Rationally Designed Interfacial Peptides Are Efficient In Vitro Inhibitors of HIV-1 Capsid Assembly with Antiviral Activity

Abstract

Virus capsid assembly constitutes an attractive target for the development of antiviral therapies; a few experimental inhibitors of this process for HIV-1 and other viruses have been identified by screening compounds or by selection from chemical libraries. As a different, novel approach we have undertaken the rational design of peptides that could act as competitive assembly inhibitors by mimicking capsid structural elements involved in intersubunit interfaces. Several discrete interfaces involved in formation of the mature HIV-1 capsid through polymerization of the capsid protein CA were targeted. We had previously designed a peptide, CAC1, that represents CA helix 9 (a major part of the dimerization interface) and binds the CA C-terminal domain in solution. Here we have mapped the binding site of CAC1, and shown that it substantially overlaps with the CA dimerization interface. We have also rationally modified CAC1 to increase its solubility and CA-binding affinity, and designed four additional peptides that represent CA helical segments involved in other CA interfaces. We found that peptides CAC1, its derivative CAC1M, and H8 (representing CA helix 8) were able to efficiently inhibit the in vitro assembly of the mature HIV-1 capsid. Cocktails of several peptides, including CAC1 or CAC1M plus H8 or CAI (a previously discovered inhibitor of CA polymerization), or CAC1M+H8+CAI, also abolished capsid assembly, even when every peptide was used at lower, sub-inhibitory doses. To provide a preliminary proof that these designed capsid assembly inhibitors could eventually serve as lead compounds for development of anti-HIV-1 agents, they were transported into cultured cells using a cell-penetrating peptide, and tested for antiviral activity. Peptide cocktails that drastically inhibited capsid assembly in vitro were also able to efficiently inhibit HIV-1 infection ex vivo. This study validates a novel, entirely rational approach for the design of capsid assembly interfacial inhibitors that show antiviral activity.

Biophysical Characterization of the Feline Immunodeficiency Virus p24 Capsid Protein Conformation and In Vitro Capsid Assembly

“…The obtention of structural data for FIV p24, or at least its CTD domain (which is the main determinant of p24 oligomerization for HIV) will probably be the next step towards the understanding of FIV capsid assembly. Moreover, getting structural informations on FIV p24 and its dimeric interface would allow the rational design of peptidic inhibitors of FIV capsid assembly, as it has been described for HIV[21]–[23], [59], [61]–[65]. Such compounds could be screened as a first approach in our DLS-based monitoring system of in vitro capsid assembly.…”

“…Disruption of any part of these interactions may be sufficient to suppress the infectivity of HIV [17]. To date, only a few studies have attempted to identify such molecules in HIV-1 and assess their ability to inhibit virus assembly[18]–[23].…”

HIV-1 Capsid Inhibitors as Antiretroviral Agents

“…These 2 peptides bind CA-CTD with binding affinities lower than CAC1, with a K d of 19 ± 8 μM and 8 ± 1μM for CAC1-C and CAC1-M, respectively. NMR spectroscopy on monomeric CA-CTD mutant W184A revealed that CAC1 and CAC1-derivative peptides bind essentially in the same region of the CA-CTD overlapping extensively with helix 9 of the CA-CTD dimerization interface [77]. Both peptides are able to efficiently inhibit in vitro assembly of the mature capsid.…”

“…In order to assess their antiviral activity, CAC1, CAC1-M and H8 were transported into cells using a cell-penetrating peptide. Both peptides showed poor antiviral activity, but when used in combination with other CA-binding peptides (CAC1/CAC1M+H8; CAC1/CAC1M+CAI; CAC1/CAC1M+H8+CAI), they additively inhibited HIV-1 infection [77]. …”

The Antimicrobial and Antiviral Applications of Cell-Penetrating Peptides

“…Peptide-targeting HIV-1 capsid formation has been successfully applied using a commercial CPP-based transfection reagent, Chariot. What makes this development especially interesting is that this is one of the rare occasions upon which the molecule of interest is not conjugated to the transfection peptide covalently but employs non-covalent bonding strategy instead [ 139 ]. The approach to target the capsid of the virus has also been used in HCV inhibition-the capsid has been targeted by R7 in fusion with nucleocapsid binding subunits.…”