Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2
- The Rockefeller University, United States
- University of Colorado School of Medicine, United States
- Howard Hughes Medical Institute, United States
- Aaron Diamond AIDS Research Center, United States
Figures
Figure 1 with 3 supplements
MX2 sensitivity is affected by the viral CA, the cell-cycle, and CypA/CsA in a cell-type dependent manner.
Wild-type (WT) or CA-mutant HIV-1-GFP reporter virus infection of dividing or non-dividing (aphidicolin treated) HeLa or HT1080 cells expressing doxycycline-inducible MX2 in the presence (white …
Figure 1—figure supplement 1
Western blot analysis of doxycycline inducible MX2 and tubulin loading control in HeLa and HT1080 cells.
https://doi.org/10.7554/eLife.35738.003
Figure 1—figure supplement 2
MX2 sensitivity is altered by the viral CA, the cell-cycle, and cyclophilin A in a cell-type dependent manner.
Infection of dividing or non-dividing (aphidicolin treated) HeLa or HT1080 cells expressing doxycycline-inducible MX2 in the presence (white bars) or absence (black bars) of pretreatment with …
Figure 1—figure supplement 3
CsA sensitivity of HIV-1 CA mutants in HOS cells.
WT, CA-mutant HIV-1, or HIV-2-GFP reporter virus infection of dividing or non-dividing (aphidicolin treated) HOS cells expressing doxycycline-inducible MX2 in the presence or absence of CsA. Titers …
Figure 2 with 3 supplements
Nup and NTR expression in immortalized cell lines.
(A) Western blot analysis of expression of Nups (color-coded by subcomplex as indicated in panel B), selected NTRs, and CPSF6 in T cell (MT4, CEM X174, Jurkat, SupT1, H9), adherent cell (HeLa, HOS, …
Figure 2—figure supplement 1
Nup and NTR expression in immortalized cell lines.
Heat map representing Nup, selected NTRs, and CPSF6 protein levels in T cell (MT4, CEM X174, Jurkat, SupT1, ) adherent cell (HeLa, HOS, HT1080, 293T), or monocytic cell (THP-1, K-562, U937) lines …
Figure 2—figure supplement 2
Nup and NTR expression in primary cells.
Western blot analysis of expression of Nups, selected NTRs, and CPSF6 in MT4, THP-1, primary CD4+ T cells, and macrophages from two donors (color coded by subcomplex as in Figure 2B). Arrowheads …
Figure 2—figure supplement 3
Nup and NTR expression in primary cells.
(A) Heat map representing Nup, selected NTRs, and CPSF6 protein levels in MT4, THP-1, primary CD4+ T cells, and macrophages from two donors (color coded by subcomplex as in Figure 2) from the …
Figure 3 with 1 supplement
Interactions between CA tubes and cellular Nups.
(A) CA tubes were assembled in vitro and incubated with lysates of HeLa cells without (lanes 1–5) or with (lanes 6–10) Dox dependent expression of MX2. The reaction mixtures were subjected to …
Figure 3—figure supplement 1
Specificity of CA binding assay.
(A) Coomassie stained SDS-PAGE image for pull-down experiments with in vitro assembled CA tubes and HeLa cell lysates. Lanes 1–10 correspond to those in Figure 3. In addition, molecular weight …
Figure 4
An siRNA-based knockdown approach to investigate the role of Nups and NTRs in MX2 localization and anti-viral activity.
(A) Schematic representation of the nuclear pore complex and genes included in siRNA library color coded by subcomplex. (*NUP98 is listed as a member of Nup107 subcomplex, however. NUP98 and NUP96 …
Figure 5 with 8 supplements
Efficient siRNA-mediated knockdown of Nups and pleiotropic effects of Nup depletion in HeLa cells.
Heat map representing Nup and NTR protein levels (color coded by subcomplex as in Figure 4A and according to the included schematic) in dividing (top) and non-dividing (bottom) HeLa cells 64 hr …
Figure 5—figure supplement 1
Efficient siRNA-mediated knockdown of Nups and pleiotropic effects of Nup depletion in HT1080 cells.
