1. Cell Biology
  2. Microbiology and Infectious Disease
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HIV-1 nuclear import in macrophages is regulated by CPSF6-capsid interactions at the nuclear pore complex

  1. David Alejandro Bejarano
  2. Ke Peng  Is a corresponding author
  3. Vibor Laketa
  4. Kathleen Börner
  5. K Laurence Jost
  6. Bojana Lucic
  7. Bärbel Glass
  8. Marina Lusic
  9. Barbara Müller
  10. Hans-Georg Kräusslich  Is a corresponding author
  1. University of Heidelberg, Germany
  2. German Center for Infection Research, Germany
Research Article
Cite this article as: eLife 2019;8:e41800 doi: 10.7554/eLife.41800
9 figures, 1 video, 1 table and 2 additional files

Figures

Figure 1 with 3 supplements
CPSF6 is enriched on nuclear HIV-1 complexes in infected macrophages.

MDM were mock-infected (A) or infected with HIV-1 NL4-34059 (IN.eGFP) at an MOI of 14.5 (B–G) in the presence of 10 µM EdU (A–G), and 5 µM Efavirenz (D) or 5 µM Ral (Raltegravir) (E) for 48 hr, click-labeled and immunostained with anti-CPSF6 antibody (magenta). Z-stacks were acquired by spinning disk confocal microscopy (SDCM). RTC/PIC were identified via co-localization of IN.eGFP (green) and EdU (red) signals. Images show representative z-sections of the nucleus of infected cells. Solid arrowheads indicate nuclear complexes. Open arrowheads indicate lack of co-localization for the respective stain. Scale bars represent 5 µm. (A, B) Distribution of CPSF6 in the nucleus of a mock-infected or infected cell. Immunostaining against lamin A/C was performed to visualize the nuclear envelope. (C–E) As in A,B, but immunostaining against CA (cyan) was performed instead of lamin staining. (F) Distribution and mean intensities of CPSF6 signals co-localizing with individual RTC/PIC in the cytoplasm, close to the nuclear envelope and inside the nucleus; each dot represents one RTC/PIC. The graph summarizes data from two donors from two independent experiments. 62 cells were analyzed in total. (G) Distribution and mean intensities of CA signals co-localizing with individual EdU-positive RTC/PIC. Numbers of total RTC/PIC detected are given in parentheses. The graph summarizes data from four donors from three independent experiments. 92 cells were analyzed in total. Statistical significance was assessed with two-tailed non-paired Mann-Whitney test; *p=0.013.

https://doi.org/10.7554/eLife.41800.003
Figure 1—source data 1

Mean CPSF6 and CA signal intensities of individual HIV-1 EdU positive subviral complexes at different subcellular localizations.

Data corresponds to Figure 1F and G.

https://doi.org/10.7554/eLife.41800.007
Figure 1—figure supplement 1
Effects of cytoplasmic CPSF6 on HIV-1 reverse transcription.

(A) Association of CPSF6 with cytoplasmic RTC/PIC in HeLa P4-derived TNPO3 K/D cells. Cells were infected with HIV-1NL4-3 (IN.eGFP) at an MOI of 25 in the presence of 10 µM EdU for 4.5 hr, fixed, click-labeled and immunostained with anti-CPSF6 (cyan) and anti-laminA/C (white) antibodies. A z-section of a representative infected cell is shown. The arrowhead indicates a cytoplasmic RTC/PIC enlarged (i) on the right side. An additional enlargement from another region of the same cell (ii) is also shown. IN.eGFP and EdU signals in the merged panel are displayed in green and red, respectively. Scale bar represents 5 µm. (B) Comparison of EdU signal intensities of single CPSF6 negative (neg) or positive (pos) cytoplasmic RTC/PIC in infected TNPO3-K/D cells. Statistical significance was assessed by a two-tailed unpaired Mann Whitney test; **p<0.01. a.u.: arbitrary units. (C) MDM were transduced with AAV expressing shRNAs targeting CPSF6 (K/D) or non-targeted shRNA (NS) as described in Materials and Methods. Subsequently, cells were infected with HIV-1 NL4-3 4059-WT (IN.eGFP). At 48 h p.i., cells were fixed, click-labeled and immunostained. The graph shows the number of RTC/PIC detected per cell under the indicated conditions.

https://doi.org/10.7554/eLife.41800.004
Figure 1—figure supplement 2
Replication kinetics of HIV-1 in primary macrophages and induction of CPSF6 enrichment by lentiviral transduction.

