Journal: Retrovirology

Article Title: TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm

doi: 10.1186/1742-4690-10-20

Figure Lengend Snippet: The effect of CPSF6-358 on the infectivity of HIV-1 CA mutants correlates with the effect of TNPO3 KD. ( A ) Schematic representation of the protein domains of WT CPSF6 and the truncated mutant CPSF6-358. RNA recognition motif (RRM), proline-rich domain (P-rich), arginine/serine rich domain (RS). ( B ) Expression levels of CPSF6 in TZM-bl cells transduced with empty or CPSF6-358 vectors. Cell lysates were probed in western blots with anti-CPSF6 antibody (upper panel) and anti-β-actin antibody (lower panel). The upper panel shows the endogenous CPSF6 and the truncated form. ( C ) TZM-bl cells transduced with an empty vector or with a vector encoding CPSF6-358 were challenged with a panel of 27 HIV-1-GFP reporter vectors bearing either WT CA or the indicated CA mutants. At 72 hrs the percent GFP + cells was determined by flow cytometry as an indication of infectivity. The ratio of HIV-1 infectivity in CPSF6-358 expressing cells and empty vector cells is shown. White bars show CA mutants inhibited to a similar extent as the WT virus by CPSF6-358, black bars shows CA mutants insensitive or slightly sensitive to CPSF6-358, and gray bars show CA mutants hypersensitive to the presence of CPSF6-358 in the cell. ( D ) Correlation between the infectivity ratios of the 27 CA mutants when infecting Ctrl KD vs TNPO3 KD and Empty vector vs CPSF6-358 (R 2 = 0.8528).

Article Snippet: Cells were then incubated for 1 hour either with anti-HA (Covance) or anti-CPSF6 antibodies (Novus Biologicals) diluted in PBS with 1% BSA.

Techniques: Infection, Mutagenesis, Expressing, Transduction, Western Blot, Plasmid Preparation, Flow Cytometry, Virus

Journal: Retrovirology

Article Title: TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm

doi: 10.1186/1742-4690-10-20

Figure Lengend Snippet: CPSF6-358 inhibits HIV-1 replication when localized to the cytoplasm. ( A ) CPSF6 protein in TZM-bl cells transduced with empty, CPSF6-358, CPSF6-358 NLS and CPSF6-358 NES vectors. Cell lysates were probed in western blots with anti-CPSF6 antibody (upper panel) and anti-β-actin antibody (lower panel). The upper panel shows the endogenous CPSF6 and the truncated form. ( B ) Indirect immunofluorescence showing localization of the different forms of CPSF6-358. The TZM-bl cells, transduced as in ( A ), were stained with an anti-HA antibody (green) for the detection of the CPSF6-358 proteins. DAPI was used to mark the nuclear compartment (blue). ( C ) TZM-bl stably expressing the different forms of CPSF6-358 were challenged with WT or CA mutant HIV-1 NL4-3 GFP reporter viruses. After 72 hrs, GFP reporter expression was assessed by flow cytometry. Data represent one of at least three independent experiments. Error bars represent ± SEM (n = 3).

Article Snippet: Cells were then incubated for 1 hour either with anti-HA (Covance) or anti-CPSF6 antibodies (Novus Biologicals) diluted in PBS with 1% BSA.

Techniques: Transduction, Western Blot, Immunofluorescence, Staining, Stable Transfection, Expressing, Mutagenesis, Flow Cytometry

Journal: Retrovirology

Article Title: TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm

doi: 10.1186/1742-4690-10-20

Figure Lengend Snippet: HIV-1 replication is inhibited when full-length CPSF6 is targeted to the cytoplasm. ( A ) Expression levels of CPSF6 in TZM-bl cells transduced with empty, CPSF6, CPSF6-NLS and CPSF6-NES vectors. Cell lysates were probed in western blots with anti-CPSF6 antibody (upper panel) and anti-β-actin antibody (lower panel). The upper panel shows the endogenous and exogenous full-length CPSF6 with an HA tag. ( B ) Localization of different forms of CPSF6 in TZM-bl cells stably expressing CPSF6, CPSF6 NLS or CPSF6 NES. The cells were stained with an anti-HA antibody (green) for the detection of the CPSF6 proteins. DAPI staining (blue) was used to mark the nuclear compartment. ( C ) TZM-bl stably expressing the different forms of CPSF6 were challenged with WT or CA mutant HIV-1 NL4-3 GFP reporter viruses. After 72 hours, GFP reporter expression was assessed by flow cytometry. Data represent one of at least three independent experiments. Error bars represent ± SEM (n = 3).

