Figure 4

Influence of the human APOBEC3G protein on viral replication. (A) Transiently (white bars) or stably human APOBEC3G transfected (light and dark grey bars) MDCK cells were infected with avian influenza virus A/FPV/Bratislava/79 (H7N7) (FPV) (MOI = 0.05) for 9 hours or A/Puerto Rico/8/34 H1N1 (PR8) (MOI = 0.1) virus for 16 hours. Both virus strains were originally taken from the collection of the Institute of Virology, University of Giessen, Germany. Stably transfected A549 cells (black bars) were infected with PR8 for 16 hours (MOI = 0.01). Supernatants were taken, and virus titres were determined by means of plaque assay. (B and C) MDCK cells or (D) A549 cells were transfected with 3 μg DNA/6 well dish pcDNA-APOBEC3G or pcDNA3.1 empty vector using L2000 (Invitrogen) according to manufacturer's instructions. To generate a bulk amount of stable expressing cells, MDCK cells (C) or A549 cells (D) were treated with G418 300 μg/ml for selection of APOBEC3G expression for four weeks. Thereafter, cells were infected with FPV at MOI = 0.05 (B) or at MOI = 0.1 for 9 hours (C) or with PR8 at MOI = 0.001 for 16 hors (D). Expression of HA-tagged human-APOBEC3G was detected by anti-HA 3F10 (Roche) antibody. To control equivalent protein loading, ERK2 or JNK were detected by anti-ERK2 (Santa Cruz) or anti-JNK antibody (Santa Cruz). Viral replication was monitored by detection of the viral polymerase protein using an anti-PB1 antibody (Santa Cruz).