Heat map representing protein levels of Nups or NTRs (color coded by subcomplex as in Figure 4A) in dividing (top) and non-dividing (bottom) HT1080 cells 64 hr after transfection with the indicated …
Figure 5—figure supplement 2
Efficient siRNA-mediated knockdown of Nups and pleiotropic effects of Nup depletion in HOS cells.
Heat map representing protein levels of Nups or NTRs (color coded by subcomplex as in Figure 4A) in dividing (top) and non-dividing (bottom) HOS cells 64 hr after transfection with the indicated …
Figure 5—figure supplement 3
Knockdown of Nups and NTRs in dividing HeLa cells.
Western blot analysis of Nup and NTR expression levels in HeLa cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as …
Figure 5—figure supplement 4
Knockdown of Nups and NTRs in non-dividing HeLa cells.
Western blot analysis of Nup and NTR expression levels in growth arrested (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the …
Figure 5—figure supplement 5
Knockdown of Nups and NTRs in HT1080 cells.
Western blot analysis of Nup and NTR expression levels in A) dividing and B) non-dividing (aphidicolin treated) HT1080 cells stably transduced with doxycycline-inducible MX2 64 hr after transfection …
Figure 5—figure supplement 6
Knockdown of Nups and NTRs in HOS cells.
Western blot analysis of Nup and NTR expression levels in A) dividing and B) non-dividing (aphidicolin treated) HOS cells 64 hr after transfection with the indicated siRNA color coded by subcomplex …
Figure 5—figure supplement 7
Cell-cycle profile of HOS cells following transfection with Nup/NTR targeting siRNA.
Cell-cycle profile of HOS cells 64 hr after transfection with the indicated siRNA or following aphidicolin treatment, color coded by subcomplex as in Figure 4A. DNA content was determined by flow …
Figure 5—figure supplement 8
Cell-cycle profile of HeLa cells following transfection with Nup/NTR targeting siRNA.
Cell-cycle profile of HeLa cells 64 hr after transfection with the indicated siRNA or following aphidicolin treatment, color coded by subcomplex as in Figure 4A. DNA content was determined by flow …
Figure 6 with 20 supplements
Effects of depleting individual Nups on NPC integrity and function, and MX2 localization.
Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), …
Figure 6—figure supplement 1
Effects of depleting individual Nups on NPC integrity and function, and MX2 localization.
Summary of the localization of Nups, MX2-RFP, CPSF6-RFP, or NLS-GFP-LacZ fusions upon siRNA transfection of HeLa or HT1080 cells shown in Figure 6, and Figure 6—figure supplements 1–18. Aberrant …
Figure 6—figure supplement 2
Localization of MX2, NUP153, and RANBP2 in HeLa cells following transfection with Nup/NTR targeting siRNA.
Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), …
Figure 6—figure supplement 3
Localization of MX2, NUP153, and RANBP2 in HeLa cells following transfection with Nup/NTR targeting siRNA continued.
Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), …
Figure 6—figure supplement 4
Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA.
Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 …
Figure 6—figure supplement 5
Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued.
Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 …
Figure 6—figure supplement 6
Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued.
Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 …
Figure 6—figure supplement 7
Localization of MX2 and NUP62 following transfection with Nup/NTR targeting siRNA.
Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP62 (green), …
Figure 6—figure supplement 8
Localization of MX2 and NUP62 following transfection with Nup/NTR targeting siRNA continued.
Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP62 (green), …
Figure 6—figure supplement 9
Localization of MX2 and NUP214 following transfection with Nup/NTR targeting siRNA.
Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP214 (green), …
Figure 6—figure supplement 10
Localization of MX2 and NUP214 following transfection with Nup/NTR targeting siRNA continued.
Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP214 (green), …
Figure 6—figure supplement 11
Localization of CPSF6 and NUP98 in HeLa cells following transfection with Nup/NTR targeting siRNA.
Deconvolution microscopic images (single optical sections) of HeLa cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), …
Figure 6—figure supplement 12
Localization of CPSF6 and NUP98 in HeLa cells following transfection with Nup/NTR targeting siRNA continued.
