replication characteristics of herpes simplex virus type-1 (hsv-1) recombinants in 3 types of tissue cultures

a complication in the analysis of the role of icp34.5 gene in the herpes simplex virus type-1 (hsv-1) lifecycle is the presence of overlapping antisense gene, open reading frame p (orf p), which is also deleted in hsv-1 icp34.5 negative mutants. a hsv-1 wild type strain (17+) icp34.5/orf p deletion mutant (1716) is totally avirulent in animal models and impaired in a number of in vitro functions: replication in 3t6 cells and replication and shutoff of cellular protein synthesis in sk-n-sh cells. to attribute characteristics of 1716 to each of these two genes (icp34.5 or orf p), a number of hsv-1 recombinant viruses that express icp34.5 and orf p independently were constructed, purified and characterized. the parent of these recombinants is 1716 and they are (so called): 1622, expressing icp34.5 1624/24.5, expressing orf p and 1625, expressing both icp34.5 and orf p in separate loci. using homologous recombination in cell culture, the recombinants were constructed and their dna were analyzed by southern-blotting. expression of icp34.5 and orf p from the recombinants was checked by western-blotting. using cell culture, titration and plaque assay techniques, replication kinetics of the recombinants were compared with 17+ and 1716 in 3 cell lines, bhk, 3t6, and sk-n-sh. the results showed that (i) icp34.5 restored the ability to replicate and prevent host shutoff similar to wild type in sk-n-sh cells (ii) icp34.5 restored the replication phenotype to near wild type levels in 3t6 cells and (iii) orf p expression had no effect on the replication of these mutants. the characteristics of 1716 is obviously due to the lack of icp34.5 and orf p has no role in the characteristics studied. thus, the function of orf p still has to be determined.