Article Snippet: Cells were then incubated for 1 hour either with anti-HA (Covance) or anti-CPSF6 antibodies (Novus Biologicals) diluted in PBS with 1% BSA.

Techniques: Expressing, Transduction, Western Blot, Stable Transfection, Staining, Mutagenesis, Flow Cytometry

Journal: Retrovirology

Article Title: TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm

doi: 10.1186/1742-4690-10-20

Figure Lengend Snippet: TNPO3 KD inhibits HIV-1 replication by shifting CPSF6 to the cytoplasm. ( A ) Expression level of CPSF6 and TNPO3 in TZM-bl cells stably transduced with control or CPSF6 KD vectors and transfected with scrambled or TNPO3 specific small interfering RNA (siRNA). Cell lysate was probed in western blots with anti-TNPO3 antibody (upper panel), anti-CPSF6 antibody (middle panel) and anti-β-actin antibody (lower panel). ( B ) Immuno-fluorescence localization of endogenous CPSF6 (green) in control (Ctrl) KD or TNPO3 KD TZM-bl cells. DAPI staining (blue) was used to mark the nuclear compartment. ( C ) Cell fractionation to identify the cellular localization of endogenous CPSF6. Expression of tubulin in the cytoplasm and histone 3 (H3) in the nucleus was assessed to verify the fractionation. ( D ) Stable ctrl KD and CPSF6 KD cells transfected with scrambled or TNPO3 siRNA were challenged with WT or CA mutant HIV-1 NL4-3 GFP reporter viruses. After 72 hours GFP expression was checked by flow cytometry. The fold inhibition of infectivity due to TNPO3 KD is shown. Data represent one of at least three independent experiments. Error bars represent ± SEM (n = 3).

Article Snippet: Cells were then incubated for 1 hour either with anti-HA (Covance) or anti-CPSF6 antibodies (Novus Biologicals) diluted in PBS with 1% BSA.

Techniques: Expressing, Stable Transfection, Transduction, Control, Transfection, Small Interfering RNA, Western Blot, Fluorescence, Staining, Cell Fractionation, Fractionation, Mutagenesis, Flow Cytometry, Inhibition, Infection

Journal: Retrovirology

Article Title: TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm

doi: 10.1186/1742-4690-10-20

Figure Lengend Snippet: TNPO3 depletion does not inhibit HIV-1 if CPSF6 is independently targeted to the nucleus. ( A ) CPSF6 and TNPO3 protein in TZM-bl cells stably transduced with CPSF6 KD vectors, control or TNPO3 KD vectors, and rescue of CPSF6 (ntCPSF6) with or without the SV40 T-Ag NLS. Cell lysate was probed in western blots with anti-TNPO3 antibody (upper panel), anti-CPSF6 antibody (middle panel) and anti-β-actin antibody (lower panel). ( B ) Localization of the non-targetable CPSF6 (ntCPSF6) constructs (green) in control (Ctrl) KD or TNPO3 KD TZM-bl cells stably depleted of CPSF6. DAPI staining (blue) was used to mark the nuclear compartment. ( C ) The pools of stable cell lines in ( B ) were challenged with WT or CA mutant HIV-1 NL4-3 GFP reporter viruses. After 72 hrs, GFP expression was checked by flow cytometry. Data represent one of at least three independent experiments. Error bars represent ± SEM (n = 3).

Article Snippet: Cells were then incubated for 1 hour either with anti-HA (Covance) or anti-CPSF6 antibodies (Novus Biologicals) diluted in PBS with 1% BSA.

