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. 2017 Jan 3;91(2):e01854-16. doi: 10.1128/JVI.01854-16

FIG 5.

FIG 5

Y102E fails to stimulate transient BPV-1 replication. (A) C33a cells transfected in 8 replicate wells with 10 ng/well of the pCG-E2 WT or mutants (Y102F and Y102E), E2R, or mRFP-GFP (−) as well as 10 ng/well pCG-E1, 2.5 ng/well pFLORI-BPV1 (firefly luciferase reporter), and 0.5 ng/well pRL (Renilla luciferase reporter). Lysates were processed for luminescence. F/R, firefly output (in relative light units) divided by the Renilla output; **, P < 0.01; ****, P < 0.00001. Values are expressed as means ± SEM. (B) Replication assay completed as described above for panel A but with firefly and renilla luciferase levels being graphed separately. (C) Replication assay as described above for panel A. Each group had E1 transfected with either WT E2 or increasing amounts of Y102E. Fluc, firefly luciferase; Rluc, renilla luciferase (D) HEK293TT cells were transfected with 3 μg each mRFP-GFP (−), the BPV-1 E2 WT and mutants (YF and YE), and BPV-1 E1 or pCI. Lysates were immunoprecipitated with Sepharose A beads and BPV E2 antibody II-I. Western blots were probed for E1 and E2 by using rabbit anti-E1 peptide antibody 502-2 and antibody B201, respectively. *, nonspecific band.