Figure 4

MATR3 knockdown impairs Rev activity. A) Knockdown of MATR3 by siRNA. 293T cells were transfected either with siRNA targeting MATR3 (siMATR3) or with a control siRNA (siCTRL) and lysed after 72 hours for western blot analysis to assess the efficiency of MATR3 knockdown. Tubulin is the protein loading control. B) RT-PCR of spliced and unspliced HIV-1 RNA levels modulated by MATR3. Spliced (S) and unspliced (US) HIV-1 RNAs were detected (lanes 1-4, upper panel) simultaneously by RT-PCR on total RNA extracted from siRNA-treated 293T cells expressing vHY-IRES-TK, Tat and Rev-EGFP as indicated. RT-PCR amplification of an unrelated RNA was not affected (β-actin mRNA) (lanes 1-4, lower panel). Reactions without RT are shown to demonstrate lack of DNA contamination (lanes 5-8). Water (mock) was used as control of DNA contamination in the reaction. C) Quantitative analysis of unspliced HIV-1 RNA levels modulated by MATR3. Unspliced (US) viral RNA expression in siRNA treated 293T cells was assayed after transfection with vHY-IRES-TK, Tat and Rev-EGFP. Unspliced RNA levels were analyzed by quantitative real-time PCR and data normalized to β-mRNA expression. Data are presented as fold change, whereby siCTRL treated cells transfected with vHY-IRES-TK and Tat in the absence of Rev were set as 1. The results of three independent experiments are shown ± SD. The inhibition was significant (p = 0.00112). D) Rev-dependent expression of HIV-1 Gag (p17*). Western blot analysis of protein extracts from siRNA-treated 293T cells expressing vHY-IRES-TK, Tat and Rev-EGFP as indicated. p17* is the product of the truncated gag gene of the vHY-IRES-TK vector. Tubulin is the protein loading control. E) Quantitative analysis of unspliced HIV-1 RNA levels modulated by MATR3 in the nucleus and the cytoplasm. Unspliced (US) viral RNA expression in siRNA treated 293T cells was assayed after transfection with vHY-IRES-TK, Tat and Rev-EGFP. Unspliced RNA levels were analyzed by quantitative real-time PCR on nuclear (NF) and cytoplasmic fractions (CF). Data were normalized to β-mRNA expression and presented as fold changes, whereby siCTRL 293T treated cells transfected with vHY-IRES-TK and Tat and Rev-EGFP were set as 1. The results of three independent experiments are shown ± SD. The inhibition was significant (p = 0.00091). F) Quantitative analysis of spliced HIV-1 RNA levels modulated by MATR3 in the nucleus and the cytoplasm. The experiment was conducted for spliced (S) HIV-1 RNA as described above (Figure 4E).