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Cells and Virus. African green monkey kidney (Vero and CV-1) and human embryonic lung (HEL) cells were propagated in Dulbecco modified Eagle minimal essential medium containing 10% fetal calf serum, 0.03% glutamine, and 0.25% NaHCO3. Vero cells were utilized for growth of virus stocks, plaque assays, complementation tests, infectivity assays, 796

We report the construction and characterization of deletion mutants in the herpes simplex virus type 1 gene encoding the immediate-early protein ICPO. In the event that ICPO proved to play an essential role in virus replication, ICP0-transformed Vero cells were generated to serve as permissive hosts for such mutants. Two mutants, dlX0.7 and dlX3.1, were isolated in these cells by a marker rescue-transfer procedure involving the rescue of an ICP4 deletion mutant and the simultaneous insertion of a linked deletion in the ICPO gene. Mutant dlX0.7 contained a 700-base-pair deletion in both copies of ICPO. The deletion lay entirely within the transcript specified by the gene. dlXO.7 induced the synthesis of an ICP0-specific mRNA that was-0.7 kilobases smaller than the corresponding mRNA specified by wild-type virus. The 3.1-kilobase deletion in both copies of the ICPO gene in mutant dlX3.1 removed the majority of the transcriptional-regulatory signals and coding sequences, retaining only sequences at the 3' end of the gene. As expected, no ICPO-specific mRNA was detected in dIX3.1-infected Nero cells (G418-resistant Vero cells). Both mutants grew in all cells tested, although their burst sizes were 10to 100-fold lower than that of wild-type virus. Although the plaque sizes of dlXO.7 and dlX3.1 were equally small on Nero and ICPO-transformed cells, the plating efficiency of the mutants was 15to 50-fold greater on ICPO-transformed cells than on Nero cells. The mutants exhibited modest interference with the growth of wild-type virus in mixed infections, an effect that was abolished by UV irradiation of the mutants, implying that interference required viral gene expression. Polypeptide proffles generated by the mutants in Nero cells were qualitatively similar to that of wild-type virus. Quantitatively, only slight reductions in the levels of certain late viral polypeptides were observed, a phenomenon also borne out by analysis of viral glycoproteins. Both mutants induced the synthesis of significant, although reduced, levels of viral DNA relative to wild-type virus. Taken together, the results demonstrate that ICPO is not essential for productive infection in cell culture but that this protein plays a significant role in viral growth, as indicated by the impaired abilities of the mutants to replicate.

We have used suspension-cultured parsley cells (Petroselinum crispum) and an oligopeptide elicitor derived from a surface glycoprotein of the phytopathogenic fungus Phytophthora megasperma f.sp. glycinea to study the signaling pathway from elicitor recognition to (Fig. 1). Following leaf inoculation with fungal zoospores, cyst formation, germination, and formation of appressoria and infection vesicles (-4

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