William C. Summers

Exploring the active site of herpes simplex virus type-1 thymidine kinase by X-ray crystallography of complexes with aciclovir and other ligands

Antiherpes therapies are principally targeted at viral thymidine kinases and utilize nucleoside analogs, the triphosphates of which are inhibitors of viral DNA polymerase or result in toxic effects when incorporated into DNA. The most frequently used drug, aciclovir (Zovirax), is a relatively poor substrate for thymidine kinase and high‐resolution structural information on drugs and other molecules binding to the target is therefore important for the design of novel and more potent chemotherapy, both in antiherpes treatment and in gene therapy systems where thymidine kinase is expressed. Here, we report for the first time the binary complexes of HSV‐1 thymidine kinase (TK) with the drug molecules aciclovir and penciclovir, determined by X‐ray crystallography at 2.37 Å resolution. Moreover, from new data at 2.14 Å resolution, the refined structure of the complex of TK with its substrate deoxythymidine (R = 0.209 for 96% of all data) now reveals much detail concerning substrate and solvent interactions with the enzyme. Structures of the complexes of TK with four halogen‐containing substrate analogs have also been solved, to resolutions better than 2.4 Å. The various TK inhibitors broadly fall into three groups which together probe the space of the enzyme active site in a manner that no one molecule does alone, so giving a composite picture of active site interactions that can be exploited in the design of novel compounds. Proteins 32:350–361, 1998.

Structure and function of herpesvirus genomes II. EcoRI, XbaI, and Hin dIII endonuclease cleavage sites on herpes simplex virus type I DNA

Abstract Restriction endonuclease cleavage site mapping of the herpes simplex virus type I genome shows that the DNA is composed of a large segment of 80 megadaltons linked to a small segment of 16 megadaltons. The DNA molecules fall into four distinct groups, related to each other by the relative orientation of the large segment and the small segment. In addition, the large segment shows limited heterogeneity at its ends so that there is a possible total of more than 16 distinguishable populations of DNA molecules.

Construction and characterization of a recombinant plasmid encoding the gene for the thymidine kinase of herpes simplex type 1 virus

We have constructed a hybrid plasmid by insertion of the thymidine kinase (TK) gene of Herpes simplex virus (HSV) type I at the BamHI site on Escherichia coli plasmid pBR322. The restriction endonuclease cleavage site map for the viral DNA fragment was determined for ten nucleases, and the insert in the recombinant plasmid has the same restriction nuclease digestion pattern as bona fide viral DNA. This result indicates that the plasmid contains an accurate copy of the viral DNA. The viral TK gene carried on the plasmid can be introduced into mammalian cells where it is expressed. This source of DNA with a selectable marker should be of considerable practical use in gene-transfer experiments in mammalian cells.

The process of infection with coliphage T7: I. Characterization of T7 RNA by polyacrylamide gel electrophoretic analysis

Abstract Twelve T7 RNA species can be identified by electrophoresis in polyacrylamide gels. The sum of the molecular weights of the species is sufficient to account for the entire genome of T7. Some of the large species must certainly be polycistronic in nature. All identifiable T7 species are represented in infected cells prior to the onset of DNA synthesis.

The structure of herpes simplex virus type 1 DNA as probed by micrococcal nuclease digestion.

Micrococcal nuclease digestion was used to probe the structures in which herpes simplex virus type I (HSV-I) DNA is found during virus replication. Parental DNA, progeny DNA and DNA in nucleocapsids were analysed. Parental DNA was examined after infection of Vero cells with 32P- or 3H-thymidine-labelled HSV-I. Progeny DNA was examined after HSV-I-infected Vero cells were pulse-labelled with 3H-thymidine during HSV-I DNA synthesis. In both cases, nuclei were isolated and digested with micrococcal nuclease. Digestion products were analysed by agarose or polacrylamide gel electrophoresis (PAGE). Most parental DNA remained as intact molecules. However, a small amount was degraded into fragments which were heterogeneous in size or the size of nucleosomal cell DNA. These two classes of fragments were also produced upon digestion of progeny DNA. The heterogeneous fragments and nucleosomal fragments comprised major and minor fractions, respectively, of digested progeny DNA. When digested DNA from HSV-I-infected cells was transferred from composite polyacrylamide-agarose gels to diazobenzyloxymethyl paper, nucleosomal fragments hybridized to 32P-labelled HSV-I DNA as well as to 32P-labelled Vero cell DNA.. Therefore, nucleosomal fragments contained HSV-I DNA sequences. HSV-I DNA in nucleocapsids was analysed by micrococcal nuclease digestion after nucleocapsids were disrupted with PH 9.3 buffer, pyridine, Sarkosyl or NcCl/urea. Only fragments of heterogeneous size were produced. Thus, HSV-I DNA is found predominantly in structures other than nucleosomes during virus replication.

