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

Rebeca Bocanegra,Adrián Velázquez-Campoy,Javier Gómez,Olga Abián,Miguel Ángel Martínez,Inmaculada López,Alicia Rodríguez-Huete,Mauricio G Mateu,Rosa Doménech,Claudio N Cavasotto,María Nevot,José Luis Neira

doi:10.1371/journal.pone.0023877

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.

Highlights

Virus morphogenesis involves the self-assembly of the viral capsid through specific interactions between protein subunits, which makes this process an excellent target for antiviral research
We found that CAC1 includes a region (CA residues 176 to 184) with a high propensity for amyloid fibril formation, according to the ZipperDB database [58]; this prediction is in qualitative agreement with that of the AMYPbd database [59]
Peptide CAC1 as an efficient assembly inhibitor It may seem surprising that unconstrained, unstructured peptides like CAC1 and derivatives, that represent only a part of the C-terminal domain (CTD) dimerization interface, are able to bind CTD and inhibit assembly of mature human immunodeficiency virus (HIV)-1 capsid-like particles almost as efficiently as the full-length, folded CTD itself

Summary

Introduction

Virus morphogenesis involves the self-assembly of the viral capsid through specific interactions between protein subunits, which makes this process an excellent target for antiviral research. The remarkable knowledge acquired on the structure of viruses and the determinants of molecular recognition has made feasible a complementary antiviral approach, based on the rational design of inhibitors able to competitively interfere with interactions between capsid subunits. Current therapies against HIV involve the use of cocktails of drugs able to inhibit the activity of the viral reverse transcriptase, protease or integrase, or virus entry into cells. Despite the success of this combined approach, the increasing emergence of drug-resistant HIV variants is becoming a serious concern, and demands the development of new anti-HIV agents to be included in future combination therapies Of the HIV-1 capsid is being actively studied to an extraordinary level of detail (recently reviewed in refs. [2,3,4,5]), and there is a growing interest in the investigation of new anti-HIV approaches based on the inhibition of capsid assembly

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Conclusion