Role of HIV-induced CPSF6 puncta in the nuclear reverse transcription upon removal of NVP.

A) THP-1 cells, infected with VSV-G Δenv HIV-1 (NL4.3) ΔR LUC (MOI 10) in presence or not of Nevirapine (10 µM) for 5 days, or in presence of Nevirapine (10 µM) for 2 days and then the remaining 3 days without drug or in presence of Nevirapine (10 µM) for 2 days then in presence of PF74 (25 µM). Confocal microscopy images, to verify the presence of CPSF6 puncta, the cells are stained with anti-CPSF6 antibody (green). Nuclei are stained with Hoechst (blue). Scale bar 10 µm. B) Luciferase Assay, to verify luciferase expression in the aforementioned samples. Luciferase values were normalized by total proteins revealed with the Bradford kit. One-way ANOVA statistical test with multiple comparison was performed (****=p<0.0001; *=p<0.05; ns=p>0.05). Data are representative of two independent experiments. C) Western blots demonstrate CPSF6 depletion using a specific antibody against CPSF6 in THP-1 cells subjected to CRISPR Cas9 methods: CRISPR Cas9 bulk (left), and CRISPR Cas9 clones selected by limiting dilution (right). Each condition is normalized for actin labeling. The ratio between the intensity signal of CPSF6 and actin was analyzed via ImageJ and is plotted below each western blot. D) Confocal microscopy images of THP-1 ctrl CRISPR clone 2 cells (Ctrl 2) and THP-1 duplex1-2-3 CRISPR clone 4 cells (CPSF6 KO 4) infected with VSV-G / HIV-1ΔEnvINHA LAI (BRU) -vpx (MOI 10) in the presence of Nevirapine (10 µM). The cells are stained 30 h p.i. with anti-CPSF6 antibody and anti-HA antibodies to detect HA tagged integrase (IN).

Role of CPSF6 Domains in HIV-Induced CPSF6 Puncta.

A) Schema of CPSF6 isoform 588aa deletion mutants. B) Confocal microscopy images of THP-1 CPSF6 KO cells, transduced with different mutants of CPSF6, infected with VSV-G / HIV-1ΔEnvINHA LAI (BRU)-vpx (MOI 10) in presence of Nevirapine (10 µM). The cells are stained with CPSF6 and HA antibody 30 h p.i.. Scale bar 5µm. C) Analysis of the number of CPSF6 puncta in THP-1 CPSF6 KO cells transduced with different mutants of CPSF6, not infected or infected in presence of Nevirapine (10 µM) (the number of analyzed cells is shown under the x-axis). Statistical test: ordinary one-way ANOVA (****=p<0.0001; ***=p<0.001; *=p<0.05; ns=p>0.05). D) The plot compares the number of CPSF6 puncta per cell in THP-1 CPSF6 KO cells transduced with different mutants of CPSF6, infected with HIV-1 in the presence of Nevirapine (10 µM). Statistical test: ordinary one-way ANOVA (****=p<0.0001; ns=p>0.05). E) Confocal microscopy images of THP-1 CPSF6 KO clone 4, non-transduced and non-infected or transduced with WT CPSF6 and CPSF6 3xNLSΔMCD and infected with VSV-G / HIV-1ΔEnvINHA LAI (BRU) -vpx (MOI 10) in presence of Nevirapine (10 µM). Immuno-RNA FISH: the cells are stained with CPSF6 (green) antibody and with 24 probes against HIV-1 Pol sequence (gray)(RNA-FISH) 25 h p.i. Nuclei are stained with Hoechst (blue). Scale bar 10 µm. Violin plot presenting the percentage of CPSF6 puncta colocalizing with the viral RNA in THP-1 CPSF6 KO clone 4 cells transduced with LVs expressing CPSF6 WT or CPSF6 3xNLSΔMCD (respectively n=73 and n=103) and infected with VSV-G / HIV-1ΔEnvINHA LAI (BRU) -vpx (MOI 10) in presence of Nevirapine (10 µM). A total of 198 CPSF6 WT puncta and 264 CPSF6 3xNLSΔMCD puncta were counted. Statistical test: unpaired t-test, ns=p>0.05. F) Confocal microscopy images of THP-1 KO CPSF6 cells transduced with WT CPSF6 and CPSF6 ΔMCD without NLS, with 3xNLS or with PY NLS, respectively. Cells were differentiated for 3 days, transduced with CPSF6 lentiviral vectors (MOI 1) for 3 days and infected for 24 h with VSV-G / HIV-1ΔEnvINHA LAI (BRU) -vpx (MOI 10) in presence of Nevirapine (10 uM) (left panels). The panels on the right show transduced and uninfected cells. CPSF6 and the IN tagged with the HA are labeled with anti-CPSF6 (green) and anti-HA (white) antibodies, respectively. Nuclei are stained with Hoechst (blue). The arrows show CPSF6 puncta in colocalization with IN-HA. Scale bar 10µm.

