Evidence that Deletion of Coding Sequences in the 5' End of the Thymidine Kinase Gene of Herpes Simplex Virus Type 1 Affects the Stability of the Gene Products

Abstract

SUMMARYThe experiments described in the present work were designed to study the function of the N-terminal end of thymidine kinase (TK) encoded by herpes simplex virus type 1. Specifically we were interested to know whether this end was involved in binding of the enzyme to other molecules, had any influence on its subcellular localization or affected one or more of the activities associated with the enzyme. A parental enzyme and a deletion mutant, lacking the 45 N-terminal amino acids, derived from this strain, were used. Thymidine kinase from the parental virus bound to DNA-Sepharose, but the truncated enzyme did not. This was apparently not due to a specific ability to bind to DNA, since immunofluorescence studies indicated that both the normal and the deleted TK were mainly located in the cytoplasm, preferentially in the perinuclear region. Phosphorylation of thymidine as well as the amounts of TK potypeptides were markedly reduced at late times after infection with the mutant, but not to the same extent after infection with the wild-type. The deleted TK gene was efficiently transcribed as shown by hybridization of RNA to a probe specific for the gene, and this RNA directed the synthesis in vitro of TK polypeptides. Deletion of the 5' end of the gene seems to affect the stability of either the enzyme or TK-specific mRNA, or both. The TMP phosphorylating activity seems to be particularly destabilized relative to the thymidine phosphorylating activity.

A Truncated Herpes Simplex Virus Thymidine Kinase Phosphorylates Thymidine and Nucleoside Analogs and Does Not Cause Sterility in Transgenic Mice

“…These observations are in agreement with previous studies showing that some herpes simplex virus mutants synthesizing low levels of truncated TK are less sensitive to the antiviral effect of acyclovir or GCV than are wild-type viruses (13). This was correlated with the observation that the stability of truncated TK proteins could be influenced by their amounts (13,24,28). These in vitro observations translate in vivo.…”

Analysis of the Bovine Herpesvirus Type 1 Thymidine Kinase (TK) Gene from Wild-type Virus and TK-deficient Mutants

“…Furthermore deletion of 45 N-terminal amino acids of HSV-1 TK gave rise to a 39K product which (although it retained enzyme activity) was shown to be less stable than wt (Haarr & Flatmark, 1987). These workers suggested that this could be either because the truncated enzyme itself or TK-specific mRNA was more rapidly degraded.…”

Enhanced green fluorescent protein fusion proteins of herpes simplex virus type 1 thymidine kinase and cytochrome P450 4B1: Applications for prodrug-activating gene therapy

“…17 Another study described a mostly cytoplasmic distribution. 18 In 9L-TK-EGFP cells, we observed that the TK-EGFP fusion protein was expressed predominantly in the nucleus and, at significantly lower levels, also within the cytoplasm. The cytoplasmic expression of the 4B1-EGFP fusion protein in U87 cells corresponded well to the natural localization of CYP4B1.…”