Fig 2: HIV-1 dependence on Nup35.a CA-dependent use of nucleoporins by HIV-1. Wild-type (WT) or CA-mutant HIV-1-RFP reporter virus infection of control or target siRNA transfected HeLa cells. Nups targeted by siRNAs are grouped by NPC subcomplexes. WT HIV-1 infection decreases of greater than 3-fold after knockdown of nucleoporins are encircled. Percent relative infection is mean ± s.d., n = 2–5 for nucleoporins, n = 15 for TNPO3 from biologically independent experiments. b Distribution of Nup153 and Nup155 after Nup35 knockdown. Localization of Nup153 (red), Nup155 (green), and Lamin B1 (blue) in HeLa cells by 3D SIM after immunostaining. Representatives of 40 cells from two independent experiments. A z-section image is presented. Scale bars: 5 µm (overview), 1 µm (inset). c WT HIV-1 or N74D HIV-1 or MLV infection of dividing and non-dividing (2 µg/ml aphidicolin-treated) control or Nup35 siRNA transfected HeLa cells. n = 2 technical replicates, representative of two independent experiments. Western blotting confirmed knockdown efficiency. d HeLa cells stably expressing human HA-Nup35 lacking 3'-UTR or transduced with control vector (LPCX-HA), were transfected with non-target (NT) siRNA or Nup35 siRNA targeting 3'-UTR region for 48 h, then infected with VSV-G-pseudotyped viruses. n = 2 technical replicates, representative of three independent experiments. Western blot analysis confirmed Nup35 restoration. e HIV-1 CA determines Nup35 dependence in HeLa knockout cell clones. WT HIV-1 or CA-mutant HIV-1 or MLV infection of Nup35 knockout HeLa cell clones. n = 2 technical replicates, representative of three independent experiments. Western blots show the knockout efficiency of Nup35. Empty, cell lines transiently co-transfected with pX330 and a puromycin-expression vector; NT, non-targeting control lines lacking a gene-specific gRNA. f WT HIV-1 or CA-mutant HIV-1 or various retroviruses infection of control or target siRNA transfected HeLa cells. n = 2 technical replicates, representative of two independent experiments. Western blotting confirmed knockdown efficiency. Source data are provided as a Source Data file.

Fig 3: Soluble factors regulate nucleoporin dependence.a CypA enrichment at the nuclear periphery. Localization of CPSF6 (green) and CypA (red) in HeLa cells in a z-section image from deconvolution microscopy of immunostained HeLa cells. Scale bar, 15 µm. b CPSF6 aggregation at NPC. Control or Nup35 knockdown cells were infected with WT or N74D HIV-1 for 12 h. Localization of CPSF6 (green) and NPC (red) in HeLa cells by 3D SIM after immunostaining. A z-section image is presented. Scale bars: 5 µm (overview), 1.2 µm (inset). a, b Representative of 50 cells from two independent experiments. c Partial restoration of HIV-1 infection in Nup35 and POM121 knockdown cells after CPSF6 depletion. WT HIV-1 or CA-mutant HIV-1 or MLV infection of control or single-knocked down or double-knocked down HeLa cells. n = 2 technical replicates, representative of three independent experiments. Western blotting confirmed knockdown efficiency. Source data are provided as a Source Data file.

Fig 4: POM121 and Nup35 FG-motif is essential for binding to HIV-1 capsid.a HeLa cells stably expressing either wild-type or FG mutant HA-Nup35 lacking 3'-UTR or transduced with control vector (LPCX-HA), were transfected with non-target (NT) siRNA or Nup35 siRNA targeting 3'-UTR region for 48 h, then infected with VSV-G-pseudotyped viruses. n = 2 technical replicates, representative of three independent experiments. Western blot analysis confirmed Nup35 restoration. 1st FG, FG179AA; 2nd FG, FG232AA; 3rd FG, FG324AA. b Mapping of POM121 C-terminal region required for Trim5a.POM121 restriction. Left panel, schematic representation of systemic deletion constructs of POM121. Blue lines represent boundaries of each constructs, while red lines denote the locations of FG motifs. Right panel, WT HIV-1 or CA-mutant HIV-1 infection of vector or TRIMa.POM121-expressing HeLa cells. n = 2 technical replicates, representative of three independent experiments. c CypA determines the HIV-1 nuclear import pathway. Premature CPSF6 interaction with HIV-1 impairs CA interaction with FG-Nups at the NPC. CypA binding by HIV-1, however, facilitates use of Nup35, Nup153, and POM121 during infection. Subsequent CPSF6 binding may enhance release from the NPC. We propose that the HIV-1 core, comprised of multimeric CA in association with the viral nucleic acid and enzymatic proteins, directly functions as a NTR and exploits successive FG interactions to achieve transfer through the NPC but these interactions are regulated in time and space by CypA and CPSF6. CypA, azure dot; CPSF6, lilac dot. Source data are provided as a Source Data file.

Fig 5: Nup35 knockdown impairs HIV-1 nuclear entry.a CsA can restore HIV-1 infectivity in Nup35 knockdown cells 12 h after virus challenge. WT HIV-1 or CA-mutant HIV-1 or MLV infection of control or Nup35 siRNA transfected HeLa cells in the presence or absence of CsA. n = 2 technical replicates, representative of two independent experiments. b HIV-1 reverse transcription is not blocked in Nup35 knockdown cells but 2-LTR circle forms are not observed. DNase-treated WT HIV-1 or CA-mutant HIV-1 infection of control or Nup35 siRNA transfected HeLa cells in the presence or absence of CsA. Cell DNA was extracted at 3, 6, 12, and 24 h after infection and used to detect early (RU5) RT, late (2nd strand) RT, and 2-LTR circles (right). n = 2-3 technical replicates, representative of two independent experiments. The amount of DNA in each PCR assay was normalized by actin. HIV-1 infection was also measured at 48 h in parallel samples to monitor HIV-1 infectivity (Supplementary Fig. 5c). c CsA restores HIV-1 infection in Nup35, Nup153, and POM121 knockdown HeLa cells. WT HIV-1 or CA-mutant HIV-1 infection of control or target siRNA transfected HeLa cells in the presence or absence of CsA. n = 2 technical replicates, representative of three independent experiments. Western blotting confirmed knockdown efficiency. -, without CsA; +, with CsA. d–g CPSF6 distribution of Nup35, POM121, and Nup153 knockdown cells in the presence or absence of CsA. WT HIV-1 or CA-mutant HIV-1 infection of control or target siRNA transfected HeLa cells. After 12 h, cells were fixed and stained with antibodies against CPSF6 and NPC. Scale bar, 15 µm. Representatives of 100 cells from an experiment. h The number of cells with or without CPSF6 aggregates were counted from (d–g). Source data are provided as a Source Data file.