Deconvolution microscopic images (single optical sections) of HeLa cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), …
Figure 6—figure supplement 13
Localization of CPSF6 and NUP98 in HT1080 cells following transfection with Nup/NTR targeting siRNA.
Deconvolution microscopic images (single optical sections) of HT1080 cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 …
Figure 6—figure supplement 14
Localization of CPSF6 and NUP98 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued.
Deconvolution microscopic images (single optical sections) of HT1080 cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 …
Figure 6—figure supplement 15
Localization of SV40 NLS-GFP-LacZ following transfection with Nup/NTR targeting siRNA.
HeLa cells stably transduced with SV40 NLS-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection …
Figure 6—figure supplement 16
Localization of SV40 NLS-GFP-LacZ following transfection with Nup/NTR targeting siRNA continued.
HeLa cells stably transduced with SV40 NLS-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection …
Figure 6—figure supplement 17
Localization of MX2(N25)-GFP-LacZ following transfection with Nup/NTR targeting siRNA.
HeLa cells stably transduced with MX2(N25)-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection …
Figure 6—figure supplement 18
Localization of MX2(N25)-GFP-LacZ following transfection with Nup/NTR targeting siRNA continued.
HeLa cells stably transduced with MX2(N25)-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection …
Figure 6—figure supplement 19
Localization of MX2 and Nups in mitotic HeLa cells.
Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector). Representative of three independent …
Figure 6—figure supplement 20
Localization of MX2 and Nups in mitotic HT1080 cells.
Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector). Representative of three independent …
Figure 7 with 3 supplements
Localization of MX2 and Nups.
(A) Pearson’s coefficient for co-localization of MX2 with indicated Nups and (B) Nups with one another of deconvolution microscopic images of HeLa cells expressing MX2-RFP and immunoflourescently …
Figure 7—figure supplement 1
Localization of MX2 and Nups.
Deconvolution microscopic images (single optical sections approximately coincident with the dorsal surface of the nucleus) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline …
Figure 7—figure supplement 2
Localization of MX2 and Nups.
Deconvolution microscopic images (single optical sections approximately coincident with the dorsal surface of the nucleus) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline …
Figure 7—figure supplement 3
Localization of MX2 and Nups.
Deconvolution microscopic images (single optical sections approximately coincident with the dorsal surface of the nucleus) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline …
Figure 8 with 5 supplements
Effect of Nup and NTR depletion on HIV-1 infection and antiviral activity of MX2.
(A) Infectivity of HIV-1 GFP reporter virus in dividing (top) and non-dividing (bottom) HeLa cells stably transduced with doxycycline-inducible MX2 in the presence (open circles) or absence (filled …
Figure 8—figure supplement 1
Effect of Nup and NTR depletion on HIV-1 infection in HOS cells.
(A) Infectivity of HIV-1 GFP reporter virus in dividing (top) and non-dividing (bottom) HOS cells 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A). Titers are mean ±sem…
Figure 8—figure supplement 2
The amino-terminal domain of MX2 fused to a heterologous protein exhibits antiviral activity but with altered Nup requirements.
(A) Deconvolution microscopic images (single optical sections) of immunoflourescently stained myc-tagged ARFAPTIN2 or MX2(N91)-ARFAPTIN2 fusion proteins (green), NUP98 (red), and DAPI-stained DNA. …
Figure 8—figure supplement 3
Effect of ARFAPTIN2 fusion protein expression on lentivirus and HIV-1 CA mutant infection.
(A) Infection of HeLa cells stably transduced with doxycycline-inducible arfaptin2 myc-tagged fusion proteins with various GFP reporter viruses in the presence (white bars) or absence (black bars) …
Figure 8—figure supplement 4
Effect of Nup and NTR depletion on primate lentivirus infection and MX2 sensitivity.
Infectivity of (A) HIV-2 or (B) SIVmac GFP reporter virus in HeLa cells stably transduced with doxycycline-inducible MX2 in the presence (open circles) or absence (filled circles) of doxycycline. …
Figure 8—figure supplement 5
Effect of Nup and NTR depletion on non-primate lentivirus infection and MX2 sensitivity.
