FIG. 2.

Construction and confirmation of vil-17 mutants of herpesvirus saimiri C488. (A) The neo^r gene from plasmid pSV2neo was inserted in the antisense orientation into the XmnI cleavage site of plasmid x50 (14; accession no. Y13183) a few nucleotides downstream of the start codon for vIL-17. (B) Southern blots were prepared with HindIII-digested viral DNA from wild-type virus and from cloned vil-17 mutants from two independent recombination experiments. After hybridization with the radioactively labeled insert from plasmid x50, the expected band sizes for both the wild-type (given on the right) and the mutant (given on the left) viruses were observed. (C) The presence of the mutation in recombinant viruses was confirmed by PCR for vil-17, whereas the stpC gene was present in all samples. (D) In order to exclude potential artificial transcripts initiated within the SV2neo cassette, RNase protection assays were performed. The riboprobe comprised the transition region from the neo^r gene to the vil-17 open reading frame. Whereas wild-type viruses showed the protected fragment corresponding to the viral sequence (229 nt), protected fragments were not observed in RNA from mutant virus-infected OMK cells.