Fig 1.
Identification of RRP6 and MTR4 interacting proteins.
(A) SDS-PAGE analysis followed by silver staining of eluates of tandem affinity purified Dignam nuclear extracts from HeLa S3 stably expressing Flag-HA-RRP6 or Flag-HA-MTR4 as indicated. (B) Eluates described in A were analyzed by co-immunoprecipitation using the antibodies indicated on the figure. Values shown above the blots represent the intensity of the bands relative to S3 input samples, which were set to 100%. See also S1 Fig.
Fig 2.
Identification of RRP6- and MTR4- containing complexes.
(A) MTR4- or RRP6-containing complexes were separated by gradient sedimentation analysis followed by immunoblotting using the indicated antibodies. (B) Extracts of cells stably expressing Flag-HA-MTR4 were first immunoprecipitated using anti-Flag then re-immunoprecipitated using the antibodies indicated on the figure. Nuclear extract (NE), anti-Flag immunoprecipitate and the Re-IPs were analyzed by Western blotting using the antibodies indicated. Band intensities quantified by ImageJ are shown above the blots. Samples of the first IP (input IP Flag) were set to 100%. (C) Co-immunoprecipitation analysis of ZFC3H1, ZCCHC8, MTR4, RRP6 and RBM7 was performed using the antibodies indicated on the figure. Values shown above the blots represent the intensity of the bands relative to input samples, which were set to 100%. See also S2 Fig and S3 Fig.
Fig 3.
Ablation of RRP6, MTR4 and ZFC3H1/ZCCHC8 stimulates LTR-directed transcript abundance.
(A) HeLa-LTR-luc cells were transfected with siRNAs directed against RRP6, MTR4, ZFC3H1, ZCCHC8 or a control siRNA (Scr). Cell extracts were harvested at 60 hr post-transfection and analyzed by luciferase assay and immunoblotting using the antibodies indicated. Values shown above the blots represent the intensity of the bands relative to the control transfection (Scr) samples, which were set to 100%. Fold activation was calculated relative to the control transfection (Scr), which was attributed a value of 1. Data represent mean ± SEM obtained from 3 independent experiments (**P < 0.01, *P < 0.05, independent Student’s t test). (B) HeLa-LTR-luc cells were transfected with siRNAs directed against ZFC3H1, ZCCHC8 either alone or in combination, or a control siRNA (Scr). Cell extracts were harvested at 60 hr post-transfection and analyzed by luciferase assay and immunoblotting using the antibodies indicated. Values shown above the blots represent the intensity of the bands relative to the control transfection (Scr) samples, which were set to 100%. Fold activation was calculated relative to the control transfection (Scr), which was attributed a value of 1. Data represent mean ± SEM obtained from 3 independent experiments (**P < 0.01, *P < 0.05, independent Student’s t test).
Fig 4.
RRP6, MTR4, ZFC3H1/ZCCHC8 silence LTR-driven transcription.
(A) HeLa-LTR-luc cells were transfected with siRNAs directed against RRP6, MTR4, ZFC3H1, ZCCHC8, ZFC3H1+ZCCHC8 or a control siRNA (Scr). Cell extracts were harvested at 60 hr and analyzed by immunoblotting using the antibodies indicated. Values shown above the blots represent the intensity of the bands relative to the control transfection (Scr) samples, which were set to 100%. (B) Schematic diagram showing the locations of primers used to amplify sequences by Q-PCR. The luciferase reporter gene is shown as a white box. TAR RNA stem-loop is indicated at the 5’ end of the transcript. Primers used to amplify TAR and coding region sequences are indicated on the figure. (C) Total RNA isolated from cells described in A was analyzed by RT-q-PCR using the oligonucleotide pairs indicated, and GAPDH. The amount of the indicated mRNA was normalized to the amount of GAPDH mRNA in each sample, and the values were normalized to those for the control transfection (Scr), which was attributed a value of 1. Data represent mean ± SEM obtained from 3 independent experiments (**P < 0.01, *P < 0.05, independent Student’s t test). (D) HeLa-LTR-luc cells transfected with the indicated siRNAs were analyzed by nuclear run-on transcription. RT-q-PCR was performed using the indicated primer pairs. Results are shown relative to a control sample (scr), which was attributed a value of 1. Data represent mean ± SEM obtained from 3 independent experiments (**P < 0.01, *P < 0.05, independent Student’s t test). (E) HeLa-LTR-luc cells transfected with the indicated siRNAs were analyzed by chromatin immunoprecipitation for RNAPII. PCR was performed using the indicated primer pairs. Results are shown relative to an IgG control. Data represent mean ± SEM obtained from 3 independent experiments (**P < 0.01, *P < 0.05, independent Student’s t test).
Fig 5.
RRP6, MTR4, ZFC3H1 and ZCCHC8 associate with the promoter-proximal region of HIV-1 chromatin.
