6-Mercaptopurine attenuates tumor necrosis factor-α production in microglia through Nur77-mediated transrepression and PI3K/Akt/mTOR signaling-mediated translational regulation

Physical interaction between Nur77 and p65. BV-2 cells were pretreated with 6-MP (50 μM) for 16 h followed by exposure to LPS (100 ng/ml) for 60 min. Nuclear extracts were harvested for immunoprecipitation (IP) experiments using anti-Nur77 and anti-p65 antibodies. Immunoblot (IB) analyses of the immunoprecipitates were performed using these antibodies. The immunoblots are representative of three independent experiments. (TIF 280 kb

Cleavage of Dicer protein by I7 protease during vaccinia virus infection.

Dicer is the key component in the miRNA pathway. Degradation of Dicer protein is facilitated during vaccinia virus (VV) infection. A C-terminal cleaved product of Dicer protein was detected in the presence of MG132 during VV infection. Thus, it is possible that Dicer protein is cleaved by a viral protease followed by proteasome degradation of the cleaved product. There is a potential I7 protease cleavage site in the C-terminus of Dicer protein. Indeed, reduction of Dicer protein was detected when Dicer was co-expressed with I7 protease but not with an I7 protease mutant protein lack of the protease activity. Mutation of the potential I7 cleavage site in the C-terminus of Dicer protein resisted its degradation during VV infection. Furthermore, Dicer protein was reduced dramatically by recombinant VV vI7Li after the induction of I7 protease. If VV could facilitate the degradation of Dicer protein, the process of miRNA should be affected by VV infection. Indeed, accumulation of precursor miR122 was detected after VV infection or I7 protease expression. Reduction of miR122 would result in the suppression of HCV sub-genomic RNA replication, and, in turn, the amount of viral proteins. As expected, significant reduction of HCVNS5A protein was detected after VV infection and I7 protease expression. Therefore, our results suggest that VV could cleave Dicer protein through I7 protease to facilitate Dicer degradation, and in turn, suppress the processing of miRNAs. Effect of Dicer protein on VV replication was also studied. Exogenous expression of Dicer protein suppresses VV replication slightly while knockdown of Dicer protein does not affect VV replication significantly

Suppression of vaccinia virus replication slightly by exogenously expressed Dicer protein.

<p>(A, B) HeLa cells were transfected with empty vector (2 ug, lanes 1 and 2), with the plasmid expressing T7-Dicer protein (2 ug, lanes 3 and 4) or with the plasmid expressing T7-Dicer with mutations in a.a. 1816 and 1817 (2 ug, lanes 5 and 6). Twenty-four hrs after transfection, the cells were mock-infected (lanes 1, 3 and 5) or infected with VV (M.O.I. = 10) (lanes 2, 4 and 6). Twenty-four hrs after infection, cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against the Dicer, A type inclusion protein and β-actin (A) while the secreted virus particles from the supernatants (lanes 2, 4 and 6) were analyzed by the plaque assay (B). (p<0.01, **). (C) HeLa cells were transfected with scramble siRNA as a control or siRNA against Dicer. Twenty-four hrs after transfection, cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against the Dicer (upper panel) and β-actin proteins (bottom panel) as the loading control. (D) HeLa cells were transfected with scramble or Dicer siRNA. Twenty-four hrs after transfection, the cells were infected with VV (M.O.I. = 10). Twenty-four hrs after infection, the secreted viral particles from the supernatants were analyzed by plaque assay. NS: not significant.</p

Dicer protein was reduced after the induction of I7 protease expression during recombinant virus vI7Li infection.

<p>HeLa cells were mock-infected, infected with wild-type vaccinia virus (WT) or recombinant virus vI7Li (M.O.I. = 5). IPTG was also added as the indicated amount. Twenty-four hrs after infection, cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against the Dicer, A type inclusion protein, I7, β-actin for the loading control or GFP which is constitutively expressed during vI7Li infection.</p

Inhibition of miR122 function either by VV infection or by I7 protease expression.

