The Raji line of human lymphoblastoid cells, which does not show expression of Epstein-Barr virus, was made resistant to 5-bromodeoxyuridine. Within several weeks after removal of the drug, Epstein-Barr virus particles were detected in the cells. The
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)
The sequence of spontaneous EpsteinBarr virus activation was studied in P3HR-1 carrier cells and in P3HR-1(BrdU) cells made resistant to 5-bromodeoxyuridine. Virus activation was initiated during the normal cell cycle, and recruitment of additional virusactivated cells was prevented by the DNA inhibitors, I-/-D-arabinofuranosylcytosine and hydroxyurea. Virus activation was followed by synthesis of the early antigen complex in the absence of additional detectable DNA synthesis. Early antigen synthesis was followed by hydroxyurea-resistant synthesis of new DNA, which in the case of P3HR-l(BrdU) cells was characterized by the appearance of thymidine kinase. The newly synthesized DNA banded in neutral cesium chloride at peaks corresponding to normal human DNA and Epstein-Barr viral DNA. Synthesis of viral antigen was seen only in cells that had undergone hydroxyurea-resistant DNA synthesis.The Epstein-Barr herpesvirus (EB virus) persists in human lymphoblastoid cells in a repressed state (1). In some cell lines, the repressed viral genome undergoes spontaneous activationt resulting in a carrier culture where virus is synthesized in at least a portion of the cell population at any one time. In other cell lines ("virus negative"), the viral genome remains repressed (2, 3), although activation with resultant virus synthesis can be effected by treatment of the cells with 5-bromodeoxyuridine (BrdU) (4, 5) or 5-iododeoxyuridine (6).The studies reported here concern the relationship between DNA synthesis and synthesis of virus-associated intracellular antigens after spontaneous activation of the EB viral genome. MATERIALS AND METHODSCells and Media. The P3HR-1 cell line that is EB virus positive and made resistant to BrdU (1), P3HR-1(BrdU), was propagated in 250-ml plastic flasks (Falcon Plastics, Inc.) with growth medium (1) containing 100 ug of BrdU per ml. Manipulations were performed in subdued light. Control P3HR-1 cells were maintained in drug-free medium. Total and viable cell counts were determined visually by the trypan blue exclusion method. Cell cultures were tested routinely and found free of contamination by pleuropneumonia-like organisms and other contaminants. Immunofluorescence. The indirect immunofluorescence technique and cells fixed for 10 min in acetone at -20°were used.Two human sera were used after determination of their specificity for the two major-EB virus-associated-intracellular antigen complexes detected by immunofluorescence. The specificity of the sera for the early antigen (EA) complex (6) was determined in two ways. The first used "virus-negative" Raji and NC-37 cells treated with BrdU for activation of the viral genome (4). The EA complex is the first virus-specific intracellular antigen complex to appear, as detected by immunofluorescence, in BrdU-activated cells (manuscript in preparation). The second method, performed by Dr. G. Pearson, used Raji cells infected with EB virus. The predominant intracellular antigen(s) produced under these conditions is EA (7). The specificity of t...
The antibody activity against herpes-type virus (HTV) of heterophile-positive infectious mononucleosis sera and "normal" sera from humans was determined with immunoferritin. The antiviral activity of the mononucleosis sera was associated with the IgM antibodies, while the antiviral activity of the "normal" human sera was associated with the IgG antibodies. It was concluded from these findings that the appearance of antibodies to herpes-type virus in heterophile-positive sera represents a primary immunogenic exposure to this virus, or to a serologically related virus. This conclusion, in turn, suggests that herpes-type virus or a serologically related virus is the etiologic agent of heterophile-positive mononucleosis.A serologic relationship has been proposed between the herpes-type virus (HTV) associated with human lymphoblastoid cell lines derived from "normal" buffy coats and from Burkitt tumors (1) and the virus of infectious mononucleosis. This relationship is based on a correlation between the onset of heterophile-positive mononucleosis with (a) the detection of antibodies to HTV (2-4) and (b) the ability of buffy-coat cells to undergo blastoid transformation and establishment in vitro (2). The available evidence, however, is not sufficient to allow conclusions as to whether the antibodies to HTVt that have been detected in heterophilepositive sera from patients with mononucleosis are caused by a primary immunogenic exposure to this virus. Information relating to this question is important in attempts to ascribe an etiologic role for HTV in the onset of heterophile-positive mononucleosis.Our studies were based on the assumption that after a primary antigenic stimulation, 19S or IgM antibodies should be detectable before 7S or IgG antibodies (5, 6), provided that the antigen in question (e.g., HTV) induces synthesis of both classes of antibody (7). The specificity of human antibodies for antigens of the HTV virion was determined by the immunoferritin technique, which allows visual observation of the reaction between antibody and virus. This technique was previously shown effective for determination of the specificity of 7S and 19S antibodies of the rabbit for herpes-simplex virion antigens (8).Abbreviations: HTV, herpes-type virus; MSV, herpes simplex virus.
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