(A, B) MDM from two donors in two independent experiments were infected with 15 ng CA (MOI 3.5) of HIV-1 NL4-3 4059-WT. To prevent secondary infection, 5 µM Maraviroc was added to the medium at 24 h p.i.. 5 µM Efavirenz (solid lines) or DMSO (dashed lines) was added at the time of infection (0 h p.i.) or at the indicated time points. The proportion of infected cells was determined at day 6 p.i. by immunostaining using antiserum against CA. Infections were performed in triplicates per condition in each experiment. Error bars represent SEM of the technical replicates. (C) MDM, from two different donors than the ones from (A) and (B), were transduced with lentiviral vectors expressing a non-targeted shRNA (NS control) or shRNAs against CPSF6, as described in Materials and Methods. At 7d post transduction cells were fixed and immunostained with anti-CPSF6 (magenta) antibody. The nuclear envelope was visualized by immunostaining with anti-laminA/C antibody (white). The solid arrow indicates CPSF6 enrichment in the nucleus observed after lentiviral transduction.

https://doi.org/10.7554/eLife.41800.005
Figure 1—figure supplement 3
Effect of CPSF6 knockdown on CA signals.

MDM were transduced with AAVs as described in Materials and Methods. Subsequently, cells were infected with 100 ng CA (MOI 14.5) of HIV-1 NL4-3 WT (IN.eGFP) (A) or A77V (IN.eGFP) (B) in the presence of 10 µM EdU. 48 hr later, cells were fixed and stained with anti-CA and anti-laminA/C antibodies. The plots show the mean intensities of CA signals co-localizing with individual RTC/PIC at the indicated subcellular localization in cells displaying normal (NS control, black dots) or low (K/D, gray dots) CPSF6 levels. Red lines represent the mean of each data set. Data displayed were combined from two independent experiments using from MDM from different donors. Statistical significance was assessed by a two-tailed unpaired Mann Whitney test; n.s.: not significant. a.u.: arbitrary units.

https://doi.org/10.7554/eLife.41800.006
Figure 1—figure supplement 3—source data 1

Data includes mean CA signals of individual WT and A77V HIV-1 subviral complexes at different subcellular localization after CPSF6 knock-down.

https://doi.org/10.7554/eLife.41800.008
Figure 2 with 2 supplements
Characterization of the composition of nuclear HIV-1 replication complexes.

(A) MDM from three donors were infected with HIV-1 NL4-34059 at an MOI of 8 for 72 hr. Viral DNA (green) was detected by FISH as described in Materials and Methods. CPSF6 (magenta) and CA (cyan) were detected by immunostaining and images were recorded by SDCM. The maximum projection of three focal planes acquired with an axial spacing of 0.2 µm is shown. Arrowheads indicate nuclear complexes. (B) MDM from three donors were infected with 100ng p24 (MOI 14.5) HIV-1 NL4-34059 (IN.eGFP) for 48 hr, fixed and click-labeled as in Figure 1. CPSF5 (cyan) and CPSF6 (magenta) (top panel) or CPSF7 (white) and CPSF5 (cyan) (bottom panel) were detected by immunostaining. Images show a section through the nuclei of infected cells. Arrowheads indicate nuclear complexes. 112 cells were analyzed in total. (C,D) MDM from two donors in two independent experiments were infected with 100ng p24 (MOI 14.5) HIV-1 NL4-3ΔTat4059 (IN.eGFP) (C) or HIV-1 NL4-34059 (IN.eGFP) (D). At 96 h p.i., a final concentration of 5 µM Flavopiridol (D) was added to the medium and infection was continued for 12 hr. Cells were fixed, click-labeled and CPSF6 (magenta) and laminA/C (white) were detected by immunostaining. Images show a section through the nucleus of representative infected cells. Arrowheads indicate nuclear complexes. 22 cells were analyzed in (C) and 37 cells in (D). (E,F) MDM from three donors, in two independent experiments, were infected at an MOI of 8 with HIV-1 NL4-34059 (E) or HIV-1 NL4-3ΔTat4059 (F). At 108 h p.i. cells were fixed. CPSF6 (magenta) was detected by immunostaining and nucleus (blue) with Hoechst. Viral RNA (green) was detected by RNA FISH as explained in Materials and Methods. Arrowhead indicates nuclear complexes. 42 cells were analyzed in total in (E) and 49 cells in (F). (G) MDM from the same donors as in (E,F) were infected with HIV-1 NL4-34059 using the same conditions. At 96 h p.i. 5 µM Flavopiridol was added to the medium for 12 hr. Viral RNA (green) was detected by RNA FISH. CPSF6 (magenta) was detected by immunostaining and nucleus (blue) with Hoechst. Arrowheads indicate nuclear CPSF6 enrichments. 36 cells were analyzed in total. Scale bars in A-G: 5 µm.