Infectivity of (A) EIAV or (B-D) FIV GFP reporter virus in (A-B) HeLa or (C) HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) …
Figure 9 with 1 supplement
Effect of Nup and NTR depletion on HIV-1 CA mutant infection and MX2 sensitivity.
Infectivity of HIV-1 CA mutant GFP reporter viruses in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) of …
Figure 9—figure supplement 1
Effect of Nup and NTR depletion on HIV-1 CA mutant infection and MX2 sensitivity.
Infectivity of HIV-1 CA mutant GFP reporter viruses in HOS cells or HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) of …
Figure 10
Effect of Nup and NTR depletion on HIV-1 infection and MX2 sensitivity in the presence of CsA.
(A–B) Infectivity of HIV-1 GFP reporter virus in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the absence of doxycycline and in the presence (asterisks) or absence …
Figure 11
Effect of Nup and NTR depletion on HIV-1 A92E and N57S CA mutant infection in the presence of CsA.
(A–C) Infectivity of HIV-1 A92E or N57S GFP reporter viruses in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the absence of doxycycline and in the presence (asterisks) or …
Figure 12
MX2 can inhibit nuclear transport of nonviral cargos in an import pathway dependent manner.
(A) HeLa cells stably transduced with vectors expressing the indicated NLS-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP, were untreated or treated with doxycycline to induce …
Tables
Table 1
Nuclear localization signals fused to GFP-LacZ
https://doi.org/10.7554/eLife.35738.058| Parent protein | NLS-type | Reference(s) | Cloning method |
|---|---|---|---|
| SV40 Large T Antigen | Monopartite class 1 | (Mattaj and Englmeier, 1998) | oligonucleotide annealing |
| C-MYC | Monopartite class 2 | (Kosugi et al., 2009) | oligonucleotide annealing |
| DDX21 | Monopartite class 3 | (Kosugi et al., 2009) | oligonucleotide annealing |
| Nucleoplasmin | Bipartite | (Mattaj and Englmeier, 1998) | oligonucleotide annealing |
| HNRNP K (KNS) | n/a | (Michael et al., 1997) | PCR amplification |
| HTLV-1 Rex | n/a | (Siomi et al., 1988; Slamon et al., 1988) | oligonucleotide annealing |
| MX2 | n/a | (Melén et al., 1996) | PCR amplification |
Table 2
ON-TARGET SMARTpool siRNA utilized in this investigation
https://doi.org/10.7554/eLife.35738.059| Gene symbol | Gene ID |
|---|---|
| AAAS | 8086 |
| AHCTF1 | 25909 |
| CPSF6 | 11052 |
| GLE1 | 2733 |
| IPO11 | 51194 |
| IPO13 | 9670 |
| IPO4 | 79711 |
| IPO5 | 3843 |
| IPO7 | 10527 |
| IPO8 | 10526 |
| IPO9 | 55705 |
| KPNA1 | 3836 |
| KPNA2 | 3838 |
| KPNA3 | 3839 |
| KPNA4 | 3840 |
| KPNA5 | 3841 |
| KPNA6 | 23633 |
| KPNB1 | 3837 |
| MX2 | 4600 |
| NDC1 | 55706 |
| NUP107 | 57122 |
| NUP133 | 55746 |
| NUP153 | 9972 |
| NUP155 | 9631 |
| NUP160 | 23279 |
| NUP188 | 23511 |
| NUP205 | 23165 |
| NUP210 | 23225 |
| NUP214 | 8021 |
| NUP35 | 129401 |
| NUP37 | 79023 |
| NUP43 | 348995 |
| NUP50 | 10762 |
| NUP54 | 53371 |
| NUP62 | 23636 |
| NUP85 | 79902 |
| NUP88 | 4927 |
| NUP93 | 9688 |
| NUP98 | 4928 |
