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Inhibition of HIV-1 infection by TNPO3-depleted cells requires expression of <t>CPSF6.</t> 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.

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

Journal: Retrovirology

doi: 10.1186/1742-4690-10-46

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.

Figure Legend 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.

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

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

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

$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.$
Figure Legend 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.

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

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.

Figure Legend 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.

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

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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

CPSF6 clusters host HIV-1 viral complexes and are hubs of nuclear reverse transcription. ( A ) Confocal microscopy images of HIV-1-infected THP-1 cells (MOI = 10, 30 h p.i.) compared to uninfected cells. The graph shows the percentage of IN associated with CPSF6 per nucleus ± SD (40 cells, 132 IN foci). Three independent experiments were performed. ( B ) Confocal microscopy images of immuno-RNA FISH in HIV-1-infected THP-1 cells (MOI = 20, 2 days p.i.) treated with or without NEV (10 µM). The graph shows the percentage of vRNA foci associated with CPSF6 per cell per condition ± SD (number of cells: 56 (–NEV), 34 (+NEV); number of vRNA foci: 151 (–NEV), 61 (+NEV)). Unpaired t -test, ** P ≤ 0.01. Two independent experiments were performed. ( C ) ChIP of endogenous CPSF6 coupled to real-time qPCR of THP-1 cells infected with HIV-1 (MOI = 5, 2 days p.i.). Different reverse transcription products were amplified and normalized on the input ± SD. NI, non-infected; RT, reverse transcription. One-way ANOVA followed by Tukey's multiple comparison test, * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001. On the bottom, western blot of CPSF6 of ChIP products in HIV-1-infected cells compared to the input. Scale bar, 5 µm.

Journal: Journal of Molecular Cell Biology

Article Title: HIV-induced membraneless organelles orchestrate post-nuclear entry steps

doi: 10.1093/jmcb/mjac060

Figure Lengend Snippet: CPSF6 clusters host HIV-1 viral complexes and are hubs of nuclear reverse transcription. ( A ) Confocal microscopy images of HIV-1-infected THP-1 cells (MOI = 10, 30 h p.i.) compared to uninfected cells. The graph shows the percentage of IN associated with CPSF6 per nucleus ± SD (40 cells, 132 IN foci). Three independent experiments were performed. ( B ) Confocal microscopy images of immuno-RNA FISH in HIV-1-infected THP-1 cells (MOI = 20, 2 days p.i.) treated with or without NEV (10 µM). The graph shows the percentage of vRNA foci associated with CPSF6 per cell per condition ± SD (number of cells: 56 (–NEV), 34 (+NEV); number of vRNA foci: 151 (–NEV), 61 (+NEV)). Unpaired t -test, ** P ≤ 0.01. Two independent experiments were performed. ( C ) ChIP of endogenous CPSF6 coupled to real-time qPCR of THP-1 cells infected with HIV-1 (MOI = 5, 2 days p.i.). Different reverse transcription products were amplified and normalized on the input ± SD. NI, non-infected; RT, reverse transcription. One-way ANOVA followed by Tukey's multiple comparison test, * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001. On the bottom, western blot of CPSF6 of ChIP products in HIV-1-infected cells compared to the input. Scale bar, 5 µm.

Article Snippet: The LV plasmid pSICO-CPSF6-mNeonGreen encoding CPSF6 fluorescent fusion protein was a gift from Zandrea Ambrose (Addgene plasmid #167585; http://n2t.net/addgene:167585 ; RRID:Addgene_167585 ).

Techniques: Reverse Transcription, Confocal Microscopy, Infection, Amplification, Comparison, Western Blot