https://doi.org/10.7554/eLife.41800.009
Figure 2—figure supplement 1
Inhibition of HIV-1 transcription and LEDGF localization.

MDM from two donors were infected with HIV-1 NL4-34059 or HIV-1 NL4-3ΔTat4059 as described in Materials and Methods and Figure 2, in the presence or absence of Raltegravir. At 96 h p.i., DMSO or 5 µM Flavopiridol was added to the medium and infection was continued for 12 additional hours. (A) RNA was extracted and HIV-1 gag transcripts were quantitated by qRT-PCR as described in Materials and Methods. The graph shows the mRNA levels of gag upon Flavopiridol treatment relative to the DMSO control. Error bars represent SD of technical replicates. (B) At 6d p.i., cells were fixed and immunostained with anti-CA antiserum to determine the proportion of infected cells. Error bars represent SEM of biological replicates. (C) Co-localization of LEDGF with CPSF6 on a nuclear PIC. MDM were infected for 48 hr as above, fixed and click-labeled. LEDGF (p52 and p75; white) and CPSF6 (magenta) were detected by immunostaining. Green, IN.eGFP; red, EdU. Arrowheads indicate nuclear complexes displayed in the enlargements on the right (i, ii; note that the structure in (ii) is in a different focal plane and not clearly visible in the overview). The table shows the total number of CPSF6- or LEDGF-positive nuclear complexes detected in samples from two donors. Scale bars: 5 µm (overview), 1 µm (enlargements).

https://doi.org/10.7554/eLife.41800.010
Figure 2—figure supplement 2
FISH analysis of cells infected with a CPSF6-binding defective HIV-1 variant.

(A) MDM from three donors were infected with the A77V variant for 72 hr. Cells were fixed and immunostained using anti-CPSF6 (magenta) and anti-CA (cyan) antibodies. viral DNA (green) was detected by FISH as described in Materials and Methods. Arrows indicate nuclear viral DNA signals shown enlarged at the right. Please note that the harsh treatment of the immuno-FISH procedure compromised the quality of CA signals. Image shows a representative cell. Scale bar: 5 µm (overview) or 1 µm (enlargement). (B) Numbers of nuclear viral DNA signals per cell in cells infected with WT HIV-1 or the A77V variant at 72 h p.i. The graph shows data from three independent experiments. Whiskers represent minimum and maximum. Statistical difference was assessed with a two-tailed non-paired Mann Whitney test; ***p<0.0001.

https://doi.org/10.7554/eLife.41800.011
Figure 3 with 2 supplements
Effect of CPSF6 depletion on HIV-1 infection in primary macrophages.

