No abstract

No abstract

No abstract

The Epstein-Barr virus (EBV) DNA polymerase catalytic subunit (BALF5 protein) and its accessory subunit (BMRF1 protein) have been independently overexpressed and purified (T. Tsurumi, A. Kobayashi, K. Tamai, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:4651-4658, 1993; T. Tsurumi, J. Virol. 67:1681-1687, 1993). In an investigation of the molecular basis of protein-protein interactions between the subunits of the EBV DNA polymerase holoenzyme, we compared the DNA polymerase activity catalyzed by the BALF5 protein in the presence or absence of the BMRF1 polymerase accessory subunit in vitro. The DNA polymerase activity of the BALF5 polymerase catalytic subunit alone was sensitive to high ionic strength on an activated DNA template (80% inhibition at 100 mM ammonium sulfate). Addition of the polymerase accessory subunit to the reaction greatly enhanced DNA polymerase activity in the presence of high concentrations of ammonium sulfate (10-fold stimulation at 100 mM ammonium sulfate). Optimal stimulation was obtained when the molar ratio of BMRF1 protein to BALF5 protein was 2 or more. The DNA polymerase activity of the BALF5 protein along with the BMRF1 protein was neutralized by a monoclonal antibody to the BMRF1 protein, whereas that of the BALF5 protein alone was not, suggesting a specific interaction between the BALF5 protein and the BMRF1 protein in the reaction. The processivity of nucleotide polymerization of the BALF5 polymerase catalytic subunit on singly primed M13 single-stranded DNA circles was low (approximately 50 nucleotides). Addition of the BMRF1 polymerase accessory subunit resulted in a strikingly high processive mode of deoxynucleotide polymerization (> 7,200 nucleotides). These findings strongly suggest that the BMRF1 polymerase accessory subunit stabilizes interaction between the EBV DNA polymerase and primer template and functions as a sliding clamp at the growing 3'-OH end of the primer terminus to increase the processivity of polymerization.

We have investigated the pathogenicity of a US3 protein kinase-deficient mutant (L1 BR1) of herpes simplex virus type 2 (HSV-2) for 4-week-old ICR mice to define the role of the viral protein kinase in virus-host interaction. When mice were intraperitoneally infected with 10(5)PFU of L1 BR1, the virus disappeared from the peritoneal cavity by 2 days postinfection and failed to induce any significant histopathological changes in the liver and spleen although viral antigens were occasionally detected in the epithelial cells of small bile ducts and small vascular wall. The parental virus (HSV-2 186) and a revertant of the mutant (L1 B-11) both caused severe hepatitis, and viral antigens were clearly detected in the hepatocytes and Kupffer cells in the focal necrotic areas. Both of the virulent viruses, unlike L1 BR1, could produce infectious progeny and cytopathic effects in freshly harvested peritoneal macrophages. The growth of L1 BR1 in peritoneal macrophages was restricted at a stage of or prior to viral DNA synthesis but after the induction of viral DNA polymerase. In addition, the production and/or the spread of mutant in mouse embryo fibroblasts (MEF) was found to be much more effectively suppressed by cocultivation of peritoneal macrophages than that of 186. An almost complete inhibition of L1 BR1-plaque formation was observed at a macrophage-to-MEF ratio of 4:1. These results suggest that the attenuation of L1 BR1 following intraperitoneal infection is primarily due to its high sensitivity to intrinsic and extrinsic inhibition of peritoneal macrophages and that the US3 protein kinase may play a role in viral DNA replication in peritoneal macrophages.