Techniques: Stable Transfection, Transduction, Control, Western Blot, Construct, Staining, Mutagenesis, Expressing, Flow Cytometry

Journal: Retrovirology

Article Title: TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm

doi: 10.1186/1742-4690-10-20

Figure Lengend Snippet: CPSF6 stabilizes the HIV-1 CA core. ( A ) Env- HIV-1, pseudotyped with VSV G, and bearing either WT or A105T mutant CA, was incubated with TZM-bl cells stably transduced with CPSF6-358 (+) or empty vector (−), for 4, 10 and 16 hours. As a control, virions lacking VSV G (No VSVG) were also incubated with CPSF6-358 expressing cells. The target cells were lysed and the cytoplasmic fraction (Input) was pelleted through a 50% sucrose cushion to separate the particulate (Pellet) and soluble fractions (Supernatant). The fractions were then analyzed by western blot with an antibody anti-p24. ( B ) WT or A105T CA mutant viruses, and no VSV G virus, were incubated with control (Ctrl) or TNPO3 KD cells for 12 hours. Separation and analysis of the three fractions was described in ( A ). ( C ) WT or A105T CA mutant viruses and Env- virus were incubated with TZM-bl cells stably transduced with rhTRIM5α expressing vector or with an empty vector for 12 hours. Separation and analysis of the three fractions was described in ( A ). All experiments are representative of at least 2 repetitions.

Article Snippet: Cells were then incubated for 1 hour either with anti-HA (Covance) or anti-CPSF6 antibodies (Novus Biologicals) diluted in PBS with 1% BSA.

Techniques: Mutagenesis, Incubation, Stable Transfection, Transduction, Plasmid Preparation, Control, Expressing, Western Blot, Virus

Journal: eLife

Article Title: HIV-1 nuclear import in macrophages is regulated by CPSF6-capsid interactions at the nuclear pore complex

doi: 10.7554/eLife.41800

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-hCPSF6 , Atlas Antibodies; Cat# HPA039973 , RRID: AB_10795242 , IF(1:250).

Techniques: Enzyme-linked Immunosorbent Assay, Imaging, Reverse Transcription, Recombinant, Plasmid Preparation, Expressing, shRNA, Control, Software

Journal: Retrovirology

Article Title: TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm

doi: 10.1186/1742-4690-10-20

Figure Lengend Snippet: The effect of CPSF6-358 on the infectivity of HIV-1 CA mutants correlates with the effect of TNPO3 KD. ( A ) Schematic representation of the protein domains of WT CPSF6 and the truncated mutant CPSF6-358. RNA recognition motif (RRM), proline-rich domain (P-rich), arginine/serine rich domain (RS). ( B ) Expression levels of CPSF6 in TZM-bl cells transduced with empty or CPSF6-358 vectors. Cell lysates were probed in western blots with anti-CPSF6 antibody (upper panel) and anti-β-actin antibody (lower panel). The upper panel shows the endogenous CPSF6 and the truncated form. ( C ) TZM-bl cells transduced with an empty vector or with a vector encoding CPSF6-358 were challenged with a panel of 27 HIV-1-GFP reporter vectors bearing either WT CA or the indicated CA mutants. At 72 hrs the percent GFP + cells was determined by flow cytometry as an indication of infectivity. The ratio of HIV-1 infectivity in CPSF6-358 expressing cells and empty vector cells is shown. White bars show CA mutants inhibited to a similar extent as the WT virus by CPSF6-358, black bars shows CA mutants insensitive or slightly sensitive to CPSF6-358, and gray bars show CA mutants hypersensitive to the presence of CPSF6-358 in the cell. ( D ) Correlation between the infectivity ratios of the 27 CA mutants when infecting Ctrl KD vs TNPO3 KD and Empty vector vs CPSF6-358 (R 2 = 0.8528).

Article Snippet: We used rabbit anti-TNPO3 antibody (ab71388, abcam), rabbit anti-CPSF6 antibody (Novus biological), human anti-p24 (NIBSC) and mouse anti-actin antibody (Sigma).