(A) MDM from six donors in three independent experiments were transduced with either lentiviral vectors or AAV expressing a non-targeted shRNA (NS control) or shRNAs against CPSF6 (K/D) as described in Materials and Methods. Twelve days after initial transduction, cells were infected with 15 ng CA (MOI 3.5) of HIV-1 NL4-3 WT or A77V. To prevent secondary infection, 5 µM Maraviroc was added to the medium at 24 h p.i. The proportion of infected cells was determined by immunostaining using antiserum against CA at 6d p.i. Three independent experiments each, using cells from different donors, were conducted for lentivirally and AAV transduced cells, and infections were performed in triplicate per condition in each experiment. The graph shows mean values and SEM for the proportion of infected cells from all data sets. Statistical significance was assessed with two-tailed non-paired Mann-Whitney test; **p=0.0008, ***p<0.0001, n.s.: not significant, a.u.: arbitrary unit. Please refer to Figure 3—figure supplement 1 for data sets from individual experiments. (B) Correlation between CPSF6 levels and infectivity. MDM were transduced with AAV and infected as in (A). CPSF6 signal intensities from individual infected and non-infected cells were quantified as described in Materials and Methods. CPSF6 intensities per cell from six randomly selected wells were used to separate the population into quartiles (q1 <62. q2 <95. q3 <117. q4 <353 a.u.) and the proportion of infected cells was determined for each quartile. Symbols represent mean values calculated for samples from three individual donors; lines indicate the mean value from each data set. For each condition,>10,000 cells were analyzed, per donor. (C) MDM were transduced with lentiviral vectors and infected with HIV-1 NL4-3 4059-WT, as described in (A). CPSF6 signal intensities from individual infected and non-infected cells were quantified as described in Materials and Methods. The graph shows CPSF6 intensity values from n individual NS or K/D cells for the total (left) and infected (right) cell population. Median values are indicated by red lines. Statistical significance was assessed with two-tailed non-paired Mann-Whitney test; ***: p<0.0001. a.u.: arbitrary unit. (D) MDM were infected with HIV-1 NL4-3 WT or A77V, as described above, in the presence of 2.5 µM PF74. At 24 h p.i. 5 µM Maraviroc was added to the medium. Cells were fixed 6d p.i. and the proportion of infected cells was determined by immunostaining using antiserum against CA. Two independent experiments, using cells from four donors, were performed. Infections were performed in triplicate per condition in each experiment. The graph shows mean values and SEM for the proportion of infected cells from all data sets. Statistical significance was assessed with two-tailed non-paired Mann-Whitney test; **p=0.0013, ***p<0.0001, n.s.: not significant.

https://doi.org/10.7554/eLife.41800.012
Figure 3—source data 1

Effect of CPSF6 depletion on HIV-1 infectivity in primary macrophages.

Data corresponds to raw infectivity data of macrophages infected with WT or A77V HIV-1 after CPSF6 knock-down (Figure 3A), percentage of infected cells in populations separated by quartiles (Figure 3B), and to mean CPSF6 signal intensities of infected and non-infected cells under non-silencing (NS control) or knock-down conditions (k/d) (Figure 3C). Raw infectivity data of cells infected in the presence of 2.5 µM PF74 is also included in the file.

https://doi.org/10.7554/eLife.41800.015
Figure 3—figure supplement 1
Knockdown efficiency and HIV-1 infectivity in primary macrophages from different donors.

MDM were transduced with lentiviral (+LV; Donors 1–3) or AAV vectors (+AAV; Donors 4–6) and subsequently infected with HIV-1 NL4-3 4059-WT or HIV-1 NL4-3 4059-A77V as in Figure 3. (A) Cells were fixed and stained with anti-CPSF6 antibody and Hoechst stain. Box plots show the distribution of CPSF6 signal intensities from n cells subjected to non-silencing (black) or knock-down (gray) conditions. Whiskers represent minimum and maximum values. Statistical significance was assessed by two-tailed unpaired Mann Whitney test; ***p<0.0001. (B) At 6d p.i., cells were fixed and proportions of infected cells were determined as described in Figure 3. The graph shows mean values of infectivity and SEM from all the donors.

https://doi.org/10.7554/eLife.41800.013
Figure 3—figure supplement 1—source data 1

Mean CPSF6 signal intensities of individual cells from multiple donors after CPSF6 knock-down.

https://doi.org/10.7554/eLife.41800.016
Figure 3—figure supplement 2
Relative infectivity of the N74D CA variant.

MDM from the same donors as in Figure 3 were infected with HIV-1 NL4-3 4059-N74D as described in Materials and Methods. (A) At 6d p.i., cells were fixed and proportions of infected cells were determined as described in Figure 3. Statistical significance was assessed by two-tailed unpaired Mann Whitney test; **p=0.0008, ***p<0.0001. (B) Cells from four donors were infected with HIV-1 NL4-3 4059-N74D in the presence of DMSO or 2.5 µM PF74 as described in Figure 3. At 6d p.i., cells were fixed and proportions of infected cells were determined as in (A). Statistical significance was assessed by two-tailed unpaired Mann Whitney test; n.s.: not significant.

https://doi.org/10.7554/eLife.41800.014
Figure 3—figure supplement 2—source data 2

Raw infectivity data of primary macrophages from multiple donors infected with N74D HIV-1.

https://doi.org/10.7554/eLife.41800.017
Figure 4 with 1 supplement
Effect of CPSF6 knock-down on nuclear entry.

