 contains a bicistronic transcription unit of the UL48 gene and the neomycin resistance gene (NeoR), which are separated by an intron (IVS) and an internal ribosomal entry site (IRES) and which are flanked by the human cytomegalovirus immediate-early promoter (PHCMV-IE) and a polyadenylation signal [Poly(A)].