Figure 3. Knockdown of TRIM22 by RNA interference abrogates interferon-induced inhibition of HIV replication.

A) HOS-CD4/CXCR4 cells were transfected with the empty vector control plasmid pLKO.1 or pLKO.1/TRIM22 shRNA_#1 followed by treatment with 0, 50, 500 or 1000 units/ml of IFNβ (represented by triangles at the top of the Northern blots). Total RNA was extracted and subjected to Northern blot analysis to detect TRIM22 RNA transcripts. TRIM34 (variant 3) RNA transcript levels were also detected for a comparison (middle row). Input RNA was normalized using Northern blots probed with an actin fragment (bottom row). B) Quantitation of the Northern blots in A, indicating the fold induction of TRIM22 RNA in response to the IFNβ treatment. C) Cells were transfected with the empty vector pLKO.1 or pLKO.1/TRIM22_shRNA#1 and treated with 1000 units/ml of IFNβ. Cells were then transfected with pR9 and Western blot analysis of HIV capsid antigen in cells and supernatants was analyzed using an anti-p24 antibody. D) Cells were transfected with the control plasmids pLKO.1, pLKO.1/eGFP_shRNA or pLKO.1/scrambled_shRNA or TRIM22 shRNA plasmids pLKO.1/TRIM22_shRNA#1 and pLKO.1/TRIM22_shRNA#2 and treated with 1000 units/ml of IFNβ. Following transfection with pR9, HIV capsid antigen released into the supernatant was detected by Western blot and quantified densitometrically. Data represents the averages (+/−StdDev) of at least 3 experiments performed in triplicate. E) A comparison of the average fold inhibition in particle release of the pooled control shRNAs and the pooled TRIM22 shRNAs (+/−StdDev). *, P = 0.0002 (Mann-Whitney).