MDM were transduced with AAVs as described in Materials and Methods. Subsequently, cells were infected with 100 ng CA (MOI 14.5) of HIV-1 NL4-3 WT (IN.eGFP) (A, B) or A77V (IN.eGFP) (C) in the presence of 10 µM EdU. (A) At 24 h p.i., cells were fixed, and immunostained for CA (cyan), CPSF6 (magenta) and laminA/C (white). Filled arrowheads indicate IN.eGFP signals co-localizing with CA in cells displaying WT (NS control) or low (K/D) CPSF6 signal intensity. Open arrowheads indicate no co-localization for the respective marker. See Figure 4—figure supplement 1 for corresponding data obtained with HIV-1NL4-3 A77V. (B, C) At 60 h p.i., cells were fixed, click-labeled and immunostained against CPSF6 (magenta) and laminA/C (white). Solid arrowheads indicate IN.eGFP positive objects co-localizing with EdU in infected cells displaying WT (NS control) or low (K/D) CPSF6 signal intensity. Open arrowheads indicate lack of co-localization for the respective marker. Images in A-C show representative z-sections through the nuclear region of infected cells. IN.eGFP and EdU signals in the merged panels are represented in green and red, respectively. (D) Analysis of individual objects detected in cells from four donors at 60 h p.i. Tables summarize the subcellular distribution of IN.eGFP positive objects in n cells from four donors in three independent experiments, infected with HIV-1 NL4-3 WT (IN.eGFP) or the A77V variant (IN.eGFP) at 60 h p.i. with or without knock-down of CPSF6. Numbers of detected objects are given in parentheses. Proportions of nuclear or close to the nuclear envelope IN.eGFP objects with and without CPSF6 knock-down were compared using a two-tailed Z-test (α = 0.05); **: p=0.0002. ***: p<0.0001. (E) Numbers of nuclear IN.eGFP positive objects per cell in WT HIV-1 infected cells displaying normal (NS control) or low (K/D) CPSF6 signal intensity. Whiskers represent 5 and 95 percentile. Statistical significance was assessed with two-tailed non-paired Mann Whitney test; ***: p<0.0001. (F) Mean intensities of CPSF6 signals co-localizing with individual IN.eGFP positive objects at the indicated subcellular localization in cells infected with HIV-1 NL4-3 WT (IN.eGFP) or the A77V variant (IN.eGFP) and displaying normal (NS control) or low (K/D) CPSF6 signal intensity (related to panel D). (G) Analysis of individual objects detected in cells from two donors at 60 h p.i. Tables summarize the subcellular distribution of IN.eGFP positive objects in n cells from two donors infected with HIV-1 NL4-3 WT (IN.eGFP) or A77V (IN.eGFP) at 60 h p.i. in the presence of DMSO or 2.5 µM PF74. Numbers of detected objects are given in parentheses. Proportions of IN.eGFP objects in the absence or presence of PF74 located in the nucleus or close to the nuclear envelope were compared using a two-tailed Z-test (α = 0.05); ***p<0.0001, n.s.: not significant. See Figure 4—figure supplement 1 for corresponding imaging data.

https://doi.org/10.7554/eLife.41800.018
Figure 4—source data 1

Effect of CPSF6 knock-down on nuclear entry.

Data corresponds to number of nuclear IN.eGFP signals per cell after CPSF6 depletion in primary macrophages (Figure 4E) and mean CPSF6 signal intensities of individual WT and A77V HIV-1 subviral complexes at 60 h p.i. at different subcellular localizations in cells under non-silencing or CPSF6 knock-down conditions (Figure 4F).

https://doi.org/10.7554/eLife.41800.020
Figure 4—figure supplement 1
Effect of CPSF6 knockdown on trafficking of RTC/PIC to the nuclear envelope and effect of addition of PF74 on nuclear entry.

