MUTAGENESIS AND CHARACTERIZATION OF A 41-KILOBASE-PAIR REGION OF THE PSEUDORABIES VIRUS GENOME - TRANSCRIPTION MAP, SEARCH FOR VIRULENCE GENES, AND COMPARISON WITH HOMOLOGS OF HERPES-SIMPLEX VIRUS TYPE-1

We mutagenized and characterized a 41-kilobase-pair subgenomic cloned fragment from the unique long (UL) region of pseudorabies virus (PRV). Forty mutant clones, each carrying a single inserted oligonucleotide, were used for cotransfection with overlapping cloned viral DNA fragme nts. More than half of these transfections yielded viable virus mutant s. Short viral DNA fragments, flanking the oligonucleotide insertions, were cloned and used as probes on Northern blots with RNA isolated fr om cells infected with wild-type PRV. In this way we were able to cons truct a partial transcriptional map of this region of the PRV genome. In addition, we used these probes in cross-hybridization studies with cloned genomic fragments from the prototype alphaherpesvirus herpes si mplex virus type 1 (HSV-1). This allowed us to define homology between the corresponding regions of these viruses. Most viable PRV mutants w ere assayed for virulence in mice. Mutagenesis of the identified homol ogs of HSV-1 genes UL39 (encoding the large subunit of ribonucleotide reductase), UL40 (small subunit of ribonucleotide reductase), UL42 (DN A polymerase accessory factor), UL23 (thymidine kinase), UL21 (a capsi d-associated protein), and UL12 (alkaline nuclease) completely abrogat ed or strongly reduced virulence. (C) 1994 Academic Press, Inc.