Figure 8

Importance of the SD/SA sequences and of the SP1-specific ATG for HBZ protein synthesis (A) 293T cells were co-transfected with 5 μg K30-3'-asLUC or K30-3'-asLUC mSA and 2 μg pActin-β-gal. Luciferase activities represent the mean value of three measured samples ± S.D and are expressed as normalised RLU for 5 × 10⁶ cells. ( B ). 293T cells were co-transfected with 5 μg K30-3'-asLUC or K30-3'-asLUC mSA and 2 μg pActin-βgal. RNA samples from transfected cells were analysed by a modified RT-PCR protocol (see Materials and Methods). Controls for DNA contamination (lanes 2 and 5) and autopriming (lanes 3 and 6) were included. M = 100 bp marker (the asterisk indicates the 600 bp band). ( C ) The K30-3'/4089 construct was mutated at the splice acceptor (mSA), the splice donor of SP1 (mSD1), the splice donor of SP2 (mSD2), the presumed ATG initiation codon of SP1 (mATG/e1) or the initially identified ATG initiation codon (mATG/int). Comparison of sequences between wild-type and mutated versions of K30-3'/4089 are depicted. (D) 293T cells were transfected with 2 μg pActin-β-gal and 5 μg pcDNA3.1-Myc-His HBZ, wild-type K30-3'/4089 or versions mutated for SA, SD1, SD2, ATG/e1 or ATG/int and nuclear extract from samples transfected with equal efficiency (based on β-gal read-outs) were analysed by Western blot using anti-HBZ antiserum. The position of the SP1-specific HBZ isoform is indicated by an arrow.