Figure 1: Depletion of Nup153 and Tpr followed by reconstitution of the nuclear basket by complementation and in vitro CA–NC binding assay.

(a,b) We used the pTrip.GFP.H1shRNA vector to knockdown (KD) the expression of Nup153 and Tpr in HeLa P4CCR5 cells^13,14. Viral particles produced using the pTrip.GFP.H1shRNA vector containing the specific shRNA against Nup153 and Tpr were used to transduce HeLa P4CCR5 at the indicated multiplicity of infection (MOI). Control cells were transduced with the empty pTrip.GFP.H1shRNA vector. The efficiency of the KD was monitored by western blotting using antibodies against the endogenous Nup153 proteins and Tpr. As a loading control, samples were also blotted using antibodies against actin and lamin A/C. (c) Nup153-depleted cells at MOI 50 were complemented with a plasmid, pC2GFP, GFP-Nup153w/o FG and GFP-Nup153. The efficiency of the complementation was monitored by western blotting using antibodies against the endogenous Tpr, GFP and actin. (d, e) Nup153 and Tpr -depleted cells were challenged with HIV-1 containing luciferase as a reporter of infection. HIV-1-Luc was normalized by p24 as described in Methods. Infectivity was determined 48 h p.i. by measuring luciferase activity normalized to the amount of protein. 24 h p.i. total genomic DNA from infected cells was used to measure 2LTR circles by real-time PCR normalized to actin. The percentage of 2LTR circles with respect to the control is shown in the histograms of Nup153 and Tpr-depleted cells at MOI 50 and 100, respectively. The s.d. of three independent experiments are shown. (f) The binding of GFP-Nup153 and GFP-Tpr proteins to in vitro assembled HIV-1 capsid–nucleocapsid (CA–NC) complexes was performed by transfecting human 293T cells with plasmids expressing GFP-Nup153, GFP-Tpr, GFP and Trim5α_rh-GFP. The assay is described in Methods. Quantification of BOUND versus INPUT fractions of three independent experiments was plotted with their respective standard deviations on the lower panels.