Novel approaches to inhibiting HIV-1 replication

Abstract

Considerable success has been achieved in the treatment of HIV-1 infection, and more than two-dozen antiretroviral drugs are available targeting several distinct steps in the viral replication cycle. However, resistance to these compounds emerges readily, even in the context of combination therapy. Drug toxicity, adverse drug-drug interactions, and accompanying poor patient adherence can also lead to treatment failure. These considerations make continued development of novel antiretroviral therapeutics necessary. In this article, we highlight a number of steps in the HIV-1 replication cycle that represent promising targets for drug discovery. These include lipid raft microdomains, the RNase H activity of the viral enzyme reverse transcriptase, uncoating of the viral core, host cell machinery involved in the integration of the viral DNA into host cell chromatin, virus assembly, maturation, and budding, and the functions of several viral accessory proteins. We discuss the relevant molecular and cell biology, and describe progress to date in developing inhibitors against these novel targets. This article forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development, Vol 85, issue 1, 2010.

Fig. 1

Schematic representation of the HIV-1 replication cycle. Details are provided in the text. Reprinted with permission from Elsevier (Freed, 2004).

Fig. 2

Organization of the HIV-1 genome. The gene products encoded by HIV-1 include the Gag proteins matrix (MA), capsid (CA), nucleocapsid (NC) and p6 and spacer peptides SP1 and SP2; the Pol proteins protease (PR), reverse transcriptase (RT) and integrase (IN); the surface Env glycoprotein gp120 and the transmembrane Env glycoprotein gp41; the regulatory proteins Tat and Rev; and the accessory proteins Vif, Vpr, Vpu, and Nef. Also shown are the 5′ and 3′ long terminal repeats (LTRs).

Fig. 3

Domain organization of TRIM5α and TRIM-Cyp. The major domains of TRIM5α – RING, B-box 2, coiled-coil, and PRYSPRY (B30.2) – are indicated. In TRIM-Cyp, the PRYSPRY (B30.2) domain has been replaced with cyclophilin A (Cyp). Details are provided in the text.

Fig. 4

Structure of HIV-1 RT. The polymerase domain is in yellow, RNase H in orange, and p51 subunit in gray. The polymerase and RNase H active sites are highlighted in red and the RNase H active site is also indicated with an arrow (Das et al., 2008). We thank Kalyan Das and Eddy Arnold for providing the figure.

Fig. 5

Schematic representation of newly synthesized viral DNA tethered to chromatin by LEDGF. The preintegration complex (green) containing a tetramer of IN (yellow) and double-stranded viral DNA (vDNA) is shown. The IN-binding domain (IBD) of LEDGF is shown in association with the IN tetramer. The nuclear localization signal (NLS) and AT hooks (AT) are shown bound to DNA; the PWWP domain is depicted bound to histone proteins. Note that it remains to be established to what extent the PIC is intact at the stage of chromatin tethering.

Fig. 6

Model for MA binding to PI(4,5)P₂. (A) shows the unbound Gag monomer, with the myristic acid moiety (dark green) in the sequestered conformation. Unbound PI(4,5)P₂ is shown with both 1′- and 2′-acyl chains (yellow and purple, respectively) embedded in the inner leaflet of lipid bilayer. (B) shows MA bound to PI(4,5)P₂, with the myristic acid in the exposed conformation and embedded in the lipid bilayer, basic residues of MA (blue) engaged in electrostatic interactions with negative charges on PI(4,5)P₂, and the 2′-acyl chain extruded from the bilayer and packed into a hydrophobic groove in MA. This model is based on the NMR study of Saad et al. (Saad et al., 2006). Reprinted from (Freed, 2006).

Fig. 7

Structure of HIV-1 CA. (A) Structure of monomeric CA, with the CA_NTD (green) and CA_CTD (blue/green) indicated. The interdomain linker, N- and C-termini, and cyclophilin A binding loop are shown. Helices 1-11 and the N-terminal β-hairpin (yellow) are labeled. Binding sites for CAP1 and CAI/NYAD-1/NYAD-13 are indicated by red and black arrows, respectively. Reprinted with permission from Elsevier (Ganser-Pornillos, Yeager, and Sundquist, 2008). (B) Molecular model of the CA_NTD hexameric ring; cyclophilin A binding loop indicated with an arrow. (C) Outside view of an assembled CA tube, showing the CA hexameric lattice. One CA hexamer is shown in yellow. Scale bar = 100 Å. (D) Molecular model of an HIV-1 conical core. A line of hexamers is shown in yellow; pentamers are depicted in red at each end of the conical core. Adapted with permission from Macmillan Publishers Ltd: [Nature], (Li et al., 2000), http://www.nature.com/nature/index.html.

