Figure 1.
INrs interact with Rev-GFP and Rev-derived peptides in vitro and promote dissociation of Rev-IN complexes.
(A) Rev-GFP or GFP alone were incubated in ELISA plates coated with either INr-1 (Green full and empty diamond respectively) or INr-2 (Magenta full and empty square respectively) and binding was determined as described in Materials and Methods. IN-Rev-GFP interaction was used as positive control (Red triangle) (B) The IN protein was first bound to the ELISA plate and then incubated with Rev-GFP to obtain a Rev-IN complex. The complex was then incubated with either one or both of the INr peptides at the designated Rev-GFP∶peptide ratio. Wells were then washed and the amount of bound Rev-GFP was determined by mouse anti-GFP antibody and secondary rabbit anti-mouse HRP-conjugated antibody. (C) Rev 13–23 and Rev 53–67 (conjugated to biotinylated-BSA) were incubated in ELISA plates coated with either INr-1 (Green empty and full diamond respectively) or INr-2 (Magenta full and empty square respectively) and binding was estimated as described in Materials and Methods. All other experimental conditions were as described previously [37], [55] and in Materials and Methods. (D) Localization of INr-1 (green) and INr-2 (magenta) peptides within the IN protein was determined using a protein model (PDB: 1EX4 [35]). A clear model is shown on the left to illustrate the exact domains of INr-1 and INr-2 within the IN protein. Both models, left and right, emphasize exposure of the INr sequences on the protein surface. The models presented in this figure were generated using PyMOL v0.99 software (DeLano Scientific LLC).
Table 1.
Interaction of IN peptides derived from an IN peptide library, with Rev-GFP or Rev-derived peptides.
Table 2.
Quantitative characterization of the interaction between the INrs and Rev-GFP or Rev-derived peptides.
Figure 2.
Inhibition of IN enzymatic activity by Rev and its abrogation by the INr peptides.
(A) Rev-GFP (dark green) or GFP (turquoise) were incubated at different molar ratios with IN (390 nM) and the IN enzymatic activity was determined as described previously [22] and in Materials and Methods. (B) Rev-GFP was preincubated with the indicated INr peptides at a molar ratio of 5∶1 (peptide∶Rev-GFP) and the resultant mixture was added to a solution containing IN to give a molar ratio of 1∶200 and 1∶400 (IN∶Rev-GFP). Following an incubation period, IN activity was estimated as described previously [22]. All other experimental conditions are described in Materials and Methods.
Figure 3.
Inhibition of IN activity by Rev- and LEDGF-derived peptides: specific abrogation by the INr peptides.
IN (390 nM) was incubated with 50 µM of the Rev-derived peptides (1∶300 IN∶Rev-derived peptide) [22] (Rev13–23 (A) and Rev 53–67 (B)) or LEDGF-derived peptides [36] (LEDGF 361–370 (C)) and LEDGF 402–411 (D)) and then the INr peptides were added at different molar ratios ranging from 10∶1 to 1∶10 (Rev or LEDGF peptide∶INr peptide). (E) IN (390 nM) was incubated with the INr peptides (50 µM).
Figure 4.
Cell penetration and toxicity of INr peptides.
(A) Fluorescein-labeled INr-1, INr-2, and IN-3 (a known nonpermeable peptide [37]) each at 10 µM, were incubated for 2 h at 37°C with HeLa cells. The cells were then washed three times with PBS and visualized by confocal microscopy. (B) Cell toxicity was determined by MTT assay in HeLa TZM-bl cells. (C) Same as (B) but in H9 lymphocytes. Experimental conditions are described in Materials and Methods.
Figure 5.
Inhibition of HIV-1 infection by the Rev-derived peptides and abrogation of inhibition by INrs.
HeLa MAGI TZM-bl cells were incubated with Rev-derived (12.5 µM of Rev 13–23 or Rev 53–67) and INr (62.5 µM of INr-1 or INr-2) peptides and infected with HIV-1 Delta-env/VSV-G. at different orders of addition as indicated by arrows, A) Rev peptide>INr>HIV-1; (B) INr>Rev peptide>HIV-1; (C) Rev peptide>HIV-1>INr; (D) INr>HIV-1>Rev peptide; (E) HIV-1>Rev peptide>INr; (F) HIV-1>INr>Rev peptide. Control cells were incubated with none or only one of the indicated peptides. The length of the incubation period with each of the above-described components (HIV-1, Rev and INr peptides) was 2 h at 37°C and at the end of the total 6-h incubation, the infected cells were left for a further 48-h incubation. At the end of the incubation period, the number of blue cells per well was estimated as described in Materials and Methods. (A summary of the results is presented in Table 3).
Table 3.
Inhibition of HIV-1 infection by the Rev-derived peptides and its abrogation by the INr peptides are not affected by order of addition: a summary of the results described in Figure 5.
Figure 6.
INr peptides stimulate HIV-infection of LC5-RIC cultures.
LC5-RIC reporter cells contain an integrated reporter gene for HIV-dependent production of a red fluorescent protein. LC5-RIC cultures were incubated with HIV-1 IIIB and peptide INr-1 (A) or INr-2 (B) at the indicated concentrations and the fluorescent intensities of the cultures measured after the indicated time periods. The graph shows the overall fluorescent intensities measured in peptide-containing cultures relative to HIV-exposed cultures incubated without peptide (solvent only) for the same time period.
Figure 7.
Stimulation of HIV-1 replication by the INrs.
The INrs (12.5 µM) were incubated with H9 cells (A and C) and Sup-T1 lymphocyte cells (B and D) which were then infected with HIV-1 (MOI 0.01) as described in Materials and Methods. The amount of viral P24 (A and B) and virus titer (C and D) were determined every 2 days by ELISA and MAGI assay [49], respectively. All other experimental conditions are described in Materials and Methods. The concentration of AZT used was 2 µM.
Figure 8.
Virus-cell adsorption and viral reverse-transcriptase activity are not affected by the INr peptides.
(A and B) Estimation of viral DNA in virus-infected cells: Sup-T1 cells were incubated with the indicated peptides or with AZT for 2 h and then were infected with HIV. Following another 6 h incubation, the viral Gag (A) or Nef (B) DNA sequences were amplified using specific primers. The reaction was terminated after 30 cycles. Lanes: 1) INr-1 (12.5 µM); 2) INr-2 (12.5 µM); 3) AZT (2 µM); 4) untreated cells; 5) uninfected cells. Note that in the AZT-treated cells, reverse-transcribed viral DNA is absent. (C) Influence of the time of addition on P24 production [59]: Sup-T1 cells were infected with HIV-1 at a MOI of 2, and the indicated constituents were added at different time points PI (0, 2, 4,…, 24 h). Viral p24 was determined at 48 h PI. No addition (Blue X); no virus (Gray diamond); dextran sulfate 20 µM (Brown asterisk); AZT 2 µM (Red empty circle); INr-1 12.5 µM (Green diamond); INr-2 12.5 µM (Magenta square); LEDGF 402–411 (Black full circle). All other experimental conditions are described in Materials and Methods.
Figure 9.
Stimulation of viral DNA integration events by the INr peptides.
H9 T-lymphoid cells were incubated with the indicated peptides (12.5 µM) or AZT (2 µM) and following HIV-1 infection, the integrated viral DNA/cell was assessed by real-time PCR following: (A) a single cycle of infection at a MOI of 5 of Delta-env/VSV-G HIV-1 or (B) 8 days PI at a MOI of 0.01 of wild-type HIV-1. All other experimental conditions are described in Materials and Methods and in Ref. [22].