Journal: Journal of Molecular Cell Biology

Article Title: HIV-induced membraneless organelles orchestrate post-nuclear entry steps

doi: 10.1093/jmcb/mjac060

Figure Lengend Snippet: Phase-separation properties of HIV-1 MLOs. ( A ) Confocal microscopy images of THP-1 cells infected with HIV-1 (MOI = 5, 24 h p.i.) compared to uninfected cells. On the right, mean intensity of CPSF6 per nucleus ± SD (number of cells in the NI group = 49, number of cells in the HIV-1-infected group = 55). Unpaired t -test, **** P ≤ 0.0001. Two independent experiments were performed. ( B ) THP-1 cells were infected with HIV-1 (MOI = 5) and treated with NEV (10 µM). Confocal microscopy images of these cells at different time post-infection were compared (24 h p.i. vs. 72 h p.i). On the right, box plots of the volume (median: ∼0.19 vs. ∼0.37 µm 3 ) and percentages of CPSF6 clusters that show ≥95% or <95% sphericity value (number of cells: 125 (24 h p.i.), 95 (72 h p.i)). Unpaired t -test, **** P ≤ 0.0001. Results of two independent experiments were analyzed from 3D acquisitions. ( C ) Frames extracted from a 10-h time-lapse microscopy in 2D of THP-1 cells expressing CPSF6 mNeonGreen infected with HIV-1 (MOI = 10). The graph shows the circularity (normalized ratio between the contour and interior expressed as a percentage) along the MLO fusion–fission event. Representative of six independent experiments. ( D ) Frames extracted from a FRAP time-lapse microscopy in THP-1 cells expressing CPSF6 mNeonGreen infected with HIV-1 (MOI = 10, 3 days p.i.). Representative of three independent experiments. The graph shows the recovery of the signal curve. Pre-bleach signal is set to 1 and bleach signal is set to 0. ( E ) Confocal microscopy images of THP-1 cells infected with HIV-1 (MOI = 10, 3 days p.i.). On the bottom, graphs show the intensity profile of Hoechst, CPSF6, and SC35 signals along the segment crossing the MLO (yellow in top right image) and the percentage of CPSF6 colocalizing with SC35 per nucleus ± SD. Results of two independent experiments were analyzed from 3D acquisitions. Scale bar, 5 µm.

Techniques: Confocal Microscopy, Infection, Time-lapse Microscopy, Expressing

Complete reverse transcribed products are released from HIV-1 MLOs. ( A ) Confocal images of THP-1 cells expressing OR-GFP infected with HIV-1 ANCH3 (MOI = 10, 30 h p.i.) compared to uninfected cells. The graph shows the percentage of vDNAs associated with IN per nucleus ± SD (13 cells, 100 vDNAs). Results of two independent experiments were analyzed from 3D acquisitions. ( B ) Cropped frames from a time-lapse microscopy of vDNA (green) and IN (red) dynamics in THP-1 cells (HIV-1 ANCH3 GIR, 80 h p.i.). Pearson's correlation of the two signals per 2D frame along the time. Representative of three independent experiments. Scale bar, 1 µm. ( C ) Confocal images of THP-1 cells expressing OR-GFP infected with HIV-1 ANCH3 (MOI = 10, 3 days p.i.). The graphs display the percentage of vDNA associated with CPSF6 clusters per nucleus ± SD (17 cells, 146 vDNAs) and the distance of each vDNA from the closest NE point or SC35-marked NS border ± SD (7 cells, 83 vDNAs). Results of two biological replicates were analyzed from 3D aquisitions. Unpaired t -test, **** P ≤ 0.0001. Scale bar, 5 µm.

Journal: Journal of Molecular Cell Biology

Article Title: HIV-induced membraneless organelles orchestrate post-nuclear entry steps

doi: 10.1093/jmcb/mjac060

Figure Lengend Snippet: Complete reverse transcribed products are released from HIV-1 MLOs. ( A ) Confocal images of THP-1 cells expressing OR-GFP infected with HIV-1 ANCH3 (MOI = 10, 30 h p.i.) compared to uninfected cells. The graph shows the percentage of vDNAs associated with IN per nucleus ± SD (13 cells, 100 vDNAs). Results of two independent experiments were analyzed from 3D acquisitions. ( B ) Cropped frames from a time-lapse microscopy of vDNA (green) and IN (red) dynamics in THP-1 cells (HIV-1 ANCH3 GIR, 80 h p.i.). Pearson's correlation of the two signals per 2D frame along the time. Representative of three independent experiments. Scale bar, 1 µm. ( C ) Confocal images of THP-1 cells expressing OR-GFP infected with HIV-1 ANCH3 (MOI = 10, 3 days p.i.). The graphs display the percentage of vDNA associated with CPSF6 clusters per nucleus ± SD (17 cells, 146 vDNAs) and the distance of each vDNA from the closest NE point or SC35-marked NS border ± SD (7 cells, 83 vDNAs). Results of two biological replicates were analyzed from 3D aquisitions. Unpaired t -test, **** P ≤ 0.0001. Scale bar, 5 µm.

