F72L-containing GFP-Vpr proteins fail to incorporate into forming virions, which show decreased infectivity of non-dividing MAGI (Multinuclear Activation of a Galactosidase Indicator) cells correlating with reduced importin-β3 binding. (A) Virus was derived from 293T cells cotransfected with pEPI-GFP-Vpr and proviral plasmid pUC18-NL4.3 and subjected to Western blot analysis, revealing the absence of GFP-Vpr protein in F72L containing samples. Control staining for p24 capsid protein indicates the presence of virus in all samples (*denotes lack of virion incorporation). (B) Virus derived from 293T cells cotransfected with the ΔVpr pro-viral plasmid pUC18-NL4.3(FS) and pEPI-GFP-Vpr (1–96, A6-2, A6-3 or F72L) was purified, normalized using an RT assay and used to infect growth arrested (γ-irradiated, 2 cycles at 30 Gy) MAGI (CD4+, integrated HIV-1-LTR-β-gal) cells. 48 hours post infection cells were fixed (1% formaldehyde/0.2% glutaraldehyde/PBS), stained (4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, 2 mM MgCl2, 0.4 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside [X-gal]) and scored for viral infectivity. Infected cells display X-gal stained (blue) nuclei due to expression of the early HIV protein Tat, which binds the HIV-LTR promoter of the integrated β-gal gene, resulting in expression of the β-gal protein. Non-infected cells remain colourless due to the lack of Tat expression and subsequent activation of the β-gal gene. Data presented depicts relative levels of infectivity (% +/- SEM) compared to wildtype Vpr1–96. Virus derived from F72L-cotransfected 293T cells displayed a significant (p < 0.0001) 5-fold reduction in viral infectivity of non-dividing cells compared to wildtype Vpr1–96. (C) Native PAGE gel-shift mobility assay; 2 μM GFP-Vpr1–96 or GFP alone was incubated with 10 μM importin-α2, -β1, -α2/β1 or -β3 as indicated. (D) Native PAGE gel-shift mobility titration assay; (i) 2 μM GFP-Vpr1–96 or GFP-VprF72L was incubated with increasing concentrations of Importin-β3 protein as indicated. (ii) Fluorimetric analysis of gel-shift assays from D(i), was performed as per  with the binding curves generated used to calculate dissociation constants (Kd). (E)i Typical CLSM images of fixed COS-7 cells expressing the indicated GFP-fusion proteins alone or in the context of c-myc-tagged-human Importin-β3. Cells were permeabilised and stained 14 hours post transfection with anti-c-myc antibody (Sigma) and visualized with Alexa-Fluo-568 (Molecular Probes). (E)ii Analysis of CLSM images (as per E(i)) with ImageJ was performed to determine the Fn/c. Exogenous Importin-β3 was found to significantly (p = 0.0378) increase the nuclear accumulation of wildtype GFP-Vpr1–96, but not that of GFP-VprF72L or F72L containing GFP-VprA6-3.