MDM were transduced with AAV as described in Materials and Methods. (A) Cells were infected with 100 ng CA (MOI 14.5) HIV-1 NL4-3 4059-A77V (IN.eGFP) in the presence of 10 µM EdU. At 24 h p.i., cells were fixed and immunostained with anti-CPSF6 (magenta), anti-CA (cyan) and anti-laminA/C (white) antibodies. Solid arrowheads indicate IN.eGFP signals co-localizing with CA signals in cells displaying wt (NS control) or low (K/D) CPSF6 signal intensity. Open arrowheads serve as a spatial reference. Images show representative z-sections through the nuclear region of infected cells. IN.eGFP signals in the merged panels are represented in green. Scale bars: 5 µm. (B) Tables on the left summarize the distribution of IN.eGFP positive objects detected at 24 h.p.i. in n cells from two donors infected with HIV-1 NL4-3 4059-WT (IN.eGFP) or HIV-1 NL4-3 4059-A77V (IN.eGFP) under non-silencing or knock-down conditions. Proportions of IN.eGFP objects localized in the nucleus were determined based on micrographs and data were analyzed using a two-tailed Z-test (α = 0.05); ***p<0.0001. (C) Mean intensities of CPSF6 signals associated with single IN.eGFP positive objects, detected at the indicated subcellular localization in the data from the tables in (B). (D, E) MDM from two donors in two independent experiments were infected with HIV-1 NL4-3 4059-WT (IN.eGFP) (D) or HIV-1 NL4-3 4059-A77V (IN.eGFP)(E) in the presence of 2.5 µM PF74 and 10 µM EdU as described in Figure 4. At 60 h p.i., cells were fixed, click-labeled and immunostained with anti-CA antiserum (cyan) and anti-laminA/C (white) antibody. IN.eGFP and EdU signals in the merged panels are represented in green and red, respectively. Arrows in (D) indicate HIV-1 RTC/PIC. Open arrows serve as a spatial reference. Box in (E) marks a large number of EdU negative VLP in the cytoplasmic area, a predominant phenotype observed under the given conditions. See main Figure 4G for quantification. Scale bars: 5 µm.

https://doi.org/10.7554/eLife.41800.019
Figure 4—figure supplement 1—source data 1

Mean CPSF6 signal intensities of individual WT and A77V HIV-1 subviral complexes after 24 h p.i. at different subcellular localizations in cells under non-silencing or CPSF6 knock-down conditions (Figure 4—figure supplement 1).

https://doi.org/10.7554/eLife.41800.021
Visualization of RTC/PIC of A77V HIV-1 at the NPC using STED nanoscopy.

MDM from two donors were infected with 100 ng CA (MOI 14.5) of HIV-1 NL4-3 A77V (IN.eGFP) for 60 hr, fixed and immunostained using antibodies against CA (magenta) and NPC proteins (FG repeats; green). (A) Confocal images of the nucleus of a representative infected cell. (B) STED images of the same infected cell. Enlargements of the four boxed regions are shown to the right of the overlay image. Arrows indicate CA positive objects co-localizing with NPC proteins. Scale bars: 2 µm (confocal images), 500 nm (STED images).

https://doi.org/10.7554/eLife.41800.022
HIV-1 subviral complexes arrested at the NPC analyzed by STED nanoscopy.

MDM were transduced with AAVs expressing shRNAs targeting CPSF6 (A, B) or non-targeted shRNA (C, D) as described in Materials and Methods. Subsequently, cells were infected with 100 ng CA (MOI 14.5) of HIV-1 NL4-3 WT (IN.eGFP) (A, B) or HIV-1 NL4-3 A77V (IN.eGFP) (C, D). At 60 h p.i., cells were fixed and immunostained against CA (magenta) and Nup153 (green). (A, C) Confocal images of the nucleus of a representative infected cell (upper panels) and super resolved images of boxed regions (lower panels). Super resolved images are shown as an overlay on the right (magenta – CA, green – Nup153). Boxed region in the overlayed images (dashed line) are shown as an enlargement. Arrows indicate CA positive objects partially co-localizing with Nup153. Scale bars: 2 µm (confocal images), 500 nm (STED images) (B, D) Averaged line profiles from (A) or (C) of selected CA positive objects (n = 30, from two independent experiments). Error bars represent SD.

https://doi.org/10.7554/eLife.41800.024
Association of CPSF6 and Nup153 analyzed by STED nanoscopy.