(A) Schematic diagram showing the location of primers used to amplify ChIP samples by Q-PCR. Primers used to amplify promoter-proximal and coding region sequences are indicated on the figure. (B) HeLa-LTR-luc cells were analyzed by ChIP for association of RRP6, MTR4, ZFC3H1 and ZCCHC8 with the promoter-proximal and coding regions of LTR-luc, Results are shown relative to an IgG control. Data represent mean ± SEM obtained from 3 independent experiments (**P < 0.01, *P < 0.05, NS indicates not significant, independent Student’s t test). (C) HeLa-LTR-luc cells were analyzed by RNA-ChIP for association of RRP6, MTR4, ZFC3H1 and ZCCHC8 with the promoter-proximal region of LTR-luc. Data represent mean ± SEM obtained from 3 independent experiments (**P < 0.01, *P < 0.05, NS indicates not significant, independent Student’s t test). (D) HeLa-LTR-luc cells transfected with the indicated siRNAs were analyzed by RNA-ChIP for association of ZFC3H1 and ZCCHC8 with the promoter-proximal region of LTR-luc. Data represent mean ± SEM obtained from 3 independent experiments (**P < 0.01, *P < 0.05, NS indicates not significant, independent Student’s t test).
Fig 6.
RRP6, MTR4, ZFC3H1 and ZCCHC8 interact and are associated with HIV-1 chromatin in J-Lat cells.
(A) Nuclear extracts of J-Lat (clone 10.6) cells treated with a combination of 20 ng/ml TNFα and 20 mM TSA were used for co-immunoprecipitation analysis of MTR4 and RRP6 using the antibodies indicated on the figure. (B) J-Lat (clone 10.6) cells were analyzed by ChIP for association of RRP6, MTR4, ZFC3H1 and ZCCHC8 or an IgG control with the promoter and coding regions of HIV-1. A schematic diagram showing the locations of primers used to amplify sequences present in chromatin immunoprecipitates by Q-PCR is shown above the graph. Data represent mean ± SEM obtained from 3 independent experiments (***P < 0.001, **P < 0.01, *P < 0.05, independent Student’s t test).
Fig 7.
Nuclear surveillance factors modulate HIV expression in J-Lat cells.
(A-C) J-Lat (clone 10.6) cells infected with the indicated shRNAs for 7 days, treated or not with 100 nM TSA for 24 hr, were analyzed by flow cytometry for GFP expression. (A) Representative profiles are shown. (B) Graphs represent mean ± SEM of GFP-positive cells obtained from 4 independent experiments (**P < 0.01, *P < 0.05, independent Student’s t test). Numbers indicated above the columns represent fold increase over the untreated control. (C) An aliquot of cells was analyzed by direct western blotting using the antibodies indicated. (D-E) J-Lat (clone 10.6) cells were transduced with lentiviral particles expressing the indicated shRNAs. Cells expressing GFP or not were separated by cell sorting 7 days later. (D) ChIP analysis was performed using the indicated antibodies on GFP expressing (+) and non-expressing (-) cells. Graphs represent mean ± SEM from 3 independent experiments (***P < 0.001, * P < 0.05, independent Student’s t test). (E) An aliquot of cells harvested prior to sorting was analyzed by direct western blotting using the antibodies indicated.
Fig 8.
Nuclear exosome factors modulate HIV-1 expression in PBMCs.
(A-C) PBMCs from healthy donors were activated using anti-CD3/CD28 beads and IL2 (30 U/ml) for 3 days then infected with HIV-1BaL. Cells were transduced with lentiviral particles expressing the indicated shRNAs 3 days post-infection. Cell extracts and culture supernatants were harvested 7 days later. (A) Graph represents mean ± SEM of p24 concentration in cell culture supernatants obtained from 3 experiments performed on different donor cells (*P < 0.05, independent Student’s t test). (B) RT-qPCR analysis of cell extracts from (A) using primers amplifying the Vif region (*P < 0.05, independent Student’s t test). (C) An aliquot of cells was analyzed by direct western blotting using the antibodies indicated. (D-F) Activated CD4+ T-cells from healthy donors were infected with HIV-1DuoFluo and latently infected cells isolated by cell sorting were transduced with lentiviral particles expressing the indicated shRNAs. Cells were analyzed by flow cytometry and p24 ELISA 7 days later. (D) Representative profiles are shown. Circled areas on each profile show mCherry single positive cells (left circle) and mCherry and GFP double positive cells (right circle). Numbers indicate the percentage of cells of the total within the circles. (E) Quantification of p24 present in culture supernatants. Graphs represent mean ± SEM obtained from 3 experiments using PBMC from different donors (***P < 0.001, independent Student’s t test). (F) An aliquot of cells was analyzed by direct western blotting using the antibodies indicated. See also S5 Fig.