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. 2019 Mar 21;11(3):285. doi: 10.3390/v11030285

Figure 2.

Figure 2

Figure 2

Protein expression, DNA replication, and progeny production of BSRF1 KO EBV in HEK293 cells. (A) Viral protein expression in HEK293. HEK293 EBV-BAC cells were transfected with the BZLF1 expression plasmid by electroporation. Cells were harvested after 2 days and subjected to immunoblotting with the indicated antibodies. (B–H) Quantitative data. Band intensity was quantified using ImageJ software. (I) Viral genome DNA replication in HEK293. Cells transfected as in (A) were harvested after 2 days and subjected to qPCR for EBV and host cell genomic DNA. The means ± SD of three independent biological replicates are shown after normalization to the value of the host control. (J) Production of progeny into the culture media. Cells transfected as in (A) were cultured for 3 days, and the medium was collected after centrifugation, treated with DNase, and subjected to DNA extraction and quantification by qPCR. The means ± SD of three independent biological replicates are shown. (K) Infectivity of the progeny in the culture media. The same medium as in Figure 2J was used to infect Akata(−) cells. The percentage of green fluorescent protein (GFP)-positive cells was determined by fluorescence-activated cell sorting (FACS) analysis and is shown as logarithmic values. The means ± SD of three independent biological replicates are shown. (L, M) Time course and comparison of extracellular- and cell-associated progeny levels. Cells were transfected as in (A) with the BZLF1 expression vector, and the medium and cells were separately harvested at the indicated time points for titration using Akata(−) cells. The means ± SD of three independent biological replicates are shown. Cell-clone numbers are also shown (#1, #2, #3, #4).