Techniques: Infection, Mutagenesis, Expressing, Transduction, Western Blot, Plasmid Preparation, Flow Cytometry, Virus

Journal: Retrovirology

Article Title: TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm

doi: 10.1186/1742-4690-10-20

Figure Lengend Snippet: CPSF6-358 inhibits HIV-1 replication when localized to the cytoplasm. ( A ) CPSF6 protein in TZM-bl cells transduced with empty, CPSF6-358, CPSF6-358 NLS and CPSF6-358 NES vectors. Cell lysates were probed in western blots with anti-CPSF6 antibody (upper panel) and anti-β-actin antibody (lower panel). The upper panel shows the endogenous CPSF6 and the truncated form. ( B ) Indirect immunofluorescence showing localization of the different forms of CPSF6-358. The TZM-bl cells, transduced as in ( A ), were stained with an anti-HA antibody (green) for the detection of the CPSF6-358 proteins. DAPI was used to mark the nuclear compartment (blue). ( C ) TZM-bl stably expressing the different forms of CPSF6-358 were challenged with WT or CA mutant HIV-1 NL4-3 GFP reporter viruses. After 72 hrs, GFP reporter expression was assessed by flow cytometry. Data represent one of at least three independent experiments. Error bars represent ± SEM (n = 3).

Article Snippet: We used rabbit anti-TNPO3 antibody (ab71388, abcam), rabbit anti-CPSF6 antibody (Novus biological), human anti-p24 (NIBSC) and mouse anti-actin antibody (Sigma).

Techniques: Transduction, Western Blot, Immunofluorescence, Staining, Stable Transfection, Expressing, Mutagenesis, Flow Cytometry

Journal: Retrovirology

Article Title: TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm

doi: 10.1186/1742-4690-10-20

Figure Lengend Snippet: HIV-1 replication is inhibited when full-length CPSF6 is targeted to the cytoplasm. ( A ) Expression levels of CPSF6 in TZM-bl cells transduced with empty, CPSF6, CPSF6-NLS and CPSF6-NES vectors. Cell lysates were probed in western blots with anti-CPSF6 antibody (upper panel) and anti-β-actin antibody (lower panel). The upper panel shows the endogenous and exogenous full-length CPSF6 with an HA tag. ( B ) Localization of different forms of CPSF6 in TZM-bl cells stably expressing CPSF6, CPSF6 NLS or CPSF6 NES. The cells were stained with an anti-HA antibody (green) for the detection of the CPSF6 proteins. DAPI staining (blue) was used to mark the nuclear compartment. ( C ) TZM-bl stably expressing the different forms of CPSF6 were challenged with WT or CA mutant HIV-1 NL4-3 GFP reporter viruses. After 72 hours, GFP reporter expression was assessed by flow cytometry. Data represent one of at least three independent experiments. Error bars represent ± SEM (n = 3).

Article Snippet: We used rabbit anti-TNPO3 antibody (ab71388, abcam), rabbit anti-CPSF6 antibody (Novus biological), human anti-p24 (NIBSC) and mouse anti-actin antibody (Sigma).

Techniques: Expressing, Transduction, Western Blot, Stable Transfection, Staining, Mutagenesis, Flow Cytometry

Journal: Retrovirology

Article Title: TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm

doi: 10.1186/1742-4690-10-20

Figure Lengend Snippet: TNPO3 KD inhibits HIV-1 replication by shifting CPSF6 to the cytoplasm. ( A ) Expression level of CPSF6 and TNPO3 in TZM-bl cells stably transduced with control or CPSF6 KD vectors and transfected with scrambled or TNPO3 specific small interfering RNA (siRNA). Cell lysate was probed in western blots with anti-TNPO3 antibody (upper panel), anti-CPSF6 antibody (middle panel) and anti-β-actin antibody (lower panel). ( B ) Immuno-fluorescence localization of endogenous CPSF6 (green) in control (Ctrl) KD or TNPO3 KD TZM-bl cells. DAPI staining (blue) was used to mark the nuclear compartment. ( C ) Cell fractionation to identify the cellular localization of endogenous CPSF6. Expression of tubulin in the cytoplasm and histone 3 (H3) in the nucleus was assessed to verify the fractionation. ( D ) Stable ctrl KD and CPSF6 KD cells transfected with scrambled or TNPO3 siRNA were challenged with WT or CA mutant HIV-1 NL4-3 GFP reporter viruses. After 72 hours GFP expression was checked by flow cytometry. The fold inhibition of infectivity due to TNPO3 KD is shown. Data represent one of at least three independent experiments. Error bars represent ± SEM (n = 3).

