Comparison of Thymidine Kinase Activities Induced in Cells Productively Infected with Herpesvirus Saimiri and Herpes Simplex Virus

Abstract

SUMMARYThe replication of unselected strains of herpesvirus saimiri (HVS) was sensitive to bromodeoxyuridine and bromovinyldeoxyuridine (BVdU) but insensitive to acycloguanosine (ACG), in contrast to the growth of herpes simplex virus (HSV) which was sensitive to all three analogues. Mutants of HVS resistant to bromodeoxyuridine and BVdU could be selected by growth in the presence of these inhibitors. Productive infections of owl monkey kidney or Vero cell cultures by unselected strains of HVS resulted in increases in a thymidine kinase (TK) activity which was deficient in cells infected with bromodeoxyuridine-resistant mutants of the virus. Induction of the virus enzyme promoted a net increase in the uptake and incorporation of exogenous labelled thymidine in the face of the progressive inhibition of the overall incorporation of [35S]methionine and [3H]uridine into productively infected cells. The TK induced in cells infected with HVS differed from the major activity of uninfected cells and resembled that encoded by HSV in its capacity to phosphorylate iododeoxyuridine and in the sensitivity of all the thymidine phosphorylating activity to competition by BVdU. However, in contrast to the HSV TK, which phosphorylated deoxycytidine and iododeoxycytidine relatively efficiently and was sensitive to ACG, the HVS enzyme did not phosphorylate deoxycytidine or iododeoxycytidine and was insensitive to ACG. Whilst HVS, therefore, shares the characteristic of other members of the herpesvirus group of inducing a novel TK, the properties of the HVS-induced enzyme differ significantly from the enzyme of the prototype herpesvirus, HSV. The properties of the HVS TK are nonetheless sufficiently distinct from those of the uninfected cell to provide a possible basis for selective antiviral chemotherapy based on preferential phosphorylation of nucleoside analogues such as BVdU by infected cells.

Intercellular delivery of thymidine kinase prodrug activating enzyme by the herpes simplex virus protein, VP22

“…Culture medium was replaced 40 h after transfection with media containing 3 H-thymidine at 20 Ci/ml and incubated for a further 2.5 h. The monolayers were washed, harvested and the amount of 3 H-thymidine incorporated into TCA precipitable material measured by binding on to GF/C glass microfibre filters (Whatman, UK) as previously described. 26 In vitro thymidine kinase assay Monolayers of tk-ve HeLa cells (70% confluent) transfected as described above were harvested 40 h after transfection and extracted in 200 l of a buffer containing 400 mm NaCl as described previously. 10 Aliquots (5 l) were incubated in 0.2 m Tris pH 8.2, 5 mm MgCl 2 , 5 mm ATP, 1 Ci 3 H-thymidine, for 10 min at 37°C and the reaction stopped by boiling.…”

“…Duplicate samples of the mix (20 l) were dried on to DE81 paper discs (Whatman) and counted directly (unwashed control), or washed under vacuum with 3 × 20 ml washes of 1 mm ammonium formate (pH 3.6) and dried for a further 1 h. Bound, phosphorylated, 3 H-thymidine counts were measured in a scintillation counter. 26 Fluorescent viability/cell death assay This assay examines cell viability by measuring calcein-AM uptake and esterification, which results in green fluorescence, and cell death by measuring uptake of ethidium bromide dimers which results in red fluorescence and was performed according to the manufacturer's instructions (Molecular Probes, Leiden, The Netherlands). …”

Asynchronous Expression of the Immediate-Early Protein of Herpesvirus Saimiri in Populations of Productively Infected Cells

“…51K and 110K non-structural DNA-binding proteins and virus-specific thymidine kinase and DNA polymerase activities) and finally by the synthesis of progeny virus DNA and proteins of the third phase, the late gene products (e.g. 160K, 130K and 150K structural proteins : Randall et al, 1983Randall et al, , 1984aHoness et al, 1982;O'Hare & Honess, 1983a, b;Blair & Honess, 1983). However, the growth cycle of the virus and the synthesis of independently regulated virus proteins is relatively protracted in populations of productively infected cells (e.g.…”

Evidence for a Herpesvirus Saimiri-specified DNA Polymerase Activity which is Aphidicolin-resistant and Phosphonoacetate-sensitive

“…This virus stock, HVS [SMB1] pr clone 1, was assayed by plaque titration, in parallel with its sensitive parent virus, in the presence of concentrations of PAA ranging from 0 to 125 Ixg/ml (Table 1). In comparative experiments the herpes simplex virus strains used were HSV-1 [HFEM] P 18 cl [PAA-resistant, HSV-I (pr)] and HSV-I [HFEM/STH2] [PAA-sensitive, HSV-1 (ps)] (Honess & Watson, 1977b); the other HVS strains, HVS [HOT] and HVS [11Att] ts 98B r, have been described previously (Honess et al, 1982).…”

“…Discs were then dried, placed in scintillation fluid and radioactivity counted in a Beckman LS7000 liquid scintillation spectrophotometer. Thymidine kinase activity was estimated as previously described (Honess et al, 1982).…”