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. 2014 Nov;88(21):12825–12838. doi: 10.1128/JVI.02139-14

FIG 2.

FIG 2

BFRF2 is necessary for virion production. (A) Raji cells were incubated with supernatants from HEK293EBVΔBFRF2 cells transfected with an expression plasmid encoding the EB1 protein in order to induce the productive cycle together (or not) with an expression plasmid encoding the Flag.BFRF2 protein to complement BFRF2 deficiency. (Left side) The GFP fluorescence of Raji cells was analyzed 48 h after infection under UV light. (A, sub panels a and b) Phase contrast; (A, sub panels c and d) UV light. (Right side) Immunoblot analysis of the EB2, Flag.BFRF2, and EB1 proteins expressed in HEK293EBVΔBFRF2 cells that either had not been transfected (lane 1), had been transfected with an EB1 expression plasmid (lane 2), or had been cotransfected with expression plasmids for both EB1 and BFRF2 (lane 3). Western blots were revealed using a polyclonal anti-Flag antibody, an anti-EB2 polyclonal rabbit antibody, or an anti-EB1 MAb. * indicates an unspecified protein recognized by the anti-EB2 serum. (B) BFRF2 is not required for viral DNA replication. Viral DNA in HEK293EBV and HEK293EBVΔBFRF2 cells transfected with EB1 and Flag.BFRF2 expression plasmids, as indicated in the figure, was quantitated by qPCR. The amount of viral DNA in the induced cells (transfected with the EB1 expression plasmid) is expressed relative to the amount of viral DNA present in the uninduced cells. (C, top) BFRF2 enhances expression of the late viral genes. Total RNA from HEK293EBV (wild type [WT]) or HEK293EBVΔBFRF2 (ΔF2) cells that either had not been transfected (−), had been transfected with an EB1 expression plasmid (EB1), or had been cotransfected with expression plasmids for EB1 and Flag.BFRF2 (EB1 + BFRF2) was purified, reverse transcribed, and analyzed by qPCR using specific primers for either the BMRF1 early gene or the BDLF1 and BcLF1 late genes. (Bottom) Western blot of HEK293EBV or HEK293EBVΔBFRF2 cells either not transfected (−) or transfected with an EB1 expression plasmid or with expression plasmids for both EB1 and Flag.BFRF2. Protein extracts were analyzed by Western blotting using a polyclonal anti-Flag antibody and an anti-EB1 MAb. The asterisk indicates an unspecified protein detected by the anti-Flag antibody.