Peptides derived from the HIV-1 integrase promote HIV-1 infection and multi-integration of viral cDNA in LEDGF/p75-knockdown cells

Abstract

Background: The presence of the cellular Lens Epithelium Derived Growth Factor p75 (LEDGF/p75) protein is essential for integration of the Human immunodeficiency virus type 1 (HIV-1) cDNA and for efficient virus production. In the absence of LEDGF/p75 very little integration and virus production can be detected, as was demonstrated using LEDGF/p75-knockdown cells.

Results: Here we show that the failure to infect LEDGF/p75-knockdown cells has another reason aside from the lack of LEDGF/p75. It is also due to inhibition of the viral integrase (IN) enzymatic activity by an early expressed viral Rev protein. The formation of an inhibitory Rev-IN complex in virus-infected cells can be disrupted by the addition of three IN-derived, cell-permeable peptides, designated INr (IN derived-Rev interacting peptides) and INS (IN derived-integrase stimulatory peptide). The results of the present work confirm previous results showing that HIV-1 fails to infect LEDGF/p75-knockdown cells. However, in the presence of INrs and INS peptides, relatively high levels of viral cDNA integration as well as productive virus infection were obtained following infection by a wild type (WT) HIV-1 of LEDGF/p75-knockdown cells.

Conclusions: It appears that the lack of integration observed in HIV-1 infected LEDGF/p75-knockdown cells is due mainly to the inhibitory effect of Rev following the formation of a Rev-IN complex. Disruption of this inhibitory complex leads to productive infection in those cells.

Figure 1

INS and INS-derived peptides bind LEDGF/p75 and promote partial dissociation of the IN-LEDGF/p75 complex. (A) LEDGF/p75 was incubated in ELISA plates coated with the indicated peptide or with BSA as a negative control, and binding was determined as described in Methods. Wells containing the buffer carbonate (BC) in the absence of peptide were used as a background control. (B) The IN protein was first bound to the ELISA plates which were then incubated with LEDGF/p75 to obtain LEDGF/p75-IN complex. The complex was then incubated with either the indicated peptide or BSA at the designated IN:peptide (or BSA) ratios. The amount of bound LEDGF/p75 was then determined. (C) Same as (B) but the peptides (or BSA) were added at the same time as LEDGF/p75 to determine competition. (D) Formation of Rev-IN, IN-LEDGF/p75 and Rev-LEDGF/p75 complexes and their dissociation by the INS and INS-derived peptides. Co-IP was performed in lysates obtained from virus-infected cells. All other experimental conditions are described in Methods.

Figure 2

Effect of INS concentrations on integration and total viral-DNA in infected wt and LEDGF/p75-knockdown HeLa-P4 cells. HeLa P4 and HeLaP4/shp75Cl15 (LEDGF/p75-knockdown) cells were incubated with the indicated concentration of INS and infected with wt or ΔRev HIV-1 at a MOI of 1.0. The average number of integration events per cell (A) and of total viral DNA copies per cell (B) was estimated as described in Methods. Black shading and dark grey shading are LEDGF/p75-knockdown cells infected with WT or ΔRev HIV-1, respectively; white grey shading and white shading are HeLa P4 cells infected with WT or ΔRev HIV-1, respectively. AZT was used at 2 μM concentration.

Figure 3

Effect of increasing HIV-1 MOIs on integration levels in infected wt and LEDGF/p75-knockdown HeLa P4 cells. HeLa P4 and HeLaP4/shp75Cl15 (LEDGF/p75-knockdown) cells were incubated with or without 100 μM INS and infected at the indicated MOIs with (A) WT or (B) ΔRev HIV-1. The average number of integration events per cell was estimated as described in Methods. Black shading and dark grey shading are infected LEDGF/p75-knockdown cells without or with INS treatment, respectively; light grey shading and white shading are infected HeLa P4 cells without or with INS treatment, respectively. AZT was used at 2 μM concentration.

Figure 4

INr peptides stimulate viral cDNA integration in LEDGF/p75-knockdown cells. HeLa P4 (□) and HeLaP4/shp75Cl15 (LEDGF/p75-knockdown) (■) cells were incubated with or without 100 μM INS or INr and infected with wt HIV-1 at a MOI of 1.0. AZT was used at 2 μM concentration. The average number of integration events per cell was estimated as described in Methods.

Figure 5

Stimulation of p24 and virus production in LEDGF/p75-knockdown cells by the INS and INr peptides. Sup-T1 and Sup-T1/TL3 (LEDGF/p75-knockdown) cells were incubated with or without 100 μM INS or INr and then infected with wt HIV-1 at a MOI of 0.01. The amount of viral p24 (A) and of infectious virus (B) was estimated every 2 days post-infection (PI) as described in Methods. ■, ● and ▲ represent Sup-T1 cells treated with INS, INr or not treated, respectively; □, ○ and Δ represent LEDGF/p75-knockdown cells treated with INS, INr or not treated, respectively; ◊ are non-infected cells.

Figure 6

Schematic model of the process of viral cDNA integration in wt and LEDGF/p75-knockdown cells. (A) In LEDGF/p75-knockdown cells, an early expressed Rev (green triangle), from unintegrated viral DNA (brown double line), binds and inactivate all viral IN molecules (red circle) (in both the cytoplasm (light blue) and nucleus (yellow)) before integration occurs (due to the absence of LEDGF/p75 which supports rapid integration). (B) Addition of INS or INr peptides promotes dissociation of the inhibitory Rev-IN complexes, allowing the IN, which bound to viral DNA, to mediate integration into the host genome (black double line).