Edward Littler

Identification of an Epstein-Barr virus-coded thymidine kinase.

We have demonstrated the presence of an Epstein‐Barr virus (EBV)‐coded thymidine kinase (TK) by producing biochemically transformed, TK‐positive mammalian cell lines using either microinjection of whole EBV virions or calcium phosphate‐mediated transfection of the SalI‐B restriction endonuclease fragment of EBV DNA. Analysis of these cell lines showed that: (i) EBV DNA was present in the cell lines, (ii) sequences from the SalI‐B restriction endonuclease fragment of EBV were expressed, (iii) a TK activity was present and (iv) a protein with antigenic cross‐reactivity with the herpes simplex virus (HSV) TK was produced. The identity of the EBV TK gene was determined by demonstrating that a recombinant plasmid, which expressed the protein product of the BXLF1 open reading frame as a fusion protein, could complement TK‐ strains of E. coli. A comparison of the predicted amino acid sequences of the TK proteins of EBV and HSV‐1 revealed significant regions of homology.

Herpes simplex virus non-structural proteins. III. Function of the major DNA-binding protein.

The herpes simplex virus type 2 major DNA-binding protein has been functionally characterized using temperature-sensitive mutants in the complementation group 2-2. The mutants were shown to be defective in the DNA-binding protein gene by mapping the mutants to the area of the genome known to code for the protein, and by demonstrating alterations in the major DNA-binding protein induced in mutant-infected cells. The mutants were shown to be defective in the replication of virus DNA. The nature of this defect was examined by studying virus DNA synthesis in vitro and by the examination of virus enzymes. An effect of mutation in the DNA-binding protein was to destabilize both the DNA polymerase and the alkaline exonuclease.

Antigenic and Structural Conservation of Herpesvirus DNA-binding Proteins

Previously, we have shown a common antigen of several herpesviruses (pseudorabies virus, equine abortion virus and bovine mammillitis virus) to be antigenically related to the major DNA-binding proteins of herpes simplex virus types 1 and 2. In this study we have purified the cross-reacting polypeptide from cells infected with pseudorabies virus, equine abortion virus and bovine mammillitis virus and shown the cross-reacting protein to be a major DNA-binding protein for each virus. Tryptic peptide analysis of the cross-reacting DNA-binding proteins of all five viruses has shown structural similarities. The proteins thus were shown to share common antigenic sites, to have similar biological properties and to have a highly conserved amino acid sequence. This unexpected similarity between proteins from diverse herpes viruses suggests an essential and fundamental role of the major DNA-binding protein in herpes virus replication.

Antibodies to Epstein‐Barr virus thymidine kinase: A characteristic marker for the serological detection of nasopharyngeal carcinoma

Patients suffering from nasopharyngeal carcinoma (NPC) generally exhibit elevated serum IgA antibody titres to Epstein‐Barr virus (EBV) early antigen (EA) and virus capsid antigen (VCA). This property is frequently used as a diagnostic aid. Preliminary experiments suggested that an ELISA for IgA antibodies against the EBV‐encoded thymidine kinase (TK) could form the basis of a more reliable diagnostic test. Here, we describe the construction of a recombinant baculovirus that expresses the EBV TK and present a full analysis of its use in serological surveys of NPC patients. Baculovirus‐derived TK was used to develop a simple ELISA for serum IgA against this antigen. ELISA reactivity was strongly associated with NPC compared with an EBV‐positive, normal control population. Comparison with the existing IgA‐VCA and EA assays showed that the TK ELISA had higher sensitivity whilst the specificity was similar or higher. We conclude that the TK ELISA presents a strong predictor of NPC and, in its refined form, has improved pickup rates. In addition, results from patients with chronic nasopharyngitis (CNP) suggest that individuals with both symptoms of CNP and an elevated TK ELISA value may be at increased risk for the development of head‐and‐neck cancer.

Immunological Conservation between Epstein-Barr Virus and Herpes Simplex Virus

We have analysed Epstein-Barr virus (EBV)- and herpes simplex virus (HSV)-infected cells for evidence of antigenic conservation of virus-coded proteins. Immunofluorescence and Western blot analyses of EBV-transformed cell lines demonstrated the presence of proteins that are antigenically related to the HSV alkaline DNase, infected cell-specific protein 34/35, glycoprotein B, thymidine kinase and the major DNA-binding protein. These proteins were characterized on the basis of Mr and possible kinetic class.

Diagnosis of Nasopharyngeal Carcinoma Using the Epstein-Barr Virus Coded Alkaline Dnase, Membrane Antigen and Thymidine Kinase

Nasopharyngeal carcinoma (NPC) is a tumour of epithelial origin and is the most common form of nasopharyngeal cancer in man (1,2). In most countries of the world it is a rare disease with rates of less than 1 per 100,000 per year. However in several Chinese populations (Southern provinces of China, Hong Kong, Taiwan, Singapore and Malaysia) the malignancy occurs with a high frequency (15–30 per 100,000 per year)(3,4).