Inhibition of HIV-1 integrase nuclear import and replication by a peptide bearing integrase putative nuclear localization signal

Abstract

Background: The integrase (IN) of human immunodeficiency virus type 1 (HIV-1) has been implicated in different steps during viral replication, including nuclear import of the viral pre-integration complex. The exact mechanisms underlying the nuclear import of IN and especially the question of whether it bears a functional nuclear localization signal (NLS) remain controversial.

Results: Here, we studied the nuclear import pathway of IN by using multiple in vivo and in vitro systems. Nuclear import was not observed in an importin alpha temperature-sensitive yeast mutant, indicating an importin alpha-mediated process. Direct interaction between the full-length IN and importin alpha was demonstrated in vivo using bimolecular fluorescence complementation assay (BiFC). Nuclear import studies in yeast cells, with permeabilized mammalian cells, or microinjected cultured mammalian cells strongly suggest that the IN bears a NLS domain located between residues 161 and 173. A peptide bearing this sequence -NLS-IN peptide- inhibited nuclear accumulation of IN in transfected cell-cycle arrested cells. Integration of viral cDNA as well as HIV-1 replication in viral cell-cycle arrested infected cells were blocked by the NLS-IN peptide.

Conclusion: Our present findings support the view that nuclear import of IN occurs via the importin alpha pathway and is promoted by a specific NLS domain. This import could be blocked by NLS-IN peptide, resulting in inhibition of viral infection, confirming the view that nuclear import of the viral pre-integration complex is mediated by viral IN.

Figure 1

Immunostaining experiments for intracellular localization of IN in transfected cells. HeLaP4/IN-expressing cells were generated by stable transfection into HeLaP4 cells using pcDNA3.1 plasmid bearing the full wt IN gene. Cells were fixed and immunostained using 1:100 rabbit a-IN and second antibody, Cy3-conjugated anti-rabbit antibody as described in Methods. Staining of IN (red) and DAPI (blue) was observed under confocal microscope. Bar 10 μm.

Figure 2

Sub-cellular localization of the full-length and truncated IN fused to GFP in transformed yeast cells. (A) Schematic presentation of the various expressed GFP-IN fusion proteins used in this experiment. (B) W303 yeast cells were transformed, using lithium acetate method, with expression vectors coding for the following: GFP-IN, GFP-180-IN, GFP₂-NLS-IN, GFP-152-IN and GFP. Left panel, GFP fluorescence (green); middle panel, DAPI staining (blue); merged fluorescence is shown in the right panel. Bottom, a line profile through the overlay image showing that maximum GFP fluorescence and DAPI staining are co-localized (in the nucleus). Yeast cells were grown to exponential phase in selective minimal medium. After induction with galactose, cells were harvested and GFP fluorescence was observed under confocal microscope; all other conditions were as described in Methods. Bar 7 μm.

Figure 3

Nuclear import mediated by recombinant HIV-1 IN protein: studies with microinjected and permeabilized mammalian cells. Solutions containing the following conjugates: (A) FITC-BSA-IN, (B) FITC-BSA-180-IN, (C) FITC-BSA-NLS-IN, (D) FITC-BSA-152-IN and (E) FITC-BSA*, were microinjected into the cytoplasm of cultured COS-7 cells. All other experimental conditions were as described in Methods. (F) Nuclear import was quantitatively estimated by an ELISA-based assay system. Digitonin-permeabilized Colo-205 cells were incubated for 1 h with Bb-IN, Bb-NLS-IN or Bb-152-IN (4 μg) in a final volume of 40 μL of transport buffer containing ATP regeneration system. The nuclear import experiments were repeated at least three times; data given in the figure represent results obtained from a single experiment. Error bars represent standard deviation which is about +/-5%. Bar 10 μm.

