Inhibition of Translation Initiation by Protein 169: A Vaccinia Virus Strategy to Suppress Innate and Adaptive Immunity and Alter Virus Virulence
Fig 5
Protein 169 inhibits cap-dependent and FMDV IRES-dependent translation.
(A) HeLa cells were transfected in duplicate with plasmids encoding the indicated proteins or with empty vector (EV) control together with a plasmid expressing GFP.FLAG. After 16 h, EV-transfected cells were treated with cycloheximide (CHX) (10 μg/ml) for 7 h. Cell lysates were resolved by SDS-PAGE and immunoblotted using antibodies against α-tubulin, 169, rabbit anti-FLAG (FLAG (R)) and mouse anti-FLAG (FLAG (M)). The positions of molecular size markers in kDa are indicated on the left. (B) Performed as in (A) except that mRNAs were extracted from cells, cDNAs were synthesized and GFP mRNA levels were measured by RT-q-PCR. Results are expressed as CT values compared to HPRT mRNA levels ± SD. Statistical analysis was performed using a two-tailed Student’s t-test with Welch’s correction where necessary, *** p < 0.001. Data shown are from one representative experiment (n = 3). (C, D) HEK 293T cells were transfected with a plasmid encoding a bicistronic RNA expressing firefly luciferase (FLuc, C) by a cap-dependent translation and renilla luciferase (RLuc, D) by foot and mouth disease virus (FMDV) IRES-dependent translation, together with plasmids for expression of the indicated proteins or EV control in quadruplicate. After 16 h the cells were treated with CHX (1 μg/ml) for 7 h. The relative amount of FLuc and RLuc was determined by luminescence and results are presented as the fold increase in luciferase expression normalized to the EV control ± SD. Data shown are one representative experiment (n = 4). (E, F) Performed as in (C), but in triplicate. mRNAs were extracted, cDNAs were prepared and mRNA levels of for 169 and RLuc were determined by RT-q-PCR. Results are expressed as CT values compared to GAPDH levels ± SD. Data shown are one representative experiment (n = 4). Statistical analysis was performed using a two-tailed Student’s t-test with Welch’s correction where necessary, ** p < 0.01, *** p < 0.01, **** p < 0.001. (G) Cell lysates from (A) were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. A shorter exposure of FLAG is shown to improve visualization of N1.TAP. The positions of molecular size markers in kDa are indicated on the left.