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