Journal: Journal of Virology

Article Title: Capsid-CPSF6 Interaction Is Dispensable for HIV-1 Replication in Primary Cells but Is Selected during Virus Passage In Vivo

doi: 10.1128/jvi.00019-16

Figure Lengend Snippet: Figure 3. The A77V mutation in the CPSF6 binding pocket relieves the 1180

Article Snippet: The pellets were fractionated by SDS-PAGE and 208 immunoblotted with antibodies against CA (183-H12-5C) and CPSF6 (Novus 209 on June 18, 2016 by U N IV E R S IT Y O F LE E D S http://jvi.asm .org/ D ow nloaded from 10 Biologicals).

Techniques: Mutagenesis, Binding Assay

Journal: Journal of Virology

Article Title: Truncated CPSF6 Forms Higher-Order Complexes That Bind and Disrupt HIV-1 Capsid

doi: 10.1128/JVI.00368-18

Figure Lengend Snippet: Purification of CPSF6-358 with an albumin tag from the mammalian secretory expression system. (A) SDS-PAGE and Western blot analysis of His6-albumin–CPSF6-358 expression and purification. Samples taken from untransfected cells (U), transfected cells (T), the flowthrough (FT) and elution (E) from Ni-NTA resin, and peaks (P1 and P2) from the Superdex 200 26/60 column (shown in panel B) were stained with Coomassie blue (top) or processed with anti-His (middle) or anti-CPSF6 (bottom) antibody, following Western blotting. (B) Gel filtration profile of the protein eluted from the Superdex 200 26/60 column. The two His6-albumin–CPSF6-358 peaks are labeled P1 and P2. (C) Representative EM images of negatively stained His6-albumin–CPSF6-358 samples from fractions P1 (left) and P2 (right), as shown in panel B. Scale bars, 100 nm.

Article Snippet: HeLa cells stably expressing CPSF6-358–eGFP (deposited in Addgene; no. 110693) or CPSF6-358-iRFP670 (deposited in Addgene; no. 110694) were seeded in MatTek dishes overnight in Fluorobrite medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals) and penicillin, streptomycin, and glutamine (Thermo Fisher Scientific) at 37°C and 5% CO 2 .

Techniques: Purification, Expressing, SDS Page, Western Blot, Transfection, Staining, Filtration, Labeling

Journal: Journal of Virology

Article Title: Truncated CPSF6 Forms Higher-Order Complexes That Bind and Disrupt HIV-1 Capsid

doi: 10.1128/JVI.00368-18

Figure Lengend Snippet: Characterization of CPSF6-358 oligomerization states. (A) SEC-MALS analysis of His6-albumin–CPSF6-358 samples from P1 (black) and P2 (red) samples, shown in Fig. 1; the estimated molecular mass of the monomeric form of the protein should be 110 kDa. (B) Superdex 200 gel filtration of CPSF6-358 after TEV cleavage of the His6-albumin tag of P2 (top) with an EM image of the purified CPSF6-358 fraction from the position of the peak indicated by the arrow (inset) and SDS-PAGE of the corresponding peaks, stained with Coomassie blue (bottom). (C) Analytical ultracentrifugation analysis of His6-albumin–CPSF6-358 from P1 (blue), P2 (black), and CPSF6-358 (red) at 1.0 mg/ml. The expected oligomeric state for each peak is indicated.

Article Snippet: HeLa cells stably expressing CPSF6-358–eGFP (deposited in Addgene; no. 110693) or CPSF6-358-iRFP670 (deposited in Addgene; no. 110694) were seeded in MatTek dishes overnight in Fluorobrite medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals) and penicillin, streptomycin, and glutamine (Thermo Fisher Scientific) at 37°C and 5% CO 2 .

Techniques: Filtration, Purification, SDS Page, Staining

Journal: Journal of Virology

Article Title: Truncated CPSF6 Forms Higher-Order Complexes That Bind and Disrupt HIV-1 Capsid

doi: 10.1128/JVI.00368-18

Figure Lengend Snippet: Estimated molecular masses of the CPSF6-358 proteins from the c(s) analysis a

Article Snippet: HeLa cells stably expressing CPSF6-358–eGFP (deposited in Addgene; no. 110693) or CPSF6-358-iRFP670 (deposited in Addgene; no. 110694) were seeded in MatTek dishes overnight in Fluorobrite medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals) and penicillin, streptomycin, and glutamine (Thermo Fisher Scientific) at 37°C and 5% CO 2 .

