Partial purification of the Epstein-Barr virus and some properties of its DNA

187

mentioning

confidence: 64%

Synthesis of Epstein-Barr Virus After Activation of the Viral Genome in a “Virus-Negative” Human Lymphoblastoid Cell (Raji) Made Resistant to 5-Bromodeoxyuridine

Hampar

Derge

Martos

et al. 1972

187

“…2C). The ratio of [14C]Ade counts to [3H]dT counts was 325 in the major peak (density 1.69 g/cm3), which corresponded to human DNA, and 9 in the minor peak, which banded at a density of 1.71 g/cm3, corresponding to that of EB virus DNA (13,14). The absence of a detectable radioactive "viral" DNA peak from P3HR-1 cells (Fig.…”

Section: Brdu-resistant P3hr-1 Cellsmentioning

confidence: 99%

Persistence of a Repressed Epstein-Barr Virus Genome in Burkitt Lymphoma Cells Made Resistant to 5-Bromodeoxyuridine

Hampar

Derge

Martos

et al. 1971

The P3HR-1 line of human lymphoblastoid cells that is Epstein-Barr virus positive was made resistant to 5-bromodeoxyuridine. Epstein-Barr virusassociated antigens, but not virus particles, were produced in P3HR-1(BU) cells maintained on 5-bromodeoxyuridine. However, virus particles did appear within 4 days after removal of the drug. Thymidine kinase activity was limited to P3HR-1(BU) cells producing viral antigen, whereas all control P3HR-1 cells showed thymidine kinase activity regardless of viral antigen synthesis.Cellular DNA in most P3HR-1(BU) cells was made via pathways that did not involve thymidine kinase. In cells having a pathway that involved thymidine kinase, a second DNA of density 1.71 g/cms, corresponding to EpsteinBarr virus, was detected.It was concluded that: (a) a repressed Epstein-Barr virus genome persists in P3HR-1(BU) cells that do not contain thymidine kinase, with activation of the viral genome being accompanied by productive infection and the appearance of enzyme, and (b) thymidine kinase activity in P3HR-1(BU) cells could be used as a marker for viral genome expression.Some human lymphoblastoid cell lines show persistent infection with the human Epstein-Barr herpesvirus (EB virus), which is synthesized in at least a portion of the cell population at any one time. The persistence of EB virus may be due either to a low-grade infection with transmission of infectious virus or to derepression of an integrated viral genome. Cloning experiments with lymphoblastoid cells favor the derepression mechanism (1-3).The studies reported here concern the properties of EB virus-negative cells in a virus-positive cell population made resistant to 5-bromodeoxyuridine (BrdU (v/v); the slides were pressed lightly between the pages of a bibulous paper pad. The cells were scanned by darkfield fluorescence microscopy, and appropriate areas were photographed and their locations were recorded.The coverslips were removed, the cells were washed in water and air dried. Stripping film (Kodak AR-10) was applied and the slides were exposed for 1-3 weeks at 4VC before developing. The cells were stained for 30 see in 0.2% crystal violet in 95% methanol (w/v) and the localized areas were scanned under brightfield microscopy. microscopy)

“…However, abortive infection and the appearance of early antigen are accompanied by inhibition of cell DNA synthesis (7) and by impaired ability of the superinfected cells to form colonies in soft agar (8 A major obstacle to comparative study of EB virus strains has been that most producer cell lines release minute quantities of extra-cellular virus, and there is yet no completely permissive culture system. For most studies heretofore, purified extracellular EB virions and EB viral DNA have been prepared from the P3J-HR-1 line of Burkitt lymphoma cells (9,10). Recently it was demonstrated that high titers of EB virus, biologically active by the immortalization assay, were found in the extracellular fluid of marmoset cells converted into lines following exposure to EB virus (11).…”

mentioning

confidence: 99%

Differences Between Laboratory Strains of Epstein-Barr Virus Based on Immortalization, Abortive Infection, and Interference

Miller

Robinson

Heston

et al. 1974

253

171

Biologic activities of extracellular EpsteinBarr virus (EB virus) from two laboratory strains, namely, PJ-HR-I (P-H) from Burkitt lymphoma and B95-8 (B95) from infectious mononucleosis, were compared. Virus stocks from both sources contained approximately the same number of virions. Virus from the P-H line induced "early antigen" in six nonproducer EB virus genome carrier cell lines; virus from B95 did not induce "early antigen." Extracellular virus from B95 regularly caused lymphocytes from human umbilical cords to form continuous lines (immortalization); P-H virus did not cause primary cultures of human lymphocytes to grow continuously. B95 virus stimulated DNA synthesis as determined by rate of incorporation of [3H]thymidine into acid-insoluble material; P-H virus did not stimulate DNA synthesis. Pretreatment of lymphocytes with undiluted P-H virus inhibited immortalization and stimulation of DNA synthesis by B95 virus. The inhibitory properties of the P-H virus were sedimented at 100,000 X g and inactivated by heat and UV irradiation; interference by the P-H virus was neutralized by human serum with antibody to EB virus and not by antibody-negative human serum.The hypothesis most consistent with these results is that the P-H virus is defective in gene(s) needed for initiation of immortalization. We speculate that the absence of this gene allows early antigen to be expressed upon superinfection of nonproducer cell lines. The availability of two laboratory strains of EB virus which differ in biologic behavior provides starting material for analysis of the mechanism of lymphocyte immortalization by EB virus and of virus structural differences which affect immortalization.Two biologic properties of Epstein-Barr virus (EB virus) can be studied in cell culture systems. The first is transformation or, as we prefer, immortalization (1-4). The virus causes normal human lymphocytes with a limited life span in vitro to form continuous cell lines. The second activity is abortive replication. When EB virus is added to certain continuous human lymphoblastoid cell lines, an EB virus associated antigen complex termed "early antigen" (EA) appears (5). The term "early antigen" is used because superinfected cells do not produce mature virus and because production of "early antigen" occurs in the presence of levels of cytosine arabinoside which inhibit DNA synthesis (6). However, abortive infection and the appearance of early antigen are accompanied by inhibition of cell DNA synthesis (7) and by impaired ability of the superinfected cells to form colonies in soft agar (8). A major obstacle to comparative study of EB virus strains has been that most producer cell lines release minute quantities of extra-cellular virus, and there is yet no completely permissive culture system. For most studies heretofore, purified extracellular EB virions and EB viral DNA have been prepared from the P3J-HR-1 line of Burkitt lymphoma cells (9, 10). Recently it was demonstrated that high titers of EB virus, biologically active by the immort...