Evaluation of CPSF6 Deletion Mutants’ Binding Capacity to the Viral Core.

A) Ability of wild type and mutant CPSF6 proteins to bind to the HIV-1 core. Cellular extracts derived from human 293T cells expressing similar levels of the indicated CPSF6 proteins (INPUT) were incubated with HIV-1 capsid stabilized tubes for 1 hour at room temperatures in the presence and absence of 10 µM PF74, as described in materials and methods. As a carrier control, we utilized DMSO. Subsequently, HIV-1 capsid stabilized tubes were washed, and the bound proteins eluted 1X Laemmli buffer 1X. The BOUND fractions were analysed by Western blotting using antibodies against neon-GFP and the HIV-1 capsid. B) Experiments were repeated at least three times and the average BOUND fraction relative to the INPUT fraction normalized to wild type binding are shown for the different CPSF6 mutants. *** indicates a p-value < 0.001; **** indicates a p-value < 0.0001; and ns indicates no significant difference as determined by unpaired t-tests.

Comparison of second structures of ADD2 and LCR.

A) Physicochemical characteristics of the LCR-FG and ADD2-FG sequences. Intrinsic disorder predispositions evaluated by PONDR® VLXT. Position of the FR segment within the LCR-FG and ADD2-FG sequences is shown as gray shaded area. B) Linear distribution of the net charge per residue (NCPR) within the LCR-FG sequence evaluated by CIDER. C) Linear distribution of the net charge per residue (NCPR) within the ADD2-FG sequence evaluated by CIDER. D) Secondary structure propensity of the LCR-FG sequence evaluated by PSIPRED. E) Secondary structure propensity of the ADD2-FG sequence evaluated by PSIPRED. F) Analysis of the peculiarities of the amino acid compositions of the intrinsically disordered C-terminal domain (residues 261-358) of human CPSF6 and its different mutants. Relative abundance of prion-like LCR defining uncharged residues in analyzed protein segments. G) Relative abundance of proline residues in analyzed protein segments. H) Relative abundance of charged residues in analyzed protein segments. The values were calculated by dividing numbers of prion-like LCR defining uncharged (Ala, Gly, Val, Phe, Tyr, Leu, Ile, Ser, Thr, Pro, Asn, Gln), Pro, and charged (Asp, Glu, Lys, Arg) residues by the total number of amino acids in the respective protein fragments. Corresponding values for all protein sequences deposited in the UniProtKB/Swiss-Prot database, PDB Select25, and DisProt are shown for comparison.

Role of FG motif in viral replication.

A) WB showing CPSF6 protein from several single clones derived from CPSF6 KO clone obtained upon complementation with CPSF6 ΔFG and normalized with beta-actin. B) Quantification of the expression of CPSF6 ΔFG protein in different single clones compared to CPSF6 WT (value 1). C) Infectivity essay using a single round infectious virus carrying the cDNA of Luciferase as reporter gene. Values are expressed as% of RLU compared to WT cells. D) Infectivity essay of a replication competent virus: vRNA from new viruses produced after infection of WT, CPSF6 KO and CPSF6ΔFG cells was analyzed and showed in the histograms as % of vRNA copies compared to vRNA in WT THP-1 cells considered 100%.

Depletion of MCD or LCR does not affect the formation of HIV-induced CPSF6 puncta.