Techniques: Sedimentation

Journal: Journal of Virology

Article Title: Truncated CPSF6 Forms Higher-Order Complexes That Bind and Disrupt HIV-1 Capsid

doi: 10.1128/JVI.00368-18

Figure Lengend Snippet: CPSF6-358 binds and disrupts WT CA tubular assemblies. (A) SDS-PAGE of WT and N74D CA assemblies, following incubation with His6-albumin–CPSF6-358, from P1 or P2 and centrifugation. The gel was Coomassie blue stained, with supernatant (s) and pellet (p) samples indicated. (B) SDS-PAGE of WT and N74D CA assemblies following incubation with untagged CPSF6-358 and centrifugation. (C to H) Representative negative-stain EM micrographs of the samples in panel A. (C to E) WT CA tubular assemblies alone (C) or with 30 μM P1 (D) or 30 μM P2 (E) His6-albumin–CPSF6-358. (F to H) CA N74D alone (F) or with 30 μM P1 (G) or 30 μM P2 (H) His6-albumin–CPSF6-358. The arrows indicate the capsid fragments. (I to L) Representative negative-stain EM micrographs of the samples in panel B. Shown are WT CA tubular assemblies alone (I) or with 30 μM CPSF6-358 (J) and CA N74D tubular assemblies alone (K) or with 30 μM CPSF6-358 (L). Scale bars, 100 nm. (M) Dose-dependent effect of CPSF6-358 on CA tubes. Shown is binding of P1 (blue), P2 (black), and CPSF6-358 (red) to assembled WT CA tubes (left). The effects of P1 (blue), P2 (black), and CPSF6-358 (red) binding on the average length of tubes (middle) and on the number of remaining initial tubular assemblies (right) were measured. The error bars indicate the standard deviation of the values.

Article Snippet: HeLa cells stably expressing CPSF6-358–eGFP (deposited in Addgene; no. 110693) or CPSF6-358-iRFP670 (deposited in Addgene; no. 110694) were seeded in MatTek dishes overnight in Fluorobrite medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals) and penicillin, streptomycin, and glutamine (Thermo Fisher Scientific) at 37°C and 5% CO 2 .

Techniques: SDS Page, Incubation, Centrifugation, Staining, Binding Assay, Standard Deviation

Journal: Journal of Virology

Article Title: Truncated CPSF6 Forms Higher-Order Complexes That Bind and Disrupt HIV-1 Capsid

doi: 10.1128/JVI.00368-18

Figure Lengend Snippet: Dynamic interactions occur between CPSF6-358 and WT HIV-1 particles. (A) Images were obtained by live-cell frustrated TIRF imaging 10 min after synchronized infection with WT HIV-1 of HeLa cells stably expressing CPSF6-358–eGFP. The arrowheads indicate initial colocalization of CPSF6-358–eGFP (green) with mRuby3-IN (red) and then separation approximately 3 min later. (B) eGFP and mRuby3 colocalized particles were quantified at 10, 30, and 60 min postinfection. The error bars represent SEM. ****, P < 0.0001.

Article Snippet: HeLa cells stably expressing CPSF6-358–eGFP (deposited in Addgene; no. 110693) or CPSF6-358-iRFP670 (deposited in Addgene; no. 110694) were seeded in MatTek dishes overnight in Fluorobrite medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals) and penicillin, streptomycin, and glutamine (Thermo Fisher Scientific) at 37°C and 5% CO 2 .

Techniques: Imaging, Infection, Stable Transfection, Expressing

Journal: Journal of Virology

Article Title: Truncated CPSF6 Forms Higher-Order Complexes That Bind and Disrupt HIV-1 Capsid

doi: 10.1128/JVI.00368-18

Figure Lengend Snippet: Binding of CPSF6-358 with 14C/45C/W184A/M185A hexamer. (A to C) Gel filtration (Superdex 200) profile of CA hexamer with His6-albumin–CPSF6-358 from P1 (A) or P2 (B) or with untagged CPSF6-358 (C). Red, CA hexamer alone; blue, CPSF6-358 proteins alone; black, mixtures. (D) SDS-PAGE analysis of fractions in panels A to C.