Article Snippet: We used rabbit anti-TNPO3 antibody (ab71388, abcam), rabbit anti-CPSF6 antibody (Novus biological), human anti-p24 (NIBSC) and mouse anti-actin antibody (Sigma).

Techniques: Expressing, Stable Transfection, Transduction, Control, Transfection, Small Interfering RNA, Western Blot, Fluorescence, Staining, Cell Fractionation, Fractionation, Mutagenesis, Flow Cytometry, Inhibition, Infection

Journal: Retrovirology

Article Title: TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm

doi: 10.1186/1742-4690-10-20

Figure Lengend Snippet: TNPO3 depletion does not inhibit HIV-1 if CPSF6 is independently targeted to the nucleus. ( A ) CPSF6 and TNPO3 protein in TZM-bl cells stably transduced with CPSF6 KD vectors, control or TNPO3 KD vectors, and rescue of CPSF6 (ntCPSF6) with or without the SV40 T-Ag NLS. Cell lysate was probed in western blots with anti-TNPO3 antibody (upper panel), anti-CPSF6 antibody (middle panel) and anti-β-actin antibody (lower panel). ( B ) Localization of the non-targetable CPSF6 (ntCPSF6) constructs (green) in control (Ctrl) KD or TNPO3 KD TZM-bl cells stably depleted of CPSF6. DAPI staining (blue) was used to mark the nuclear compartment. ( C ) The pools of stable cell lines in ( B ) were challenged with WT or CA mutant HIV-1 NL4-3 GFP reporter viruses. After 72 hrs, GFP expression was checked by flow cytometry. Data represent one of at least three independent experiments. Error bars represent ± SEM (n = 3).

Article Snippet: We used rabbit anti-TNPO3 antibody (ab71388, abcam), rabbit anti-CPSF6 antibody (Novus biological), human anti-p24 (NIBSC) and mouse anti-actin antibody (Sigma).

Techniques: Stable Transfection, Transduction, Control, Western Blot, Construct, Staining, Mutagenesis, Expressing, Flow Cytometry

Journal: Retrovirology

Article Title: TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm

doi: 10.1186/1742-4690-10-20

Figure Lengend Snippet: CPSF6 stabilizes the HIV-1 CA core. ( A ) Env- HIV-1, pseudotyped with VSV G, and bearing either WT or A105T mutant CA, was incubated with TZM-bl cells stably transduced with CPSF6-358 (+) or empty vector (−), for 4, 10 and 16 hours. As a control, virions lacking VSV G (No VSVG) were also incubated with CPSF6-358 expressing cells. The target cells were lysed and the cytoplasmic fraction (Input) was pelleted through a 50% sucrose cushion to separate the particulate (Pellet) and soluble fractions (Supernatant). The fractions were then analyzed by western blot with an antibody anti-p24. ( B ) WT or A105T CA mutant viruses, and no VSV G virus, were incubated with control (Ctrl) or TNPO3 KD cells for 12 hours. Separation and analysis of the three fractions was described in ( A ). ( C ) WT or A105T CA mutant viruses and Env- virus were incubated with TZM-bl cells stably transduced with rhTRIM5α expressing vector or with an empty vector for 12 hours. Separation and analysis of the three fractions was described in ( A ). All experiments are representative of at least 2 repetitions.

Article Snippet: We used rabbit anti-TNPO3 antibody (ab71388, abcam), rabbit anti-CPSF6 antibody (Novus biological), human anti-p24 (NIBSC) and mouse anti-actin antibody (Sigma).