Techniques: Reverse Transcription, Expressing, Infection, Time-lapse Microscopy

Active proviruses are excluded from HIV-1 MLOs. ( A ) Confocal images of THP-1 cells and primary MDMs expressing OR-GFP infected with HIV-1 ANCH3 (THP-1: MOI = 20, 3 days p.i.; MDMs: MOI = 40, 4 days p.i.). Analysis was performed from 3D acquisitions. ( B ) Confocal images of immuno-RNA FISH in primary MDMs infected with HIV-1 GFP (MOI = 20) and treated with or without NEV (10 µM) 2–3 days p.i. The scatter plots show the GFP intensity per cell ± SD and the percentage of vRNA foci outside CPSF6 clusters per nucleus ± SD (number of cells: 22 (–NEV), 20 (+NEV); number of vRNA foci: 268 (–NEV), 66 (+NEV)); Unpaired t -test, **** P ≤ 0.0001. ( C ) Correlation of GFP intensity and the percentage of vRNA foci excluded from CPSF6 clusters in HIV-1-infected cells (22 cells). Pearson's r coefficient, ** P ≤ 0.01. ( D ) vRNA intensity per CPSF6 cluster per condition ± SD; unpaired t -test, ** P ≤ 0.01. All the analyses were performed in 3D from two independent experiments. Scale bar, 5 µm.

Journal: Journal of Molecular Cell Biology

Article Title: HIV-induced membraneless organelles orchestrate post-nuclear entry steps

doi: 10.1093/jmcb/mjac060

Figure Lengend Snippet: Active proviruses are excluded from HIV-1 MLOs. ( A ) Confocal images of THP-1 cells and primary MDMs expressing OR-GFP infected with HIV-1 ANCH3 (THP-1: MOI = 20, 3 days p.i.; MDMs: MOI = 40, 4 days p.i.). Analysis was performed from 3D acquisitions. ( B ) Confocal images of immuno-RNA FISH in primary MDMs infected with HIV-1 GFP (MOI = 20) and treated with or without NEV (10 µM) 2–3 days p.i. The scatter plots show the GFP intensity per cell ± SD and the percentage of vRNA foci outside CPSF6 clusters per nucleus ± SD (number of cells: 22 (–NEV), 20 (+NEV); number of vRNA foci: 268 (–NEV), 66 (+NEV)); Unpaired t -test, **** P ≤ 0.0001. ( C ) Correlation of GFP intensity and the percentage of vRNA foci excluded from CPSF6 clusters in HIV-1-infected cells (22 cells). Pearson's r coefficient, ** P ≤ 0.01. ( D ) vRNA intensity per CPSF6 cluster per condition ± SD; unpaired t -test, ** P ≤ 0.01. All the analyses were performed in 3D from two independent experiments. Scale bar, 5 µm.

Techniques: Expressing, Infection

MCP-MS2 RNA labelling system detects transcription foci only. ( A ) Confocal images of immuno-RNA FISH in THP-1 cells infected with HIV-1 and treated with or without 10 µM NEV (MOI = 20, 3 days p.i.). The graph shows the number of vRNA per nucleus, and the table shows the amount of vRNA inside or outside CPSF6 clusters (number of cells: 70 (–NEV), 68 (+NEV)). Two independent experiments were performed. ( B ) Confocal images of THP-1 cells expressing MCP-GFP infected with HIV-1 ANCH3 MS2 and treated with or without 10 µM NEV (MOI = 2.5, 3 days p.i.). The graph shows the number of vRNA per nucleus, and the table shows the amount of vRNA inside or outside CPSF6 clusters (number of cells: 40 (–NEV), 44 (+NEV)). Three independent experiments were performed. ( C ) Confocal images of THP-1 cells expressing MCP-GFP infected with HIV-1 ANCH3 MS2 (MOI = 2.5, 3 days p.i.). The graph shows the distance of each RNA focus from the closest NE point or SC35-marked NS border ± SD (31 vRNAs, 8 cells). Results of two independent experiments were analyzed from 3D acquisitions. Unpaired t -test, **** P ≤ 0.0001. Scale bar, 5 µm. Inset, 1 µm.