Site-directed mutagenesis of a nucleotide-binding domain in HSV-1 thymidine kinase: Effects on catalytic activity

The thymidine kinase encoded by herpes simplex virus type 1 contains an amino acid sequence homologous to a consensus sequence related to the ATP-binding site in many proteins. We have used site-directed mutagenesis to investigate the importance of the five highly conserved amino acids within this segment. When any one of the three glycines was changed to valine the corresponding mutant enzyme was inactive. The mutation of lysine 63 to isoleucine destroyed the enzymatic activity. When threonine 64 was changed to alanine the mutant enzyme lost its activity. However, when this threonine was changed to serine the enzyme was still active but with different apparent Michaelis constants (Km) for thymidine and ATP. The wild-type thymidine kinase has apparent Kms of 0.5 and 20 microM for thymidine and ATP, respectively, while the mutant enzyme displayed Kms of 2.3 and 60 microM for thymidine and ATP. These results indicate that this homologous segment is essential for the function of the thymidine kinase and is involved in the substrate binding domain of the enzyme.

UV-REACTIVATION OF HERPES SIMPLEX VIRUS IS MUTAGENIC AND INDUCIBLE IN MAMMALIAN CELLS

ABSTRACT We have studied the processes of UV-reactivation and mutagenesis in mammalian cells with HSV-1 as the experimental probe. The survival of plaque-forming ability was measured along with the frequency of forward mutation to the TK − (thymidine kinase deficient) viral phenotype. These studies suggest that that UVR and mutagenesis are in some way inducible, and may be tightly coupled phenomena.

DNA base sequence homology between coliphages T7 and øII and between T3 and øII as determined by heteroduplex mapping in the electron microscope

Abstract The genetic relatedness of the similar coliphages T7, T3 and oII was investigated by electron microscopic observations of the DNA heteroduplexes formed with one DNA strand from coliphage T7 (or T3) and the complementary strand from oII. The T7-oII heteroduplex is almost invariant with denaturing conditions, below the melting temperature of T7 DNA. This observation indicates that the DNAs of T7 and oII have regions of complete homology and regions of complete non-homology. In direct contrast, the T3-oII heteroduplex varies with denaturing conditions. The fraction of the T3-oII heteroduplex observed as double-stranded decreases with an increase in denaturing conditions below the melting temperature. This observation indicates that the DNAs of T3 and oII have extensive sequences of partial homology.

The process of infection with coliphage T7: III. Control of phage-specific RNA synthesis in vivo by an early phage gene☆☆☆

Abstract After infection of Escherichia coli with bacteriophage T7, 12 or 13 new phage-specific RNA species are synthesized. These are resolvable as discrete bands by polyacrylamide-agarose gel electrophoresis. Initiation of all T7 RNA species occurs to some extent before the onset of phage DNA synthesis. Only four of the normal T7 RNAs are made if phage-specific protein synthesis is blocked with chloramphenicol or kanamycin and one of these RNAs (mol. wt 1.1 × 10 6 ) is probably the messenger RNA from gene 1. RNA made after infection with T7 carrying amber mutations in gene 1 has only three discrete species of T7 messenger RNAs. The RNA from cells infected with T7 + in the presence of 400 μg chloramphenicol/ ml. is very similar to T7 am 23 (gene 1) RNA as judged by electrophoretic analysis and by RNA-DNA hybridization competition experiments. On the other hand, RNA made after infection with T7 carrying amber mutations in other genes is very similar to T7 + RNA. This result parallels that reported on the control of T7 protein synthesis which showed that the gene 1 protein controlled the production of all or nearly all other T7-induced proteins. These results suggest that while gene 1 is transcribed in the absence of phage protein synthesis, the gene 1 protein is required to transcribe the remainder of the T7 genome. The control of T7 protein synthesis is therefore mediated at the level of transcription by means of positive regulation.

Error-prone mutagenesis detected in mammalian cells by a shuttle vector containing the supF gene of Escherichia coli.

When a shuttle vector containing a tyrosine suppressor tRNA (supF) gene as a target for mutagenesis replicated in a monkey kidney cell line, the frequency of SupF+ mutations was 2.3 +/- 0.5 x 10(-3). When the host cells were treated with ethyl methanesulfonate 40 h before transfection, a 10-fold increase in SupF+ mutation frequency was observed. These results supported the hypothesis that a damage-inducible mutagenic pathway exists in mammalian cells and also demonstrated the utility of this shuttle vector for the study of mutagenesis in mammalian cells.