Techniques: Mutagenesis, Incubation, Stable Transfection, Transduction, Plasmid Preparation, Control, Expressing, Western Blot, Virus

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: Retrovirology

Article Title: The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6

doi: 10.1186/1742-4690-10-46

Figure Lengend Snippet: Inhibition of HIV-1 infection by TNPO3-depleted cells requires expression of CPSF6. HeLa TNPO3 K.D. and shRNA control human HeLa cells were transfected with a specific siRNA against CPSF6, a non-target siRNA, or left untreated. Forthy-eight hours after transfection, cells were lysed, and the expression levels of CPSF6 and TNPO3 were monitored by Western blot using antibodies against CPSF6and TNPO3, respectively ( A ). As a loading control, cell lysates were Western blotted against GAPDH. Fluorescence quantification revealed a 20 and 18 fold reduction in the expression of CPSF6 when using siRNA against CPSF6 in shRNA and TNPO3 K.D. cells, respectively. TNPO3 K.D. and shRNA control cells were challenged with increasing amounts of HIV-1 or HIV-1-N74D expressing GFP as a reporter for infection ( B ). Viruses were normalized by quantifying the particle-associated reverse transcriptase activity on viral supernatants, as described in Methods. Infectivity was determined forty-eight hours post-infection by measuring the percentage of GFP-positive cells using a flow cytometer. Similar results were obtained in three independent experiments and a representative experiment is shown.

Article Snippet: The CPSF6 protein was stained using a rabbit anti-CPSF6 antibody (Novus Biological cat# NB100-61596).

Techniques: Inhibition, Infection, Expressing, shRNA, Control, Transfection, Western Blot, Fluorescence, Reverse Transcription, Activity Assay, Flow Cytometry

Journal: Retrovirology

Article Title: The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6

doi: 10.1186/1742-4690-10-46

Figure Lengend Snippet: Binding of endogenously expressed CPSF6 to in vitro assembled HIV-1 CA-NC complexes. ( A ) Cellular extracts from human 293T cells were incubated with wild type or mutant (N74D) in vitro assembled HIV-1 CA-NC complexes for 1 h. Samples were subsequently applied onto 70% sucrose cushion and centrifuged, as described in Methods. A small fraction of each lysate was collected before centrifugation and analyzed by Western Blotting using anti-CPSF6 antibodies ( INPUT ). Pelleted fractions ( BOUND ) were analyzed for the presence of CPSF6 and HIV-1capsid (p24) by Western blotting using anti-CPSF6 and anti-p24 antibodies, respectively. ( B , C ) The ability of endogenously expressed CPSF6 from human 293T cells to bind in vitro assembled HIV-1 CA-NC complexes was measured in the presence of the small molecule PF74 (PF-3450074). ( D ) The ability of the restriction factor TRIMCyp to bind in vitro assembled HIV-1 CA-NC in the presence of PF74. ( E ) Similarly, we measured the ability of endogenously expressed CPSF6 from TNPO3 K.D. and shRNA control HeLa cells to bind in vitro assembled HIV-1 CA-NC complexes. ( F ) HeLa cells stably expressing the nuclearly localized CPSF6 and the cytoplasmicly localized NES-CPSF6 were lysed in capsid binding buffer (10 mM Tris pH 7.4, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT) or whole cell extract buffer (50 mM Tris pH 8, 2 mM MgCl 2 , 280 mM NaCl, 0.5% NP-40,10% Glycerol). Extracts were analyzed by Western blotting using antibodies against FLAG. As a control, we blotted extracts using antibodies against the nuclear marker β-laminin. Similar results were obtained in three independent experiments and a representative experiment is shown.

Article Snippet: The CPSF6 protein was stained using a rabbit anti-CPSF6 antibody (Novus Biological cat# NB100-61596).

