Viral and cellular DNA synthesis in nuclei from human lymphocytes transformed by Epstein-Barr virus.

Abstract

A DNA-synthesizing system in vitro, using nuclei prepared by treatment of human lymphocytes with the detergent Brij 58, was developed. Nuclei from cultured lymphocytes synthesized DNA for as long as 5 hr, and required ATP, deoxynucleoside triphosphates, magnesium, and a calcium chelator. In nuclei from a partially synchronized line of cultured lymphocytes carrying several hundred copies of the Epstein-Barr viral genome, synthesis in vitro was predominantly viral in early S phase and cellular in late S phase. These and other data suggested that the DNA synthesis observed in vitro was predominantly replicative.)NA synthesis has been studied in a variety of eukaryotic cell types in culture, either in intact cells or in isolated nuclei, with both normal cell lines and cell lines infected by several DNA viruses, notably tumor viruses (for example, refs. 1-11). The study of both tumor virus and host DNA synthesis in virally transformed cells (as contrasted to productively infected cells) is made difficult by the problem of distinguishing the two processes. One cell system that would be suitable for such studies is human lymphocytes transformed by Epstein-Barr virus (EBV). There are available a wide range of lymphocyte lines that carry varying numbers of EBV genomes (12, 13). Furthermore, some of these are producer lines, i.e., a small portion of the population of cells is undergoing viral macromolecular synthesis at all times; others are nonproducer lines, in which viral DNA and antigen synthesis can be induced only by chemicals or superinfection (14-16).DNA synthesis in transformed lymphocytes is of particular interest for an additional reason. Normal peripheral lymphocytes are resting cells in terms of DNA synthesis, and EBV has been shown to cause them to undergo transformation into cells which now synthesize DNA and divide for an indefinite number of generations (17)(18)(19). It is, therefore, possible that the presence of the viral genome or some viral gene product (such as the Epstein-Barr nuclear antigen, EBNA) affects the control of host DNA synthesis in order for the cel] to assume the transformed state.Study of DNA synthesis in isolated nuclei has many advantages over its study in intact cells. However, lymphocyte nuclei are difficult to prepare in an active state after mechanical disruption in the Dounce homogenizer, the usual method for preparing nuclei, probably because their relatively large size renders them sensitive to damage. In the present paper a modified procedure for making lymphocyte nuclei in the presence of detergent without mechanical disruption of the cells (20, 21) is reported. These nuclei are active in a DNAsynthesizing system in vitro over a long period of time. Some characteristics of host and viral DNA synthesis in these lreparations will be reported.

Identification of a Nuclear Targeting Domain in the Insertion between Helices C and D in Protease Inhibitor-10

“…Because the direct targeting of free PI-10 would constitute a mechanism to regulate proteases that are spatially separated from cytoplasmic protease inhibitors, the molecular species of EGFP⅐PI-10 in the nucleus was characterized to determine whether PI-10 was present in the nucleus in either a free-form or an inactive high M r protease/inhibitor complex. For this purpose, a protocol (20) for the isolation of intact and functionally active nuclei was selected. This protocol utilizes the lysis of cell membranes and other organelles with the hydrophilic detergent Brij 58, followed by isolation of nuclei by differential centrifugation.…”

“…Isolation of Nuclei and Immunoanalysis-Nuclei of EGFP⅐PI-10-transfected cells were prepared according to the procedure of Benz and Strominger (20). Briefly, washed cells (10 7 ) were resuspended in 2 ml of 0.25 M sucrose, 5 mM CaCl 2 , 25 mM Hepes, pH 8 (4°C), and incubated (4°C, 5 min) with an equal volume of 0.15 M sucrose, 5 mM CaCl 2 , 25 mM Hepes containing 0.5% Brij 58 (Sigma).…”

Effects of adenine arabinoside on lymphocytes infected with Epstein-Barr virus

“…Preparation of nuclei. Isolated nuclei were prepared from B95-8 and Raji cells and tested for ability to synthesize DNA as previously described (2).…”

Induction of a cytoplasmic activator of DNA synthesis in lymphocytes is mediated through a membrane-associated protein kinase.

“…Isolated, quiescent frog spleen nuclei were prepared by the method of Benz and Strominger (1975) as previously described (Hameed et al, 1989). Isolated nuclei (2 x 105, in 10 Al 0.25-M sucrose, 5 mM CaCI2, 25 mM HEPES, pH 7.8, 2Yo dextran) were added to 50 ,l ADR-containing or control cytoplasmic extract and 40 ,l reaction buffer containing 0.25 M sucrose, 25 mM HEPES, pH 7.8,2% dextran in microtiter wells.…”