Fig. 3.
A bipartite interaction between NUP153 and the CA lattice. (A) Top: The C-terminal 75 aa of NUP153. Bottom: percentages of various NUP153 variants, along with maltose-binding protein (MBP) and yeast NUP NSP1 negative controls, bound to CA tubes in the copelleting assays under 75 mM NaCl (black bar), 150 mM NaCl (gray bar), or 300 mM NaCl (light gray bar) conditions. Data were expressed as mean ± SD of three independent experiments. Differences were determined by one-way ANOVA and Tukey’s multiple comparisons test, compared with NUP153896–1475; NS, not significant (P ≥ 0.05); ****P < 0.0001. The schematic of the two capsid-binding motifs on NUP153CTD is shown in the Right panel. (B) Copelleting assays of CA mutant nanotubes with NUP1531411–1475. Left: percentages of NUP1531411–1475 bound to CA mutant tubes in the copelleting assays under 150 mM NaCl. Data were expressed as mean ± SD of three independent experiments. Differences were determined by one-way ANOVA and Tukey’s multiple comparisons test, compared with A14C/E45C CA tubes, NS, not significant (P ≥ 0.05), *P < 0.05, ****P < 0.0001. Right: SDS-PAGE analysis of copelleted NUP1531411–1475 alongside the positions of mutated CA residues on the tri-hexamercenter model. Negative-stain TEM validation of the formation of various CA nanotubes is shown in SI Appendix, Fig. S4. (C) Analysis of HIV-1 infection in NUP153-depleted cells. HeLa cells were treated with two separate nontargeting control small interfering RNAs (siRNAs) or with two different NUP153-targeting siRNAs. Cell monolayers were subsequently challenged with HIV-GFP reporter viruses at dilutions appropriate for detecting infection. The fraction of infected cells was analyzed by flow cytometry for GFP expression. Top, relative infectivity of the virus stocks on control siRNA-transfected cells. Shown are the individual results from infections of cells transfected with two different nontargeting siRNAs. Bottom, levels of infection in control and NUP153-depleted cells. Shown are averages of duplicate infections with error bars designating the range of the two values obtained from the two different siRNAs. Results are representative of two independent experiments. (D) The NUP1531411–1475/1425–1464△ peptide (25 aa) binds to CA tri-hexamercenter as measured by ITC. Independent experiments were performed three times, and a representative example is shown. Curve fitting errors to a single data set are shown.