Article Snippet: HeLa cells stably expressing CPSF6-358–eGFP (deposited in Addgene; no. 110693) or CPSF6-358-iRFP670 (deposited in Addgene; no. 110694) were seeded in MatTek dishes overnight in Fluorobrite medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals) and penicillin, streptomycin, and glutamine (Thermo Fisher Scientific) at 37°C and 5% CO 2 .

Techniques: Binding Assay, Filtration, SDS Page

Journal: Journal of Virology

Article Title: Truncated CPSF6 Forms Higher-Order Complexes That Bind and Disrupt HIV-1 Capsid

doi: 10.1128/JVI.00368-18

Figure Lengend Snippet: WT HIV-1 infection induces formation of CPSF6-358 higher-order complexes in HeLa cells. (A) Confocal images of HeLa cells stably expressing CPSF6-358–eGFP before or 30 min after infection with WT HIV-1 or N74D HIV-1. (B) CPSF6-358–eGFP puncta and mRuby-IN particles were quantified per cell (n ≥ 25 z-stacks) at 30 min postinfection with WT HIV-1 in the presence or absence of 10 μM PF-74, N74D HIV-1, or A77V HIV-1. The asterisks denote comparisons with P values of <0.05. (C) HeLa cells stably expressing CPSF6-358–eGFP were treated (open symbols) or not (solid symbols) with 2 μM CsA and synchronously infected with WT HIV-1 or N74D HIV-1. The number of CPSF6-358–eGFP puncta per field of view was determined. The error bars represent standard error of the mean (SEM). *, P < 0.05; **, P < 0.005; ***, P < 0.001.

Article Snippet: HeLa cells stably expressing CPSF6-358–eGFP (deposited in Addgene; no. 110693) or CPSF6-358-iRFP670 (deposited in Addgene; no. 110694) were seeded in MatTek dishes overnight in Fluorobrite medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals) and penicillin, streptomycin, and glutamine (Thermo Fisher Scientific) at 37°C and 5% CO 2 .

Techniques: Infection, Stable Transfection, Expressing

Journal: Journal of Virology

Article Title: Truncated CPSF6 Forms Higher-Order Complexes That Bind and Disrupt HIV-1 Capsid

doi: 10.1128/JVI.00368-18

Figure Lengend Snippet: Capsid permeabilization of WT HIV-1 occurs more quickly in HeLa cells expressing CPSF6-358–eGFP. HeLa cells and HeLa cells expressing CPSF6-358–eGFP were infected with WT HIV-1 (A) or N74D HIV-1 (B) and stained for viral RNA at different times. The error bars represent SEM of two (WT) or one (N74D) independent experiment. *, P < 0.05; ***, P < 0.001.

Article Snippet: HeLa cells stably expressing CPSF6-358–eGFP (deposited in Addgene; no. 110693) or CPSF6-358-iRFP670 (deposited in Addgene; no. 110694) were seeded in MatTek dishes overnight in Fluorobrite medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals) and penicillin, streptomycin, and glutamine (Thermo Fisher Scientific) at 37°C and 5% CO 2 .

Techniques: Expressing, Infection, Staining

Journal: bioRxiv

Article Title: HIV-1 Infection Regulates Gene Expression by Altering Alternative Polyadenylation Through CPSF6 and CPSF5 Delocalization