Fig. 8

Role of ESCRT and associated machinery in the sorting of cargo proteins to multivesicular bodies (MVBs) and in virus release. At the bottom is depicted the interaction of ubiquitinated (Ub) cargo protein with the STAM/Hrs complex and ESCRT-I, II, and III and the delivery of the cargo protein into a vesicles budding inwardly into the MVB. At the top is depicted the interaction of Gag with ESCRT-I and Alix and the involvement of ESCRT-III and the ATPase Vps4 in HIV-1 budding from the plasma membrane. The major Gag domains – MA, CA, NC, and p6 – are indicated. For additional details, see text, and (Fujii, Hurley, and Freed, 2007). Adapted with permission from Macmillan Publishers Ltd: [Nature Reviews Microbiology], (Fujii, Hurley, and Freed, 2007), http://www.nature.com/nrmicro/index.html.

Fig. 9

Structure of the ubiquitin enzyme 2 variant (UEV) domain of Tsg101 bound to a PTAP-containing peptide. (a) The structure of the UEV domain is shown in yellow and gray, the PTAP-containing peptide (C, C-terminus; N, N-terminus) is shown in dark green. High-resolution structure of the first Pro (b) and Ala-Pro (c) of PTAP docked in the PTAP-binding groove of Tsg101, viewed from the N-terminus of the peptide. Reprinted with permission form Macmillan Publishers Ltd: [Nature Structural and Molecular Biology], (Pornillos et al., 2002a), http://www.nature.com/nsmb/index.html.

Fig. 10

Inhibition of HIV-1 maturation by bevirimat. (A) Gag processing cascade, illustrating the order in which the Gag precursor (Pr55^Gag) is cleaved by the viral protease. Red arrow depicts the cleavage event blocked by bevirimat, leading to an accumulation of the CA-SP1 cleavage intermediate. (B) Virion morphology visualized by transmission electron microscopy. Immature (i), mature (ii), and bevirimat-treated (iii) particles are shown. (C) Structure of bevirimat. (D) Amino acid sequence at the CA-SP1 boundary region; the final residue of CA (residue 231) and the first (1) and final (14) residues of SP1 are shown. Amino acids highlighted in red indicate those at which BVM resistance arises in vitro (Adamson et al., 2006); the highly polymorphic SP1 residues 6, 7, and 8 are highlighted in green. Arrows denote the site of CA-SP1 processing. Adapted with permission from Elsevier (Adamson and Freed, 2008).

Fig. 10

Inhibition of HIV-1 maturation by bevirimat. (A) Gag processing cascade, illustrating the order in which the Gag precursor (Pr55^Gag) is cleaved by the viral protease. Red arrow depicts the cleavage event blocked by bevirimat, leading to an accumulation of the CA-SP1 cleavage intermediate. (B) Virion morphology visualized by transmission electron microscopy. Immature (i), mature (ii), and bevirimat-treated (iii) particles are shown. (C) Structure of bevirimat. (D) Amino acid sequence at the CA-SP1 boundary region; the final residue of CA (residue 231) and the first (1) and final (14) residues of SP1 are shown. Amino acids highlighted in red indicate those at which BVM resistance arises in vitro (Adamson et al., 2006); the highly polymorphic SP1 residues 6, 7, and 8 are highlighted in green. Arrows denote the site of CA-SP1 processing. Adapted with permission from Elsevier (Adamson and Freed, 2008).

Fig. 11

Schematic representation of the counteraction of APOBEC3G by Vif. In the Vif+ setting (top), Vif (yellow) induces the proteasomal degradation of APOBEC3G (red star) in the virus-producing cell (left), enabling productive infection to occur in the target cell (right). In the absence of Vif expression (bottom), APOBEC3G is packaged into virus particles, and in the next round of infection induces the deamination of cytosines to uracils, resulting in G-to-A hypermutation. The presence of APOBEC3G also impairs reverse transcription and integration. Reprinted with permission from Elsevier (Freed, 2004).

Fig. 12

Hypothetical models for the tethering of HIV-1 virions to the cell surface by CD317/BST-2/tetherin. The virion is shown in green, with core in blue and viral RNA in red. BST-2/CD317/tetherin is shown on the left anchored in the lipid bilayer of the plasma membrane, with the cytoplasmic tail (CT), transmembrane ™, and coiled-coil (CC) domains and the GPI anchor indicated. In model (i), two molecules of BST-2/CD317/tetherin are aligned in parallel, with the TM domains in the producer cell plasma membrane and the GPI anchors in the viral membrane. In model (ii), one molecule is embedded in the plasma membrane, the other in the viral membrane. The two molecules associate via their coiled-coil domains.