Figure 3
Induction of human APOBEC3G mRNA by different RNA species. (A and B) A549 cells (1.5 × 106/6 cm dish) were transfected with indicated amounts of total RNA from virally infected ("viral RNA") or uninfected ("cellular RNA") A549 cells. RNA from uninfected or virally infected cells was generated by isolation of total RNA from cells either infected with PR8 (MOI = 5) or from uninfected cells using the Qiagen RNeasy kit according to the manufacturer's instructions. For stimulation, these RNAs were transfected for 10 hours using Lipofectamine 2000 (L2000) (Invitrogen) according to the manufacturer's protocol. (C) A549 cells were transfected with either different amounts of poly (I:C) (Amersham Biosciences) or with 5'-triphosphate RNA (D). 5'-triphosphate RNA was generated by reverse transcription of a short PCR product using the MEGAshortscript kit (Ambion) according to the manufacturer's instructions. mRNA levels of IFNβ (B) or human APOBEC3G (A, C and D) were determined by qRT-PCR as described. Levels of gene induction were calculated as n-fold of the background of untreated cells, which was arbitrarily set as 1.