Techniques: Binding Assay, In Vitro, Incubation, Mutagenesis, Centrifugation, Western Blot, shRNA, Control, Stable Transfection, Expressing, Marker

Journal: Retrovirology

Article Title: The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6

doi: 10.1186/1742-4690-10-46

Figure Lengend Snippet: Depletion of TNPO3 does not change the localization of CPSF6. ( A ) TNPO3 K.D. and shRNA control HeLa cells were fixed and stained using specific antibodies against CPSF6 (red), ASF/SF2 (red) and TNPO3 (green), as described in Methods. The nuclear compartment was labeled using DAPI. Image quantification is shown in Additional file . ( B ) TNPO3 K.D. and shRNA control HeLa cells were transiently transfected using constructs expressing FLAG-tagged CPSF6 or ASF/SF2. Twenty-four hours post-transfection cells were fixed and immunostained using anti-FLAG antibodies. The nuclear compartment was labeled using DAPI. Image quantification is shown in Additional file . Similar results were obtained in three independent experiments and a representative experiment is shown.

Article Snippet: The CPSF6 protein was stained using a rabbit anti-CPSF6 antibody (Novus Biological cat# NB100-61596).

Techniques: shRNA, Control, Staining, Labeling, Transfection, Construct, Expressing

Journal: Retrovirology

Article Title: The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6

doi: 10.1186/1742-4690-10-46

Figure Lengend Snippet: Expression of a cytosolic full-length CPSF6. ( A ) The wild type CPSF6 (NCBI Reference Sequence: NP_008938.2) protein with a C-terminal FLAG epitope is depicted on top. The numbers of the amino acid residues at the boundaries of the different domains are indicated (RRM: RNA recognition motive, Pro-rich: Proline-rich domain, RS: Arginine/Serine repeats). The HIV-1 capsid binding region is shown (residues 277-285). The nuclear export signal of the protein kinase inhibitor α (NES-PKIα). The amino acid sequence of NES-PKIα is NELALKLAGLDI. The NES-PKIα was fused to the N-terminus of CPSF6. ( B ) Cf2Th cells stably transduced with the different CPSF6 variants were analyzed for expression by Western blotting using anti-FLAG antibodies. As a loading control, cell lysates were Western blotted against β-actin. ( C ) Intracellular distribution of the different CPSF6 variants stably expressed in Cf2Th was studied by immunofluorescence microscopy, as described in Methods. The different CPSF6 variants were stained using anti-FLAG antibodies (red). The cellular nuclei were stained by using DAPI (blue). Image quantification is shown in Additional file . ( D ) The ability of the different CPSF6 variants to bind in vitro assembled HIV-1 CA-NC complexes was measured. 293T cells were transfected with plasmids expressing the indicated CPSF6 variants. Thirty-six hours after transfection, cells were lysed. The lysates were incubated at room temperature for 1hour with in vitro assembled HIV-1 CA-NC complexes. The mixtures were applied to a 70% sucrose cushion and centrifuged. INPUT represents the lysates analyzed by Western blotting before being applied to the 70% sucrose cushion. The input mixtures were Western blotted using anti-FLAG antibodies. The pellet from the 70% sucrose cushion ( BOUND ) was analyzed by Western blotting using anti-FLAG and anti-p24.

Article Snippet: The CPSF6 protein was stained using a rabbit anti-CPSF6 antibody (Novus Biological cat# NB100-61596).

Techniques: Expressing, Sequencing, FLAG-tag, Binding Assay, Stable Transfection, Transduction, Western Blot, Control, Immunofluorescence, Microscopy, Staining, In Vitro, Transfection, Incubation

