A simple gel technique is described for the detection of large, covalently closed, circular DNA molecules in eucaryotic cells. The procedure is based on the electrophoretic technique of Eckhardt (T. Eckhardt, Plasmid 1:584-588, 1978) for detecting bacterial plasmids and has been modified for the detection of circular and linear extrachromosomal herpesvirus genomes in mammalian cells. Gentle lysis of suspended cells in the well of an agarose gel followed by high-voltage electrophoresis allows separation of extrachromosomal DNA from the bulk of cellular DNA. Circular viral DNA from cells which carry the genomes of Epstein-Barr virus, Herpesvirus saimiri, and Herpesvirus ateles can be detected in these gels as sharp bands which comigrate with bacterial plasmid DNA of 208 kilobases. Epstein-Barr virus producer cell lines also show a sharp band of linear 160-kilobase DNA. The kinetics of the appearance of this linear band after induction of viral replication after temperature shift parallels the known kinetics of Epstein-Barr virus production in these cell lines. Hybridization of DNA after transfer to filters shows that the circular and linear DNA bands are virus specific and that as little as 0.25 Epstein-Barr virus genome per cell can be detected. The technique is simple, rapid, and sensitive and requires relatively low amounts of cells (0.5 X 10(6) to 2.5 X 10(6)).
Microsomal membrane fraction proteins with enhanced synthesis after P3HR-1 Epstein-Barr virus (EBV) superinfection of Raji cells were identified with [35S]-methionine labeling and SDS-PAGE. One 53,000-dalton protein, which was found in both the microsomal membrane and cytosol fractions, was purified by ion-exchange chromatography, and specific rabbit antisera were prepared to it. This protein was found to be present in Raji cells, but its expression was enhanced after P3HR-1 EBV superinfection. It was more abundant in the cytosol than in the microsomal membrane fraction of the cell, and its synthesis was not affected by treatment of the cells with phosphonoacetic acid. It was present in several EBV-genome-negative cell lines and in activated B lymphocytes and consequently represents a host-cell-coded protein which is enhanced by EBV superinfection or by lymphocyte activation.
By the indirect immunofluorescence technique, IgM antibodies to the cell surface of an Epstein-Barr virus (EBV) producer cell line, P3HR-1, were detected in sera from infectious mononucleosis (IM) patients but not in sera from patients with Burkitt lymphoma or nasopharyngeal carcinoma nor in sera from healthy adult donors having antibodies to EBV-specific viral capsid antigen (VCA). Titers of the IgM antibodies were higher in the earlier stages of IM, a pattern similar to that for IgM antibodies to VCA. The IgM antibodies to the cell surface were identified as being those against the EBV-specific membrane antigen (MA) by the following criteria: (1) The antibodies were reactive to MA-positive cell preparations but to MA-negative cell preparations. (2) Titers of the IgM antibodies were not significantly affected after absorption of sera with sheep red blood cells which could completely eliminate heterophil antibodies in the same sera. Detection of the IgM antibodies to MA may have a particular diagnostic value for providing evidence of a recent EBV infection.
Summary CCL20 is expected to play a crucial role in the initiation of immune responses and tumour growth. However, expression of CCL20 in Epstein–Barr virus (EBV)‐associated diseases has not been studied. We examined the contribution of EBV infection and EBV‐encoded latent membrane protein (LMP)‐1 to CCL20 expression. EBV infection and LMP‐1 induced CCL20 mRNA expression in the EBV‐negative Burkitt lymphoma (BL) cell lines and the embryonic kidney cell line. Histone deacetylase inhibitor‐stimulated endogenous LMP‐1 also induced CCL20 expression in an EBV‐positive BL cell line. Analysis of the CCL20 promoter showed that it was activated by LMP‐1 C‐terminal activation region (CTAR)‐1 and CTAR‐2. Co‐expression of I κ B α , I κ B β , I κ B kinase (IKK) α , IKK β , IKK γ , nuclear factor (NF)‐ κ B‐inducing kinase and tumour necrosis factor receptor‐associated factor 2 dominant‐negative constructs with LMP‐1 inhibited the activation of the CCL20 promoter by LMP‐1, suggesting that LMP‐1 induces CCL20 via NF‐ κ B signalling. The requirement for the NF‐ κ B‐binding site in the CCL20 promoter in LMP‐1 responsiveness was established. Our results indicate that activation of the NF‐ κ B pathway by LMP‐1 is required for the activation of CCL20 expression.
The effect of human serum with or without Epstein-Barr virus (EBV) antibodies was characterized on virus production in P3HR-1 cells. Cell culturing with EBV-seropositive sera reduced both production of infectious virus and amounts of virion DNA in the supernat-ants. EBV DNA was also reduced in the cells. Such reductions in cell-associated EBV DNA depended upon the concentration of seropositive serum and incubation time. Decreased frequencies of productive EBV DNA-replicating cells were observed in cell populations which had reduced levels of cell-associated EBV DNA. The inhibitory effect of seropositive serum was reversed upon switching the cells to medium with seronegative serum. In serial sera of an acute infectious mononucleosis patient the EBV DNA-reducing activity arose in parallel to antibodies against EBV membrane antigen and nuclear antigen. Possible mechanisms were discussed for antibody-mediated inhibition of EBV production.
