FIG 1.

Transient infection of normal oral keratinocytes. (A) Schematic of transient infection of hTERT immortalized normal oral keratinocytes (NOK). A clonal NOK cell line was infected with a recombinant EBV (rEBV) carrying neomycin (neo) resistance and GFP cassettes in place of the BXLF1 gene. Neomycin selection pressure was applied to select and maintain EBV in the infected cell population. Removal of selection allowed loss of the viral episome (small black circles). Vector control indicates the clonal NOK cell line transfected with the PTRUF5 plasmid carrying the neomycin resistance and GFP cassettes. (B) In situ hybridization for EBV-encoded RNAs (EBERs) in uninfected, vector, EBV-positive and three EBV-negative transiently infected clones (EBV-cl1, EBV-cl3, and EBV-cl4). EBER positivity is indicated by dark nuclear staining in the EBV-positive cells. No EBER staining was detected in EBV-negative transiently infected clones. (C) DNA PCR spanning the viral genome in uninfected, vector, and EBV-positive (E+) clones and three EBV-negative transiently infected clones (EBV-cl 1, 3, and 4). The B cell line, Namalwa, with two integrated copies of EBV used as a limit of detection control at 100, 10, and 1 ng. (D) DNA PCR for the neomycin gene present in recombinant virus and vector plasmid. The EBV-positive cell line B958 was used as a positive control. Primers to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as a DNA loading control. (E) Southern blot for EBV genome using the EcoRI A fragment of the EBV genome as a probe in uninfected, vector, and EBV-positive clones and three transiently infected clones. U, uninfected; V, vector; E+, EBV-positive clone; 1, 3, and 4, transiently infected EBV-negative clones; W, water.