Journal: Retrovirology

Article Title: The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6

doi: 10.1186/1742-4690-10-46

Figure Lengend Snippet: Cytosolic CPSF6 expressed in Cf2Th cells restricts HIV-1 replication before or at the nuclear import step. ( A ) Cf2Th cells stably expressing the indicated CPSF6 constructs were challenged with HIV-1 or HIV-1-N74D expressing GFP as a reporter. Forty-eight hours post-infection the percentage of GFP-positive cells was determined by flow cytometry. As control, Cf2Th cells stably transduced with the empty vector pLPCX were challenged with HIV-1 and HIV-1-N74D. ( B ) Cf2Th cells stably expressing CPSF6 and NES-CPSF6 were challenged with the indicted viruses. Infection was determined by measuring the percentage of GFP-positive cells by flow cytometry 48 hours post-infection (upper panel). In parallel, cells from similar infections were lysed at 7 or 24 hours post-infection and total DNA was extracted. The DNA samples collected at 7 hours post-infection were used to determine the levels of late reverse transcripts by real-time PCR (middle panel). Separately, DNA samples collected at 24 hours post-infection were used to quantify HIV-1 2-LTR circles by real-time PCR in DNA (lower panel). Mock refers to control cells that were not infected. ( C ) Cf2Th cells stably expressing the different CPSF6 variants were challenged with HIV-2, SIVmac, FIV, BIV, EIAV or N-MLV. As control, Cf2Th cells stably transduced with the empty vector pLPCX were challenged with HIV-2, SIVmac, FIV, BIV, EIAV or N-MLV. Infectivity by the different viruses was determined by measuring the percentage of GFP-positive 48 hours post-infection. ( D ) Cf2Th cells stably expressing the indicated CPSF6 variant or TRIM5α rh were challenged using similar amounts of HIV-1, and performed the fate of the capsid assay to separate pelletable from soluble cytosolic capsids 16 hours post-infection as described in Methods. Input, soluble and pellet fractions were analyzed by Western blotting using antibodies against HIV-1 CA p24. Similar results were obtained in three independent experiments and a representative experiments is shown.

Article Snippet: The CPSF6 protein was stained using a rabbit anti-CPSF6 antibody (Novus Biological cat# NB100-61596).

Techniques: Stable Transfection, Expressing, Construct, Infection, Flow Cytometry, Control, Transduction, Plasmid Preparation, Real-time Polymerase Chain Reaction, Variant Assay, Western Blot

Journal: Retrovirology

Article Title: The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6

doi: 10.1186/1742-4690-10-46

Figure Lengend Snippet: Cytosolic CPSF6 expressed in human HeLa cells restricts HIV-1 replication before or at the nuclear import step. ( A ) HeLa cells were stably transduced with the indicated CPSF6 variant. Expression of the different CPSF6 variants was analyzed by Western blotting using anti-FLAG antibodies. ( B ) The cellular distribution of the different CPSF6 variants was studied by immunofluorescence using anti-FLAG antibodies (red). The nuclear compartment was stained using DAPI (blue). Image quantification is shown in Additional file . ( C ) HeLa cells stably expressing the indicated CPSF6 variants were challenged with HIV-1 or HIV-1-N74D expressing GFP as a reporter for infection. Forty-eight hours post-infection the percentage of GFP-positive cells was determined by flow cytometry. As control, HeLa cells stably transduced with the empty vector pLPCX were challenged with increasing amounts of HIV-1 and HIV-1-N74D. ( D ) HeLa cells stably expressing CPSF6 and NES-CPSF6 were challenged with the indicted viruses. Infection was determined by measuring the percentage of GFP-positive cells by flow cytometry 48 hours post-infection (upper panel). In parallel, similarly infected cells were lysed at 7 or 24 hours post-infection and total DNA was extracted. The DNA samples collected at 7 hours post-infection were used to determine the levels of late reverse transcripts by real-time PCR (middle panel). DNA samples collected at 24 hours post-infection were used to quantify HIV-1 2-LTR circles by real-time PCR (lower panel). Mock refers to control cells that were not infected. ( E ) HeLa cells expressing the different CPSF6 variants were challenged with HIV-2, SIVmac, FIV, BIV or EIAV. As control, HeLa cells stably transduced with the empty vector pLPCX were challenged with HIV-2, SIVmac, FIV, BIV or EIAV. Infectivity by the different viruses was determined by measuring the percentage of GFP-positive 48 hours post-infection. Similar results were obtained in three independent experiments and a representative experiments is shown.

Article Snippet: The CPSF6 protein was stained using a rabbit anti-CPSF6 antibody (Novus Biological cat# NB100-61596).

Techniques: Stable Transfection, Transduction, Variant Assay, Expressing, Western Blot, Immunofluorescence, Staining, Infection, Flow Cytometry, Control, Plasmid Preparation, Real-time Polymerase Chain Reaction