MDM were transduced with AAV expressing shRNAs targeting CPSF6 (B, C) or non-targeted shRNA (A) as described in Materials and Methods. Subsequently, cells were infected with 100 ng CA (MOI 14.5) of HIV-1 NL4-3 WT (IN.eGFP) (A, C) or HIV-1 NL4-3 A77V (IN.eGFP) (B). At 60 h p.i., cells were fixed and immunostained against CPSF6 (magenta) and Nup153 (green). (A–C) Confocal images of the nucleus of a representative infected cell (upper panels) and super resolved images of boxed regions (lower panels). Super resolved images are also shown as an overlay on the right (magenta – CPSF6, green – Nup153, yellow – IN.eGFP (diffraction-limited)). Boxed region in the merged image (dashed line) is shown as an enlargement above the merge. Scale bars: 2 µm (confocal images). 500 nm (STED images). Arrowheads indicate IN.eGFP positive objects at the nuclear membrane. (D) Averaged line profile from (C) of selected IN.eGFP positive objects (n = 30, from two independent experiments). Error bars represent SD.

https://doi.org/10.7554/eLife.41800.025
Model for the interaction of Nup153 and CPSF6 with the incoming HIV-1 capsid at the NPC of primary macrophages.

HIV-1 subviral complexes (depicted as a cone) at the NPC interact with Nup153 (represented as blue dots) at the nuclear basket via a capsid-derived structure. CPSF6 (represented as green dots) competitively binds to CA multimers of this structure displacing Nup153 and ultimately releasing the subviral complex from the NPC.

https://doi.org/10.7554/eLife.41800.026
Author response image 1
Relative infectivity in CPSF6 knock-down cells normalized to infectivity in non-silencing control cells vs. bulk CPSF6 knock-down efficiency determined for the respective sample.

Different symbols represent different donors. Open symbols: LV-mediated knockdown; filled symbols, AAV vector mediated knockdown.

Videos

Video 1
Related to Figure 6.

3D super resolution imaging and co-localization analysis of MDM infected with HIV-1 NL4-3 4059-A77V (IN.eGFP). The video shows a 3D reconstruction of the entire nucleus of an MDM infected with HIV-1 NL4-3 4059-A77V (IN.eGFP) at an MOI of 14.5 for 60 hr. For co-localization analysis both Nup153 and CA signals were modeled as ellipsoids with Z axis = 1.5*X,Y axis. Green – Nup153; magenta – CA; red, CA/Nup153 co-localizing structures.