doi: 10.1101/2025.08.07.669137

Figure Lengend Snippet: (A) To induce the translocation of CPSF6 to nuclear speckles, human A549 cells were challenged with HIV-1-GFP at an MOI of 2 for 24 h. Infection was assessed as the percentage of GFP-positive cells by flow cytometry. The results of three independent experiments with standard deviation are shown. (B) Cells were subsequently fixed, permeabilized, and immunolabelled using specific antibodies against SC35 (red, marker for nuclear speckles) and CPSF6 (green). Nuclei were stained with DAPI (blue). (C) The average (and standard deviation) percentage of cells containing CPSF6 in nuclear speckles (SC-35 positive compartments) from three independent experiments, as determined by visual examination of 200 cells. (D) Total RNA from three infected and three mock-infected samples was sequenced, and gene expression is displayed in a Volcano plot. The x-axis shows Log 2 fold changes in gene expression, with positive indicating upregulation and negative indicating downregulation. The y-axis shows the statistical significance, expressed as −Log 10 P. (E) Gene ontology analysis of all genes upregulated and downregulated by HIV-1 infection. The number of genes in each pathway is expressed as the size of the sphere, and the color represents the P-adjusted value. (F) Volcano plot showing PAS changes. The x-axis shows compositional fold-change, which represents the 3’UTR length for each transcript. A positive compositional fold-change indicates the presence of a longer 3’UTR (red), and a negative compositional fold-change indicates the presence of a shorter 3’UTR (green). The y-axis shows the statistical significance of the result conveyed as the −Log 10 P. APA, alternative polyadenylation; CPSF6, cleavage and polyadenylation specificity factor subset 6; DAPI, 4’6-diamino-phenylindole; GFP, green fluorescent protein; MOI, multiplicity of infection; PAS, polyadenylation signal; UTR, untranslated region.

Article Snippet: We used mouse monoclonal antibodies targeting the following proteins: SC35 (clone SC-35; Cat# ab11826, Abcam), CPSF5 (clone 3F8;Cat# H00011051-M12, Novus Biologicals), CPSF7 (clone A-9; Cat# sc-393880, Santa Cruz), CPSF6 (clone F-3; Cat# sc-376228, Santa Cruz), ISG15 (clone F-9; Cat# sc-166755, Santa Cruz), and HIV-1 p24 (clone 183-H12-5C; Cat# ARP-3537, NIH AIDS Reagent Program).

Techniques: Translocation Assay, Infection, Flow Cytometry, Standard Deviation, Marker, Staining, Gene Expression

Journal: bioRxiv

Article Title: HIV-1 Infection Regulates Gene Expression by Altering Alternative Polyadenylation Through CPSF6 and CPSF5 Delocalization

doi: 10.1101/2025.08.07.669137

Figure Lengend Snippet: (A) A549 WT, NT#H1, CPSF6-KO#B4, CPSF6-KO#B7, and CPSF6-KO#C8 cells were lysed, and proteins were analyzed by Nu-PAGE, followed by western blot using anti-CPSF6, anti-CPSF5, anti-CPSF7, and anti-GAPDH antibodies. Experiments were repeated at least three times, and a representative image is shown. Graphs show the average densitometry quantification of three replicates with standard deviation. Significance was determined using unpaired t-test; *p<0.05; **p<0.01; ***p<0.001; ns, not significant. (B) Total RNA from CPSF6-KO and WT A549 cells was prepared, and polyadenylated transcripts were identified by PAC-seq, followed by APA analysis using PolyAMiner. (C) A549 WT, NT#H1, CPSF6-KO#B4, CPSF6-KO#B7, and CPSF6-KO#C8 cells were infected with increasing amounts of HIV-1-GFP for 24 or 48 h. Infection was assessed as the percentage of GFP-positive cells by flow cytometry. (D) WT and CPSF6-KO A549 cells were infected with HIV-1-GFP at an MOI of 2 for 48 h. Cells were fixed, permeabilized, and stained using the following antibodies: (i) anti-CPSF5 (red) and anti-CPSF6 (green); (ii) anti-SC-35 (red) and anti-CPSF6 (green); (iii) anti-LEDGF/p75 (red) and anti-CPSF6 (green); and (iv) anti-LEDGF/p75 (red) and anti-SC35 (green). Nuclei were stained with DAPI (blue). Scale bar, 10 µm. (E) Percentage of A549 cells containing CPSF6, CPSF5, or CPSF7 in nuclear speckles (condensates) upon HIV-1 infection (average of three independent experiments with standard deviation). Cells containing CPSF6, CPSF5, or CPSF7 in nuclear speckles were determined by visual examination of 200 cells APA, alternative polyadenylation; CPSF5, cleavage and polyadenylation specificity factor subset 5; CPSF6, cleavage and polyadenylation specificity factor subset 6; CPSF7, cleavage and polyadenylation specificity factor subset 7; DAPI, 4’,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; KO, knockout; LEDGF/p75, lens epithelium-derived growth factor; MOI, multiplicity of infection; NT, non-targeting; PAC-Seq, Poly(A)-Click-Sequencing; UTR, untranslated region; WT, wild-type; hpi, hours post-infection.

