Thymidine Kinase Deletion Mutants of Herpes Simplex Virus Type 1

Abstract

277 SUMMARYDeletions in the cloned thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-I), strain 17 syn ÷, were produced by two methods. Removal of a 506 base pair fragment from between the unique SstI and BglII restriction endonuclease sites of pTK1 (HSV-1 BamHI p cloned in pAT153) and subsequent transformation of Escherichia coli resulted in the isolation of 50 deleted plasmids. Sequential digestion of pTK1 with BgllI and nuclease BAL 31 followed by ligation and recleavage with BglII resulted in the isolation of 31 deleted plasmids. Three clones, pTK2, pTK3 and pTK4, obtained following BglII and SstI treatment of pTK 1 were recombined with wild-type (wt) HSV-1 (17) syn ÷ DNA in baby hamster kidney (BHK) cells to produce TKdeletion mutants HSV-1 (17)TK 1301, HSV-1 (17)TK 1302 and HSV-1 (17)TK 1303 respectively. 5-Bromo-2'-deoxyuridine, 5-bromo-2'-deoxycytidine and 9-(2-hydroxyethoxymethyl)guanine were used to reduce the background of TK + virus in heterogeneous recombinant stocks analysed for the presence of TK-recombinants. All recombinant clones isolated produced a small syncytial plaque morphology in BHK cells. The mutants HSV-1 (17) TK 1301 and HSV-I (17) TK 1302 were TK-, failed to produce polypeptides of molecular weights 43 000 and 19 000 found in wt-infected cells and demonstrated one-step growth curves different from wt virus and the TK-mutant HSV-I (17)dPyk -7. Superinfection studies with HSV-1 (17)TK 1301, HSV-1 (17)TK 1302, HSV-I (MDK) and HSV-1 (17) dPyk -7 indicated that all TK-mutants except dPyK -7 produce a tram-acting gene product which can switch on the transforming HSV-1 TK gene.

Quantitative analysis of genomic polymorphism of herpes simplex virus type 1 strains from six countries: studies of molecular evolution and molecular epidemiology of the virus

“…DNA extraction from HSV-l-infected cells, RE digestion and agarose gel electrophoresis were described previously (Sakaoka et al, 1985(Sakaoka et al, , 1987a (Locker & Frenkel, 1979;Roizman, 1980: Roizman & Tognon, 1983) with a minor modification (Sakaoka et al, 1987a). The site and fragment nomenclature for A71oI and PvuII were taken from the maps of strain 17 syn + (Sanders et al, 1982) (Fig. 1).…”

“…DNA digests of a standard strain, 17 syn +, and seven isolates (S-9 to S-24) are shown. The fragment nomenclature is that of strain 17 syn + (Sanders et al, 1982). Numbers in parentheses correspond to those in Fig.…”

“…(Locker & Frenkel, 1979;Roizman & Buchman, 19791 with a minor modification (Sakaoka et al, 1987a). XhoI and PvulI maps are based on those for strain 17 syn + (Sanders et al, 1982). The numbers (1 to 63) and labels (B I to Pls) under each map indicate the position of RE sites and fragments that show variation among strains, and correspond to the sites described in Methods and Table 1. Using the 30 most variable sites, Chaney et al (1983 b) analysed 84 HSV-1 isolates obtained from people in Canada, U.K. and U.…”

DNA sequence of the herpes simplex virus type 1 gene encoding glycoprotein gH, and identification of homologues in the genomes of varicella-zoster virus and Epstein-Barr virus

“…For the sequence reported in this paper, fragments used were EcoRI j cloned in pACYC184 (from V.G. Preston) and BamHI p cloned in pAT153 (11).…”

The Effect of Triterpenoid Compounds on Uninfected and Herpes Simplex Virus-infected Cells in Culture. II. DNA and Protein Synthesis, Polypeptide Processing and Transport

“…In normal HSV infection only about 20 ~o of the virus DNA is eventually packaged (Wilkie, 1973). Thus, it seems unlikely that the reduction in virus DNA synthesis observed in some experiments employing CCX have contributed materially to the accompanying massive drug-induced To support the thesis that the triterpenoid compounds' mode of action differs from that of the commonly used anti-herpesvirus drugs that operate in this way, three HSV mutants were studied: HSV-1 (17) TK 1301 and 1302, which both have the TK gene deleted (Sanders et al, 1982), and HSV-2 52 ACG r CC 2321 which is resistant to both ACG and PAA through at least two mutations in the D N A polymerase gene (Crumpacker et al, 1980). Sensitivity of such mutants to triterpenoid drug treatment would imply a different mode of action.…”