https://doi.org/10.7554/eLife.41800.023

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional
information
AntibodyAlexa fluor
conjugated secondary antibodies
Thermo
Fisher Scientific
IF (1:1000)
AntibodyGoat anti-mouse IgG Abberior
STAR RED
Sigma-Aldrich;
Cat# 52283
IF (1:200)
AntibodyGoat anti-rabbit IgG Atto 594Sigma-Aldrich;
Cat# 77671
IF (1:200)
AntibodyGoat polyclonal HRP anti-rabbit IgGJackson ImmunoResearch Labs; Cat# 111-035-144RRID:AB_2307391ELISA (1:2000)
AntibodyMouse monoclonal anti-hCPSF5Sigma-Aldrich; Cat# SAB1404890RRID:AB_10739397IF(1:100)
AntibodyMouse monoclonal anti-hFG repeats (Nuclear Pore Complex)Abcam; Cat# ab24609RRID:AB_448181IF(1:300)
AntibodyMouse monoclonal anti-hLamin A/CSanta Cruz
Biotechnology;
Cat# sc-7292
RRID:AB_627875IF (1:100)
AntibodyMouse monoclonal anti-hLEDGFBD Biosciences;
Cat# 611714
RRID:AB_399192IF(1:100)
AntibodyMouse monoclonal anti-hNup153Abcam;
Cat# ab24700
RRID:AB_2154467IF (1:250)
AntibodyRabbit polyclonal anti-hCPSF6Atlas Antibodies;
Cat# HPA039973
RRID:AB_10795242IF(1:250)
AntibodyRabbit polyclonal anti-hCPSF7Atlas Antibodies
Cat# HPA041094
RRID:AB_10794187IF(1:200)
AntibodyRabbit polyclonal anti-HIV-1 CAIn-houseIF(1:1000)
AntibodySheep polyclonal anti-HIV-1 CAIn-houseIF (1:1200)
Cell line
(H. sapiens)
HeLaP4 TNPO3-KDZ Debyser
(University of
Leuven, Belgium); Thys et al. (2011)
Cell line
(H. sapiens)
TZM-bI indicator cellsWei et al. (2002)RRID: CVCL_B478
Cell line
(Homo sapiens)
Human embryonic kidney 293 T cells (HEK293T)Pear et al. (1993)RRID: CVCL_0063
Chemical
compound, drug
Efavirenz (EFV)AIDS Research and
Reference Reagent
Program, Division
AIDS, NIAID
Chemical
compound,
drug
FlavopiridolSigma-Aldrich; Cat#F3055
Chemical
compound,
drug
iQ SupermixBioRad; Cat#1708860
Chemical
compound,
drug
Maraviroc (MVC)Sigma-Aldrich; Cat#PZ0002
Chemical
compound,
drug
PF-3450074 (PF74)Sigma-Aldrich;
Cat#SML0835
Chemical
compound,
drug
Raltegravir (RAL)AIDS Research and
Reference Reagent
Program, Division
AIDS, NIAID
Commercial
assay or kit
Click-iT EdU
Alexa Fluor 647
Imaging kit
Thermo Fisher Scientific; Cat#C10340
Commercial
assay or kit
DIG-Nick translation mixRoche; Cat#11745816910
Commercial
assay or kit
InviTrap Spin
Universal RNA
Mini kit
Stratec; Cat#1060100300
Commercial
assay or kit
SuperScript III Reverse
transcriptase kit
Thermo Fisher
Scientific; Cat#18080093
Commercial
assay or kit
TSA Plus
system
Perkin Elmer;
Cat#NEL749A001KT
Recombinant
DNA reagent
AAV helper
plasmid
D Grimm
(University of
Heidelberg,
Germany)
AAV helper
plasmid
expressing rep
and cap genes
for transducing
MDM
Recombinant
DNA reagent
CPSF6 triple
shRNA
This studyPlasmid expressing
three shRNAs targeting CPSF6. For
packaging into
AAV vector.
Recombinant
DNA reagent
NS control AAVK Boerner
(University of
Heidelberg, Germany); Börner et al. (2010)
Plasmid expressing non-targeted shRNA. For packaging into AAV vector.
Recombinant
DNA reagent
pEnv-4059R Swanstrom
(University
of North
Carolina, USA); Schnell et al. (2011)
Plasmid expressing an R5-tropic HIV-1 Env
Recombinant
DNA reagent
pHIVSIRENG Towers
(University
College London,
UK); Rasaiyaah et al. (2013)
Plasmids expressing shRNAs targeting CPSF6 or non-targeting shRNA
Recombinant
DNA reagent
pMD2.GD Trono
(EPFL,
Lausanne, Switzerland)
RRID: Addgene_12259Plasmid expressing VSV-G
Recombinant
DNA reagent
pNL4-3Adachi et al. (1986)HIV-1 proviral
plasmid
Recombinant
DNA reagent
pNL4-3ΔEnvB Müller
(University of
Heidelberg,
Germany)
HIV-1 proviral
plasmid
Recombinant
DNA reagent
pNL4-3ΔEnv N74D/A77VThis studyHIV-1 proviral
plasmid
Recombinant
DNA reagent
pNLC4-3ΔTatT Müller
(University of
Heidelberg,
Germany)
HIV-1 proviral
plasmid
Recombinant
DNA reagent
psPAX2D Trono
(EPFL,
Lausanne, Switzerland)
RRID: Addgene_35002Lentiviral packaging vector
Recombinant
DNA reagent
pVAE2AE4-5D Grimm
(University of
Heidelberg,
Germany); Matsushita et al. (1998)
Adenoviral helper plasmid
Recombinant
DNA reagent
pVpr.IN.eGFPA Cereseto
(CIBIO, Mattareo,
Italy); Albanese et al. (2008)
Plasmid expressing Vpr.IN.eGFP fusion
Software,
algorithm
Autoquant X3Media
Cybernetics
RRID:SCR_002465
Software,
algorithm
GraphPad PrismGraphPadRRID:SCR_002798
Software,
algorithm
Image JImage JRRID:SCR_003070
Software,
algorithm
Imaris 8.1BitPlane AGRRID:SCR_007370
Software,
algorithm
ImspectorAbberior
Instruments
RRID:SCR_015249
Software,
algorithm
KNIMEKonstanz
Information Miner
RRID:SCR_006164
Software,
algorithm
VolocityPerkin ElmerRRID:SCR_002668

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files for the plots of Figures 1, 3 and 4 and supplemental material are provided.

Additional files

Source data 1

Correlation analysis.

Correlation between CPSF6 knock-down efficiency and HIV-1 infectivity. Spearman correlation of CPSF6 knock-down efficiency and K/D:NS infectivity ratio from multiple donors.

https://doi.org/10.7554/eLife.41800.027
Transparent reporting form
https://doi.org/10.7554/eLife.41800.028

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