Peptides that bind the HIV‐1 integrase and modulate its enzymatic activity – kinetic studies and mode of action

Abstract

Several peptides that specifically bind the HIV-1 integrase (IN) and either inhibit or stimulate its enzymatic activity were developed in our laboratories. Kinetic studies using 3¢-end processing and strand-transfer assays were performed to study the mode of action of these peptides. The effects of the various peptides on the interaction between IN and its substrate DNA were also studied by fluorescence anisotropy. On the basis of our results, we divided these IN-interacting peptides into three groups: (a) IN-inhibitory peptides, whose binding to IN decrease its affinity for the substrate DNA -these peptides increased the K m of the IN-DNA interaction, and were thus inhibitory; (b) peptides that slightly increased the K m of the IN-DNA interaction, but in addition modified the V max and K cat values of the IN, and thus stimulated or inhibited IN activity, respectively; and (c) peptides that bound IN but had no effect on its enzymatic activity. We elucidated the approximate binding sites of the peptides in the structure of IN, providing structural insights into their mechanism of action. The IN-stimulating peptide bound IN in several specific sites that did not bind any of the inhibitory peptides. This may account for its unique activity. Abbreviations IN, integrase; INS, integrase-stimulating peptide; LEDGF, lens epithelium-derived growth factor; LTR, long terminal repeat; PFV, prototype foamy virus; Y2H, yeast two-hybrid.

Interactions of HIV-1 Proteins as Targets for Developing Anti-HIV-1 Peptides

“…VESMNEELKKIIAQVRAQAEHLKTAY [130][131][132][133][134][135][136][137][138][139] (B) Cellular partner proteins LEDGF/p75 354-378 WIHAEIKNSLKIDNLDVNRCIEALD [69,[153][154][155] LEDGF/p75 355-377 IHAEIKNSLKIDNLDVNRCIEAL [69,[153][154][155] LEDGF/p75 361-370 NSLKIDNLDV [69,[153][154][155][156] LEDGF/p75 362-369 SLKIDNLD [69,[153][154][155] LEDGF/p75 402-413 KKIRRFVSQVIM [69,[153][154][155][156] (C) Phage display CP64 c(CVSGHPLWCGGGK) [158] CP65 c(CILGHSDWCGGGK) [158] Targeting host proteins is risky since it may affect cell viability and produce undesired toxicity. Therefore, the best strategy is to study the interactions between IN and host proteins by finding peptides derived from the INbinding region of the cellular proteins.…”

“…In addition, these peptides inhibited the integration of viral cDNA and HIV-1 replication in infected cells, by shifting the IN oligomerization equilibrium toward a stable tetramer in the cytosol. Further studies including homology modeling, alanine scan and NMR analysis revealed that all the residues of LEDGF/p75 361-370 and LEDGF/p75 402-413 are important for optimal binding and inhibition of IN ( Figure 5B) [69,[153][154][155][156]. A library of cyclic peptides (CPs) derived from LEDGF/p75 361-370 was screened for in vitro IN binding and inhibition [155].…”

“…A3G bound nine Vif-derived peptides from three distinct regions in Vif: residues 8-45 from the NTD, residues 154-192 from the CTD containing the conserved motif 161 PPLP 164 and a central region between residues 83-99 [230,231]. The A3G-derived peptides A3G [143][144][145][146][147][148][149][150][151][152][153][154][155][156][157] , A3G and A3G [263][264][265][266][267][268][269][270][271][272][273][274][275][276][277] bound fulllength Vif and Vif-CTD. The peptide array experiment revealed that peptides A3G 31-52 , A3G [166][167][168][169][170][171][172][173][174][175][176][177][178][179][180] , A3G 211-...…”

Monitoring the HIV-1 integrase enzymatic activity using atomic force microscopy in a 2LTR system

“…23,24 In our lab, we developed the LEDGF 361-370 peptide, derived from the IN-binding loop of LEDGF, which inhibited IN catalytic activity in vitro and in cells and HIV-1 replication in cells and in a mouse model. [25][26][27] We performed in vitro integration reaction in the presence of LEDGF and LEDGF 361-370. The incubation time was 2 hours at 37 1C and DNA with viral U3 and U5 LTRs was used in all cases.…”

“…S5A, ESI †), indicating that LEDGF 361-370 inhibited the IN activity, consistent with previous reports. 26,27 When the LEDGF protein was added to the reaction mixture, the 1500 bp band intensity increased (Fig. S5B, ESI †) compared to the reaction in the absence of this protein, indicating that the IN enzymatic activity was indeed stimulated.…”