Journal: Journal of Molecular Cell Biology

Article Title: HIV-induced membraneless organelles orchestrate post-nuclear entry steps

doi: 10.1093/jmcb/mjac060

Figure Lengend Snippet: MCP-MS2 RNA labelling system detects transcription foci only. ( A ) Confocal images of immuno-RNA FISH in THP-1 cells infected with HIV-1 and treated with or without 10 µM NEV (MOI = 20, 3 days p.i.). The graph shows the number of vRNA per nucleus, and the table shows the amount of vRNA inside or outside CPSF6 clusters (number of cells: 70 (–NEV), 68 (+NEV)). Two independent experiments were performed. ( B ) Confocal images of THP-1 cells expressing MCP-GFP infected with HIV-1 ANCH3 MS2 and treated with or without 10 µM NEV (MOI = 2.5, 3 days p.i.). The graph shows the number of vRNA per nucleus, and the table shows the amount of vRNA inside or outside CPSF6 clusters (number of cells: 40 (–NEV), 44 (+NEV)). Three independent experiments were performed. ( C ) Confocal images of THP-1 cells expressing MCP-GFP infected with HIV-1 ANCH3 MS2 (MOI = 2.5, 3 days p.i.). The graph shows the distance of each RNA focus from the closest NE point or SC35-marked NS border ± SD (31 vRNAs, 8 cells). Results of two independent experiments were analyzed from 3D acquisitions. Unpaired t -test, **** P ≤ 0.0001. Scale bar, 5 µm. Inset, 1 µm.

Techniques: Infection, Expressing

HIV-1 active proviruses locate in LEDGF-marked chromatin neighboring HIV-1 MLOs. ( A ) Confocal images of THP-1 cells expressing OR-GFP infected with HIV-1 ANCH3 (MOI = 10, 3 days p.i.) and expressing MCP-GFP infected with HIV-1 ANCH3 MS2 (MOI = 2.5, 3 days p.i.). On the bottom, the plots show the percentage of CPSF6 clusters associated with LEDGF per nucleus ± SD (38 cells, 161 CPSF6) and the percentage of vDNA or vRNA associated with LEDGF cluster per nucleus ± SD. Two independent experiments were performed. ( B ) Scatter plot shows the distance of vDNA or vRNA from the border of the closest HIV-1 MLO (SC35 + CPSF6) ± SD (75 vDNAs, 7 cells; 34 vRNAs, 8 cells). Unpaired t -test, ** P ≤ 0.01. Results of two biological replicates were analyzed from 3D acquisitions. Scale bar, 5 µm. Inset, 1 µm.

Journal: Journal of Molecular Cell Biology

Article Title: HIV-induced membraneless organelles orchestrate post-nuclear entry steps

doi: 10.1093/jmcb/mjac060

Figure Lengend Snippet: HIV-1 active proviruses locate in LEDGF-marked chromatin neighboring HIV-1 MLOs. ( A ) Confocal images of THP-1 cells expressing OR-GFP infected with HIV-1 ANCH3 (MOI = 10, 3 days p.i.) and expressing MCP-GFP infected with HIV-1 ANCH3 MS2 (MOI = 2.5, 3 days p.i.). On the bottom, the plots show the percentage of CPSF6 clusters associated with LEDGF per nucleus ± SD (38 cells, 161 CPSF6) and the percentage of vDNA or vRNA associated with LEDGF cluster per nucleus ± SD. Two independent experiments were performed. ( B ) Scatter plot shows the distance of vDNA or vRNA from the border of the closest HIV-1 MLO (SC35 + CPSF6) ± SD (75 vDNAs, 7 cells; 34 vRNAs, 8 cells). Unpaired t -test, ** P ≤ 0.01. Results of two biological replicates were analyzed from 3D acquisitions. Scale bar, 5 µm. Inset, 1 µm.

Techniques: Expressing, Infection

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