Epstein-Barr virus DNA synthesized in superinfected raji cells

Abstract

Raji and P3HR-1 are established Burkitt lymphoma-derived cell lines that carry the Epstein-Barr virus (EBV) genome. Superinfection of the Raji cell line, a non-virus-producer, with virus from P3HR-1 cells results in the synthesis of several thousand copies of EBV-DNA per cell with attendant inhibition of synthesis and breakdown of Raji cell DNA. The DNA synthesized in superinfected Raji cells has been characterized. Raji cells infected with P3HR-1 virus and labeled with 32P 10 hr after infection synthesized only viral DNA. As much as 90% of the 55 S, 32P-labeled material was localized in the nucleus of superinfected cells after a 10-hr labeling period. The viral DNA purified from superinfected cells had the same buoyant density as the DNA isolated from P3HR-1 virions and after purification could be recovered with a specific activity exceeding 106 cpm/μg in an amount which approached 10 wg/107 infected Raji cells. The viral DNA from superinfected cells reassociated with the DNA from Raji or P3HR-1 cells and with the DNA from virus but did not reassociate with DNA from cell line 698 (a lymphoblastoid cell line lacking the EBV genome). Nuclei isolated from superinfected Raji cells incorporated label from deoxythymidine triphosphate into an acid-insoluble product, most of which had a buoyant density identical to that of DNA from P3HR-1 virions. Digestion of the DNA from superinfected cells with the restriction endonuclease EcoRI produced a number of fragments with molecular weights which ranged from less than 1 million to approximately 30 million when analyzed by electrophoresis on agarose gels. All of the fragments produced by digestion of DNA from virus were present in the digest of DNA from superinfected Raji cells.