Figure 4

Nuclear import of HIV-1 IN is importin α-dependent. W303 and in srp1-31 yeast cells were transformed with plasmid bearing the full length of the IN fused to GFP (pYES₂yEGFP-IN for the construction of the plasmid see Methods). Following transformation using the lithium acetate method, the fusion protein GFP-IN was expressed in the yeast cells, as described in Methods. GFP fluorescence (green) and DAPI (blue) were observed under confocal microscope following growth of W303 yeast cells at 25°C or at 37°C, or of srp1-31 yeast cells at 25°C or at the non-permissive temperature, 37°C. Bar 5 μm.

Figure 5

IN interaction as observed by the BiFC assay system. EGY48 yeast cells were transformed using the lithium acetate method with plasmids encoding the following combinations: GN-IN and GC-IN, GN-IN and GC-LEDGF, GN-IN and GC-linker (control), GN-IN and GC-Impα (importin α), GN-180-IN and GC-Impα, GN-NLS-IN and GC-Impα, GN-152-IN and GC-Impα, GN-IN and GC-Impβ (importin β), GN-Rev (HIV-1) and GC-Impα. Restoration of GFP fluorescence was observed by confocal microscopy. All other experimental conditions were as described in Methods. Bar 10 μm.

Figure 6

Binding of IN to importin α as estimated by an ELISA-based system. Importin α-coated ELISA plates were incubated with increasing amounts of the following biotinylated conjugates: SV40 (black circles), IN (white diamond), NLS-IN (black squares) and 152-IN (black triangles). The degree of binding was estimated as described in Methods. Error bars represent standard deviation which is about +/-5%.

Figure 7

NLS-IN-Pen inhibits IN nuclear import by the dissociation of IN-importin α interaction in HIV-infected cells. (A) H9 lymphocytes were infected by wild-type HIV-1, and after infection half of the cells' lysate volume was subjected to SDS-PAGE, then immunoblotted with either by anti-IN, anti-importin α (anti-Impα) antibody or an anti-actin antibody. The complementary HRP-conjugated antibodies were used as the second antibody. The remaining lysate or isolated fractions were co-IP with either the anti-Impα or anti-IN antibodies and were immunoblotted with these antibodies, and the complementary HRP-conjugated antibodies as second antibodies. When peptides were used, cells were incubated with 150 μM of the indicated peptide. All others experimental details were as described in Methods. (B) HeLaP4 cells were infected and immunostained as described in Methods. IN (red); DAPI (blue); the area marked in the merge picture was magnified for a better view of IN localization within the infected cell. Bar 10 μm.

Figure 8

NLS-IN-Pen inhibits nuclear import of viral DNA. H9 lymphocytes were infected by wild-type HIV-1 at a MOI of 1; and (A) following infection, the nuclei fraction was isolated from half of the cells, and the amount of viral DNA was estimated using real time PCR method. (B) The amount of 2LTR circles was estimated using real time PCR method. All other experimental details are as described in Methods. Error bars represent standard deviation which is about +/-5%

Figure 9

NLS-IN-Pen peptide inhibits HIV-1. (A) Cell-cycle arrested TZM-b1 cells (non-dividing cells) were incubated with the designated peptides at the indicated concentrations and after HIV-1 infection were tested for β-galactosidase activity. (B) Experimental conditions were as in (A), but with dividing TZM-b1 cells. The number of integration events per cell was determined in cell-cycle arrested (non-dividing) cells (C) or dividing cells (D) following incubation with the designated peptides at different concentrations. Cells were infected with HIV-1 at a MOI of 1 as described in Methods. (E) Inhibition of HIV-1 replication by NLS-IN-Pen as well as SV40-NLS-Pen is dependent on its time of addition. Sup-T1 cells were infected with HIV-1 at a MOI of 2, and the indicated elements were added at different time points after infection (0, 2, 4,..., 24 h). Viral p24 production was determined 48 h PI. Error bars represent standard deviation which is about +/-5%. All other experimental conditions are as described in Methods.