Article Snippet: We used mouse monoclonal antibodies targeting the following proteins: SC35 (clone SC-35; Cat# ab11826, Abcam), CPSF5 (clone 3F8;Cat# H00011051-M12, Novus Biologicals), CPSF7 (clone A-9; Cat# sc-393880, Santa Cruz), CPSF6 (clone F-3; Cat# sc-376228, Santa Cruz), ISG15 (clone F-9; Cat# sc-166755, Santa Cruz), and HIV-1 p24 (clone 183-H12-5C; Cat# ARP-3537, NIH AIDS Reagent Program).

Techniques: Western Blot, Standard Deviation, Infection, Flow Cytometry, Staining, Knock-Out, Derivative Assay, Sequencing

Journal: bioRxiv

Article Title: HIV-1 Infection Regulates Gene Expression by Altering Alternative Polyadenylation Through CPSF6 and CPSF5 Delocalization

doi: 10.1101/2025.08.07.669137

Figure Lengend Snippet: (A) CPSF6-KO and control (WT and NT#H1) A549 cells were analyzed by western blot using anti-SLFN5 and anti-CPSF6 antibodies. Anti-GAPDH antibodies were used as a loading control. (B) A549 cells were challenged with HIV-1-GFP or HIV-1-Luc using an MOI of 2 for 48 hours. Subsequently, cells were lysed, and extracts were analyzed by western blot using anti-SLFN5, anti-CPSF6, anti-CPSF5, anti-p24, and anti-GAPDH antibodies. (C) A549 cells were challenged with three different HIV-1-A77V-GFP preparations using an MOI of 2 for 48 hours. Cells were lysed and analyzed by western blot using anti-SLFN5, anti-CPSF5, and anti-p24. GAPDH was used as a loading control. ( A–C ) All experiments were repeated at least three times, and a representative image is shown. Graphs show the average densitometry quantification of at least three replicates with standard deviation. Significance was determined using unpaired t-test; *p<0.05; **p<0.01; ***p<0.001; ns, not significant. CPSF5, cleavage and polyadenylation specificity factor subset 5; CPSF6, cleavage and polyadenylation specificity factor subset 6; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein KO, knockout; Luc, luciferase; MOI, multiplicity of infection; NT, non- targeting; p24, viral capsid; SLFN5, Schlafen family member 5; WT, wild-type.

Article Snippet: We used mouse monoclonal antibodies targeting the following proteins: SC35 (clone SC-35; Cat# ab11826, Abcam), CPSF5 (clone 3F8;Cat# H00011051-M12, Novus Biologicals), CPSF7 (clone A-9; Cat# sc-393880, Santa Cruz), CPSF6 (clone F-3; Cat# sc-376228, Santa Cruz), ISG15 (clone F-9; Cat# sc-166755, Santa Cruz), and HIV-1 p24 (clone 183-H12-5C; Cat# ARP-3537, NIH AIDS Reagent Program).

Techniques: Control, Western Blot, Standard Deviation, Knock-Out, Luciferase, Infection

Journal: Cell reports

Article Title: CPSF1 inhibition promotes widespread use of intergenic polyadenylation sites and impairs glycolysis in prostate cancer cells

doi: 10.1016/j.celrep.2024.115211

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies were purchased from Santa Cruz for western blot detection of CPSF1, (G-10, sc-166281, 1:100) and tubulin (B-5-1-2, sc-23948, 1:3000), Proteintech for western blot detection of NUDT21 (66335-1-Ig, 1:500) and PCF11 (23540-1-AP, 1:500), Fortis Life Sciences for western blot detection of CPSF6 (A301-356A, 1:1,000) and Origene for western blot detection of GPI (TA501171, 1:500).

Techniques: Recombinant, shRNA, Over Expression, Plasmid Preparation, Cloning, Virus, Software