Virus maturation as a new HIV-1 therapeutic target

Abstract

Development of novel therapeutic targets against HIV-1 is a high research priority owing to the serious clinical consequences associated with acquisition of resistance to current antiretroviral drugs. The HIV-1 structural protein Gag represents a potential new therapeutic target as it plays a central role in virus particle production yet is not targeted by any of the antiretroviral drugs approved at present. The Gag polyprotein precursor multimerizes to form immature particles that bud from the infected cell. Concomitant with virus release, the Gag precursor undergoes proteolytic processing by the viral protease to generate the mature Gag proteins, which include capsid (CA). Once liberated from the Gag polyprotein precursor, CA molecules interact to reassemble into a condensed conical core, which organizes the viral RNA genome and several viral proteins to facilitate virus replication in the next round of infection. Correct Gag proteolytic processing and core assembly are therefore essential for virus infectivity. In this review, we discuss new strategies to inhibit maturation by targeting proteolytic cleavage sites in Gag or CA-CA interactions required for core formation. The identification and development of lead maturation inhibitors are highlighted.

Figure 1

(A) The HIV-1 Gag proteolytic processing cascade. The Gag polyprotein is shown at the top with the matrix (MA), capsid (CA), nucleocapsid (NC) and p6 domains and two spacer peptides SP1 and SP2 indicated. The order of processing events is depicted by the flow diagram, with arrows indicating PR cleavage sites. The red arrow denotes the cleavage event blocked by 3-O-(3’,3’-dimethylsuccinyl) betulinic acid (bevirimat, PA-457, DSB, MPC-4326). (B) Immature HIV-1 particle showing donut-like morphology (i); mature HIV-1 virion with condensed CA conical core (ii); HIV-1 virion produced in the presence of bevirimat, showing aberrant core and crescent of electron density inside the viral lipid bilayer (iii). (C) Chemical structure of bevirimat. Reprinted from Drugs Discovery Today, Vol 13 (9/10), Catherine S Adamson and Eric O Freed, Recent progress on antiretrovirals – lessons from resistance, pages 424–432, Copyright (2008), with permission from Elsevier (18).

Figure 2. Location of bevirimat-resistance mutations

(A) Gag is represented at the top, with MA, CA, SP1, NC, SP2 and p6 indicated. The alignment shows a panel of six single-amino-acid substitutions that independently confer bevirimat resistance. The mutations, which were selected in vitro (Adamson 2006), cluster at or near the CA-SP1 cleavage site (arrow). (B) Amino acid sequence alignment across the CA-SP1 boundary region. The alignment was constructed from the 2004 Los Alamos HIV-1 sequence database group M consensus sequences, HIV-2 ROD and SIVmac239 (123) http://www.hiv.lanl.gov. Green arrows indicate PR cleavage sites, red arrows indicate amino acid positions to which bevirimat resistance maps in vitro and blue arrows indicate polymorphic amino acid positions correlated with variable clinical responses to bevirimat in HIV-1 infected patients. Adapted and Reprinted from Journal of Virology, Copyright (2006), vol 80 (22), pages 10957–10971 DOI:10.1128/JVI.01369-06 and reproduced/amended with permission from American Society for Microbiology (50).

Figure 2. Location of bevirimat-resistance mutations

(A) Gag is represented at the top, with MA, CA, SP1, NC, SP2 and p6 indicated. The alignment shows a panel of six single-amino-acid substitutions that independently confer bevirimat resistance. The mutations, which were selected in vitro (Adamson 2006), cluster at or near the CA-SP1 cleavage site (arrow). (B) Amino acid sequence alignment across the CA-SP1 boundary region. The alignment was constructed from the 2004 Los Alamos HIV-1 sequence database group M consensus sequences, HIV-2 ROD and SIVmac239 (123) http://www.hiv.lanl.gov. Green arrows indicate PR cleavage sites, red arrows indicate amino acid positions to which bevirimat resistance maps in vitro and blue arrows indicate polymorphic amino acid positions correlated with variable clinical responses to bevirimat in HIV-1 infected patients. Adapted and Reprinted from Journal of Virology, Copyright (2006), vol 80 (22), pages 10957–10971 DOI:10.1128/JVI.01369-06 and reproduced/amended with permission from American Society for Microbiology (50).

Figure 3

Image reconstructions of helical assemblies of HIV-1 CA. (A) Molecular model of the hexameric ring formed by CA_NTD. Structural features: Cyclophilin A-binding loop (arrow), β-hairpin (orange), helix 1 (red), helix 2 (yellow), helix 3 (green), helix 4 (cyan), helix 5 (dark blue), helix 6 (red), helix 7 (pink). (B) Exterior view of the assembled tube structure showing a single hexamer (yellow) and the hexagonal CA lattice (blue). Scale bar = 100 Å (C) Model of an HIV-1 conical core. A continuous line of hexamers is highlighted in yellow and pentamers are shown in pink. Adapted with permission from Macmillam Publishers Ltd: [Nature], Vol 407, Su Li, Christopher P. Hill, Wesley I. Sundquist & John T. Finch. Image reconstructions of helical assemblies of the HIV-1 CA protein, pages 409–413, Copyright (2000), http://www.nature.com/nature/index.html, (88) and reprinted with permission from Freed and Martin 2007 (124).

Figure 4. Binding sites of CA-based maturation inhibitors

(A) Structure of monomeric CA with the CA_NTD (green) and CA_CTD (blue) depicted and separated by the flexible linker region. The C-terminus is indicated as a dashed line to represent its disordered nature in crystal structures. Secondary structural features are labeled: N-terminal β-hairpin (yellow), cyclophilin loop and helices 1– 11 (H1-11). The site of binding of CAP1 is shown with a red arrow; the binding site of CAI/NYAD-1/NYAD-13 is indicated by a black arrow. Reprinted from Current Opinion in Structural Biology, vol 18. Barbie K Ganser-Pornillos, Mark Yeager and Wesley I Sundquist, The structural biology of HIV assembly, pages 203–217, Copyright (2008), with permission from Elsevier (28). (B) Electrostatic representation of CAP-1 bound to CA_NTD. The aromatic ring of CAP-1 is shown inserted into the pocket vacated by Phe32 upon CAP-1 binding. Reprinted from Journal of Molecular Biology, Vol 378, Brian N. Kelly, Sampson Kyere, Isaac Kinde, Chun Tang, Bruce R. Howard, Howard Robinson, Wesley I. Sundquist, Michael F. Summers and Christopher P. Hill, Structure of the antiviral assemble inhibitor CAP-1 complex with the HIV-1 CA Protein, pages 355–366, Copyright (2007) with permission from Elsevier (112). (C) Electrostatic representation of CAI bound to CA_CTD. CAI in a helical conformation is shown bound to the hydrophobic groove (white). Reprinted with permission from Ternois et al., 2005 (114).