<p>(A) HCV replicon cells were mock-infected (lane 2) or infected with VV in M.O.I. = 1 (lane 3), 5 (lane 4), 10 (lanes 5 and 6). 100 ug araC was also added (lane 6). Twenty-four hrs later, cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against Dicer protein (upper panel), NS5A and NPT proteins for the expression of HCV subgenomic RNAs, or Erk2 protein as the loading control. Cell lysates from mock-infected HuH7 cells (lane 1) were served as a negative control for the detection of NS5A and NPT. (B) HCV replicon cells were mock-transfected or transfected with the indicated amount of the plasmid expressing I7 protease (lanes 1–6). Thirty-two hrs after transfection, 10 uM MG132 was also added (lanes 5 and 6). Sixteen hrs later, cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against Dicer protein, NS5A protein for the expression of HCV subgenomic RNAs, NPT protein (the thin arrow marks the position of NPT protein expressed from the transfected plasmids while the thick arrow marks the position of core-NPT fusion protein encoding from HCV subgenomic RNAs.), V5 tag to detect the expression of I7 (The arrow marks the position of I7 in lane 6.) or Erk2 protein as the loading control.</p

Inhibition of miR122 processing either by VV infection or by I7 protease expression.

<p>(A) HuH7 cells were either mock-infected or infected with VV in different M.O.I. (1, 5, and 10). Twenty-four hrs after infection, mRNAs were extracted and converted into cDNA. Then, real-time PCR assay was performed to detect the amount of miR122 using U6 mRNA as the internal control for normalization. (B) HeLa cells were mock-transfected, transfected with empty vector (2 ug) or with the plasmid expressing pre-miR122 (2 ug). Twenty-four hrs after transfection, the cells with pre-miR122 were mock-infected or infected with VV in different M.O.I. (1, 5, and 10). Twenty-four hrs after infection, mRNAs were extracted and converted into cDNA. Then, real-time PCR assay was performed to detect the amount of miR122 using U6 mRNA as the internal control for normalization. (C) HeLa cells were mock-transfected or co-transfected with the plasmids expressing pre-miR122 and I7 protease with the indicated amount. Forty-eight hrs after transfection, mRNAs were extracted and converted into cDNA. Then, real-time PCR assay was performed to detect the amount of miR122 using U6 mRNA as the internal control for normalization. (p<0.05, *; p<0.01, **; p<0.001, ***).</p

Reduction of Dicer protein during vaccinia virus infection.

<p>(A) HeLa cells were either mock infected (lane 1) or infected with VV (M.O.I. = 10) in the absence (lane 2) or in the presence (lane 3) of AraC. Sixteen hrs after infection, cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against the extreme C-terminus of Dicer protein (upper panel), A type inclusion protein (middle panel) or β -actin protein (lower panel) as the loading control. (B) HeLa cells were either mock transfected (lane 1) or transfected with the plasmid expressing Dicer protein with a T7 tag at its N-terminus (T7-Dicer, lanes 2–6). Forty-eight hrs after transfection, these cells were either mock infected (lane 2) or infected with VV (M.O.I. = 3, lanes 3–5). After the time indicated, cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against the extreme C-terminus of Dicer protein (upper panel), NPT protein as the transfection control, A type inclusion protein or Erk2 protein (bottom panel) as the loading control. (C) HeLa cells were either mock transfected (lanes 1 and 5) or transfected with the plasmid expressing T7-Dicer (lanes 2–4 and 6–8). Forty-eight hrs after transfection, these cells were either mock infected, infected with VV (M.O.I. = 3, lanes 3, 7 and 8) or with influenza A virus (M.O.I. = 3, lane 4). MG132 (lane 7) or araC (lane 8) was also added in the culture. Twenty-four hrs after infection, cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against the T7 tag at the N-terminus of Dicer protein (upper panel), against NPT protein (middle panel) as the transfection control, or against Erk2 protein (bottom panel) as the loading control. (D) HuH7 cells were either mock infected (lane 1) or infected with VV in M.O.I = 1 (lane 2), 5 (lane 3) or 10 (lanes 4 and 5). araC was also added in the culture (lane 5). Twenty-four hrs after infection, cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against the Dicer protein (upper panel), A type inclusion protein (middle panel), β-actin protein or Erk2 protein (lower panel). Both β-actin and Erk2 proteins were served as the loading control.</p