A) Epifluorescence microscopy images of both infected and non-infected differentiated THP-1 cells showing the presence of CPSF6 puncta only in the infected condition. CPSF6 and SC35 are labelled with anti-CPSF6 (green) and anti-SC35 (red) antibodies, respectively. Nuclei are stained with Hoechst (blue). Scale bar 10µm. B) Confocal microscopy images of THP-1 KO CPSF6 cells, differentiated for 3 days, transduced with CPSF6 lentiviral vector (MOI 1) (specifically WT CPSF6, CPSF6 ΔLCRs, CPSF6 ΔMCD with 3xNLS, without NLS, or with PY NLS) for 3 days and infected for 24 h with VSV-G / HIV-1ΔEnvINHA LAI (BRU) -vpx MOI 10) in presence of Nevirapine (10 uM). CPSF6 and nuclear speckles were labelled with anti-CPSF6 (green) and anti-SC35 (red) antibodies, respectively. Nuclei are stained with Hoechst (blue). Scale bar 10µm. The percentage of CPSF6 puncta associated with SC35 per field of view is shown in the graph. N cells were counted in each condition and a one-way ANOVA statistical test with multiple comparison was performed; ns= p value > 0.05.

Dynamics of the HIV-induced CPSF6 puncta formation and their fusion with NSs.

A) Time course of infection of THP-1, 6h.p.i., 9h.p.i., 12h.p.i., 30h.p.i. or non infected. Cells were stained with antibodies against CPSF6 (green) and SC35 (red). B) The graph shows the percentage of CPSF6 puncta associated with NS or adjacent to NS or isolated from NS at different time post-infection. C) The graph shows the progression of CPSF6 puncta associated to NS during the time post-infection. N indicates the number of cells analyzed. One-way ANOVA statistical test with multiple comparison was performed; ns= p value > 0.05; **** indicates a p-value < 0.0001.

Role of SRRM2 and SON in the Formation of HIV-Induced CPSF6 puncta.

A) Depletion of SON and SRRM2 in THP-1 cells using AUMsilenceTM ASO technology. The degree of depletion is quantified by WB and the mean intensity through immunofluorescence using antibodies against SON and SRRM2, respectively. Scale bar 5µm. B) The percentage of CPSF6 puncta formation is quantified by IF in THP-1 cells knocked down for SON, SRRM2, and control (Ctrl) infected with HIV-1 (MOI 25) for 48 h. CPSF6 is stained with an antibody against CPSF6 (green), and nuclei are stained with Hoechst (blue). The graph on the right reports the percentage of CPSF6 puncta calculated from more than 100 cells. Scale bar 10µm. Experiments were performed at least twice. C) (Top panels) confocal microscopy images of ΔIDR HaloTag SRRM2 HEK293 and HaloTag SRRM2 HEK293 cells stained with the halo tag ligand (red), and nuclei (blue). Scale bar 10µm. (Bottom panels) confocal microscopy images of HaloTag SRRM2 HEK293 and ΔIDR HaloTag SRRM2 HEK293 cells, both labeled with anti-SRRM2 (red) and anti-SON (gray) antibodies. Nuclei are stained with Hoechst (blue). Scale bar 10µm. Statistical studies are summarized in the violin plot which displays the distribution of the number of SON puncta per cell in the two conditions. N cells were counted and Kolmogorov Smirnov test was performed, ns= p>0.05. D) Confocal microscopy images of HaloTag SRRM2 HEK293 and ΔIDR HaloTag SRRM2 HEK293 cells, either non-infected or infected for 24 h with VSV-G / HIV-1ΔEnvINHA LAI (BRU) (MOI 10) in the presence of Nevirapine (10 µM). CPSF6 and SC35 are labeled with anti-CPSF6 (red) and anti-SC35 (gray) antibodies, respectively. Nuclei are stained with Hoechst (blue). The plot shows the mean ± SD of the percentage of cells with CPSF6 puncta calculated in n fields of view (n=24, 29, 32); N is the number of cells analyzed for each of the three different cell lines; an unpaired t-test was performed, ****=p<0.0001, ns=p>0.05. Scale bar 10µm. Experiments were performed at list twice.