| NUPL1 | 9818 |
| POM121 | 9883 |
| RAE1 | 8480 |
| RANBP2 | 5903 |
| SEC13 | 6396 |
| SEH1L | 81929 |
| TNPO1 | 3842 |
| TNPO2 | 30000 |
| TNPO3 | 23534 |
| TPR | 7175 |
Table 3
Antibodies utilized in this investigation
https://doi.org/10.7554/eLife.35738.060| Reactivity | Species | Company | Catalog number |
|---|---|---|---|
| ALADIN | rabbit | Novus Biologicals | NBP2-21596 |
| CPSF6 | rabbit | ProteinTech | 15489–1-AP |
| CPSF6 | rabbit | Abcam | ab175237 |
| ELYS | mouse | Abcam | ab53540 |
| GAPDH | mouse | Santa Cruz | sc-32233 |
| KPNA1 | rabbit | ProteinTech | 18137–1-AP |
| KPNA2 | rabbit | Abcam | ab84440 |
| LAMIN B1 | rabbit | Abcam | ab133741 |
| MX2 | rabbit | Novus Biologicals | NBP1-8108 |
| MYCtag | mouse | Millipore | 05–724 |
| NDC1 | rabbit | Novus Biologicals | NBP1-91603 |
| NUP107 | rabbit | Abcam | ab73290 |
| NUP133 | mouse | Santa Cruz | 376763 |
| NUP153 | mouse | Abcam | ab24700 |
| NUP155 | rabbit | Abcam | ab157104 |
| NUP160 | rabbit | Abcam | ab74147 |
| NUP188 | rabbit | Abcam | ab86601 |
| NUP205 | rabbit | Abcam | ab157090 |
| NUP210 | rabbit | Novus Biologicals | NB100-93336 |
| NUP214 | rabbit | Bethyl Labs | IHC-00103 |
| NUP35 | rabbit | Bethyl Labs | A301-781A |
| NUP37 | rabbit | Abcam | ab201161 |
| NUP43 | rabbit | Bethyl Labs | A303-976A |
| NUP50 | rabbit | Bethyl Labs | A301-783A |
| NUP54 | rabbit | ProteinTech | 16232–1 AP |
| NUP62 | mouse | BD Biosciences | 610498 |
| NUP62 | mouse FITC-conjugated | BD Biosciences | 611962 |
| NUP85 | mouse | Santa Cruz | 376111 |
| NUP88 | mouse | BD Biosciences | 611896 |
| NUP93 | mouse | Abcam | ab53750 |
| NUP98 | rabbit | Cell Signaling | C39A3 |
| NUPL1 | rabbit | ProteinTech | 19907–1-AP |
| HIV-1 p24 | mouse | NIH AIDS Reagent Program | 182-H12-5C |
| POM121 | rabbit | Abcam | ab190015 |
| POM121 | rabbit | ProteinTech | 15645–1-AP |
| RAE1 | rabbit | Abcam | ab124783 |
| RANBP2 | rabbit | Abcam | ab64276 |
| SEC13 | rabbit | ProteinTech | 15397 |
| TNPO1 | mouse | Sigma-Aldrich | WH0003842M1 |
| TNPO3 | mouse | Abcam | ab54353 |
| TPR | rabbit | Abcam | wh000717m1 |
| TUBULIN | mouse | Sigma-Aldrich | T6074 |
Additional files
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Supplementary File 1
Statistical analyses.
Tab 1. Figure 1 statistics. P values generated from two-tailed paired t tests for data represented in Figure 1. Tab 2. Nup and NTR knockdown statistics 1. P values generated from two-tailed paired t tests for data represented in Figures 8–11, addressing significance within experiments. Tab 3. Nup and NTR knockdown statistics 2. P values generated from two-tailed unpaired t tests for data represented in Figures 8–11, addressing significance between experiments. Tab 4. Figure 8 additional statistics. P values generated from two-tailed paired t tests for data represented in Figure 8—figure supplements 2D and 3A–C.
- https://doi.org/10.7554/eLife.35738.061
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Transparent reporting form
- https://doi.org/10.7554/eLife.35738.062
