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

Abstract

BackgroundThe 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.ResultsHere, 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 α temperature-sensitive yeast mutant, indicating an importin α-mediated process. Direct interaction between the full-length IN and importin α 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.ConclusionOur present findings support the view that nuclear import of IN occurs via the importin α 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.

Transportin 3 and importin α are required for effective nuclear import of HIV-1 integrase in virus-infected cells

“…Several cellular nuclear-import receptors have been suggested to be involved in the process of PIC nuclear import. Among them are the importin α/β heterodimer, [14][15][16][17][18] importin 7 19,20 and transportin 3 (TPNO3/transportin-SR2, an importin β-like receptor). [21][22][23] Various viral karyophilic proteins, such as the Matrix (MA), Vpr and IN, 3,[24][25][26][27][28][29][30] have been suggested to actively translocate the PIC into the host-cell nucleus.…”

“…33,34 However, the exact mechanism governing nuclear import of the PIC remains unclear. 3,[11][12][13][14]25,27,28,30 Our previous works, 17,18 as well as that of others, 14,[35][36][37][38][39] have demonstrated that the HIV-1 IN is a karyophilic protein that IN interacts with both importin α and TNPO3. These results suggest that these two nuclear-import receptors are involved in the translocation of IN into nuclei of infected cells, either concurrently or alternately.…”

“…18 and see also Figure 1B and C) that cell-permeable peptides bearing the SV40-NLS or the putative NLS of IN (NLS-IN) can disrupt the complex formed between IN and importin α and inhibit translocation of IN into the nuclei of infected cells. As shown in Figure 1B and C, these two peptides failed to promote dissociation of the IN-TNPO3 complex, indicating that different domains mediate the interaction of IN with these two nuclearimport receptors.…”

“…14,[35][36][37][38][39] Based on the use of cell-permeable functional peptides, our work supports the view that IN has a dominant role in driving a functional PIC into infected cells' nuclei. 18 This was inferred mainly from experiments showing that a peptide bearing a sequence suggested to be the IN NLS (NLS-IN, comprised of IN residues 161-173), as well as the SV40-T-antigen NLS peptide (SV40-NLS), block nuclear import of IN in IN-transfected and HIV-infected cells. 14,17,18 Concurrent with inhibition of IN nuclear import, these two peptides blocked HIV-1 replication in virus-infected cells.…”

“…18 This was inferred mainly from experiments showing that a peptide bearing a sequence suggested to be the IN NLS (NLS-IN, comprised of IN residues 161-173), as well as the SV40-T-antigen NLS peptide (SV40-NLS), block nuclear import of IN in IN-transfected and HIV-infected cells. 14,17,18 Concurrent with inhibition of IN nuclear import, these two peptides blocked HIV-1 replication in virus-infected cells. 18 Inhibition was due to these peptides' ability to disrupt the interaction of IN with importin α, 17,18 supporting the view that this nuclear-import receptor is required for virus replication in cell cycle-arrested cells.…”