Abstract
BackgroundThe C-terminal domain (CTD) of HIV-1 capsid (CA), like full-length CA, forms dimers in solution and CTD dimerization is a major driving force in Gag assembly and maturation. Mutations of the residues at the CTD dimer interface impair virus assembly and render the virus non-infectious. Therefore, the CTD represents a potential target for designing anti-HIV-1 drugs.ResultsDue to the pivotal role of the dimer interface, we reasoned that peptides from the α-helical region of the dimer interface might be effective as decoys to prevent CTD dimer formation. However, these small peptides do not have any structure in solution and they do not penetrate cells. Therefore, we used the hydrocarbon stapling technique to stabilize the α-helical structure and confirmed by confocal microscopy that this modification also made these peptides cell-penetrating. We also confirmed by using isothermal titration calorimetry (ITC), sedimentation equilibrium and NMR that these peptides indeed disrupt dimer formation. In in vitro assembly assays, the peptides inhibited mature-like virus particle formation and specifically inhibited HIV-1 production in cell-based assays. These peptides also showed potent antiviral activity against a large panel of laboratory-adapted and primary isolates, including viral strains resistant to inhibitors of reverse transcriptase and protease.ConclusionsThese preliminary data serve as the foundation for designing small, stable, α-helical peptides and small-molecule inhibitors targeted against the CTD dimer interface. The observation that relatively weak CA binders, such as NYAD-201 and NYAD-202, showed specificity and are able to disrupt the CTD dimer is encouraging for further exploration of a much broader class of antiviral compounds targeting CA. We cannot exclude the possibility that the CA-based peptides described here could elicit additional effects on virus replication not directly linked to their ability to bind CA-CTD.
Highlights
The C-terminal domain (CTD) of HIV-1 capsid (CA), like full-length CA, forms dimers in solution and CTD dimerization is a major driving force in Gag assembly and maturation
Stapled peptides penetrate cells To show that the stapled peptides NYAD-201 and NYAD-233 penetrate cells, we examined cellular uptake of FITC-conjugated NYAD-201 and NYAD-233 by confocal microscopy
The data (Figure 3) demonstrate that the linear peptide does not Stapled peptides enhance dissociation of dimers The effect of dimer interface peptides on the dissociation of CA CTD was investigated by two independent biophysical techniques, isothermal titration calorimetry and analytical centrifugation
Summary
The C-terminal domain (CTD) of HIV-1 capsid (CA), like full-length CA, forms dimers in solution and CTD dimerization is a major driving force in Gag assembly and maturation. The formation of this mature particle is essential for HIV-1 infectivity and the capsid protein (CA) obtained from the Gag cleavage product plays a central role in post-entry steps [1,2,3,4,6,8] Mutations in both the NTD and CTD lead to defects in viral assembly and release [10,11,12,13,14,15,16,17,18,19,20,21]. The CTD plays an important role in viral assembly and maturation and is a potential target for developing a new class of anti-HIV-1 drugs [6,43]
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