The KH3 and KH4 domains are essential for IMP1 to interact with Gag. (A) Illustration of the IMP1 domain structure. The FLAG tag is attached to the N terminus of IMP1 and its mutants. (B) IMP1 binds to Gag. The FLAG-IMP1 DNA or its mutants were cotransfected with the pVRC-3900 DNA into 293T cells. The FLAG-IMP1 protein and its mutants were immunoprecipitated with anti-FLAG antibody, and the precipitates were assessed by Western blotting with anti-FLAG or anti-p24 antibodies. Expression of FLAG-IMP1 protein and Gag protein from pVRC-3900 DNA in cells was also determined by Western blotting with anti-FLAG and anti-p24 antibodies. The levels of β-actin were measured as the internal control.

Association of IMP1 with HIV-1 particles. (A) The Gag(LZ) mutant does not interact with IMP1. The Gag(LZ) mutant had the NC sequence replaced with the leucine zipper motif from the yeast transcription factor GCN4 (28). The TAP tag was attached to the C termini of Gag and Gag(LZ) in the context of HIV-1 proviral DNA BH10-PR⁻ (protease negative). After affinity purification using IgG-Sepharose and calmodulin-Sepharose, the Gag and Gag(LZ) precipitates were assessed by Western blotting with either anti-p24 antibody or anti-IMP1 antibody. The pN-TAP DNA was used in the experiments as a control. (B) IMP1 was detected in HIV-1 particles. The BH10-PR⁻ and BH10-LZ virus particles were purified by ultracentrifugation through a 15%/65% sucrose step gradient. The amount of virus particles was assessed by Western blotting with anti-p24 antibody. Virion-associated IMP1 protein was detected by Western blotting with anti-IMP1 antibody. (C) Subtilisin treatment of virus samples. The protease-negative HIV-1 particles were purified as described above and subjected to subtilisin digestion. After ultracentrifugation, the pelleted viruses were assessed in Western blot with antibodies to either IMP1, HIV-1 p24, or viral gp120.

Effects of IMP1 overexpression on Gag-membrane association and Gag processing. (A) The FLAG-IMP1 DNA or its mutants was cotransfected with the BH10 DNA into 293T cells. Membrane-associated Gag protein was isolated by membrane flotation assay and detected by Western blotting with anti-p24 antibodies. Membrane fractions 2 and 3 were indicated by the membrane marker TfR. (B) The levels of membrane-bound Gag protein (including pr55, p40, and p24) were quantified from the Western blot data by using ImageJ software and are expressed as the percentage of the total Gag protein in all eight fractions. The amounts of mature p24(CA) protein in the membrane fractions (2 and 3) were also determined and are expressed as the percentage of the total membrane-associated Gag protein (including pr55, p40, and p24). The results shown are the average of three independent flotation experiments. *, P < 0.05.

The NC sequence is important for Gag to bind IMP1. (A) Domain structures of Gag-TAP protein and Gag mutants. The amino acid number refers to the first residue of Gag. The zinc finger motifs in NC are highlighted by filled boxes. CBD, calmodulin-binding domain; TEV, tobacco etch virus protease cleavage site. (B) Gag interacts with IMP1. Gag protein was purified by using IgG-Sepharose and calmodulin-Sepharose as previously described (55). Levels of Gag and its mutants were assessed by Western blotting with anti-p24 antibody. The copurified IMP1 protein was detected by Western blotting with anti-IMP1 antibody. The pN-TAP DNA expresses the TAP protein only and is used in the transfection and purification experiments as a control (55). The lysates of 293T cells were tested in the Western blot as a control to indicate the size of endogenous IMP1.

Gag and IMP1 proteins are colocalized in cells. HeLa cells were transfected with the HIV-1 proviral DNA clone BH10 followed by immunostaining with mouse anti-p24 (red [left panels]) and rabbit anti-IMP1 antibodies (green [middle panels]). The merged images show overlapping of the red and green fluorescence signals at cell edges.

Effects of IMP1 overexpression on HIV-1 production and virus infectivity. (A) The HIV-1 proviral DNA clone BH10 was cotransfected with the wild-type FLAG-IMP1 DNA or the IMP1 mutated DNA into 293T cells. Expression of FLAG-IMP1 and its mutants was determined by Western blotting with anti-FLAG antibody. The levels of Gag protein in cells and in virus particles were assessed by Western blotting with anti-p24 antibody. The amounts of RT proteins in virus particles were also measured by Western blotting with anti-RT(HIV-1) antibody. (B) The amounts of virion p24(CA) were determined by p24 ELISA. The results are summarized in the bar graph, with the amount in the control experiment arbitrarily set as 100. The levels of virion RT products were quantified from the Western blot results by using ImageJ software. The data shown are averages of three independent transfection experiments. *, P < 0.05. (C) Infectivity of HIV-1 virions was measured by infecting TZM-bl indicator cells with 5 ng of p24 of viruses. The results shown represent those from three independent infections using viruses from three different transfection experiments. *, P < 0.05. (D) Native Northern blot to evaluate viral RNA dimerization and level of virion-associated viral RNA. The bar graph shows the average of viral RNA amounts from three independent native Northern blot assays. *, P < 0.05. (E) The stability of viral RNA dimers was measured by incubating viral RNA samples at 40, 45, 50, or 55°C for 10 min in a buffer containing 150 mM NaCl prior to native Northern blot analyses.

Effect of IMP1 overexpression on the morphology of HIV-1 particles. 293T cells were either transiently transfected with the BH10 DNA alone or together with the FLAG-IMP1 DNA or its mutants. Cells were examined by electron microscopy. Two images are shown for the IMP1 overexpression experiment. Scale bar, 200 μm.