Detailed Mechanistic Insights into HIV-1 Sensitivity to Three Generations of Fusion Inhibitors

Peptides based on the second heptad repeat (HR2) of viral class I fusion proteins are effective inhibitors of virus entry. One such fusion inhibitor has been approved for treatment of human immunodeficiency virus-1 (T20, enfuvirtide). Resistance to T20 usually maps to the peptide binding site in HR1. To better understand fusion inhibitor potency and resistance, we combined virological, computational, and biophysical experiments with comprehensive mutational analyses and tested resistance to T20 and second and third generation inhibitors (T1249 and T2635). We found that most amino acid substitutions caused resistance to the first generation peptide T20. Only charged amino acids caused resistance to T1249, and none caused resistance to T2635. Depending on the drug, we can distinguish four mechanisms of drug resistance: reduced contact, steric obstruction, electrostatic repulsion, and electrostatic attraction. Implications for the design of novel antiviral peptide inhibitors are discussed.The HIV-1 envelope glycoprotein complex (Env),³ a class I viral fusion protein, is responsible for viral attachment to CD4⁺ target T cells and subsequent fusion of viral and cellular membranes resulting in release of the viral core in the cell. Other examples of viruses using class I fusion proteins are Coronaviridae (severe acute respiratory syndrome virus), Paramyxoviridae (Newcastle disease virus, human respiratory syncytial virus, Nipah virus, Hendra virus), and Orthomyxoviridae (influenza virus), some of which cause fatal diseases in humans (1–3). The entry process of these viruses is an attractive target for therapeutic intervention.The functional trimeric Env spike on HIV-1 virions consists of three gp120 and three gp41 molecules that are the products of cleavage of the precursor gp160 by cellular proteases such as furin (4, 5). The gp120 surface subunits are responsible for binding to the cellular receptors, whereas the gp41 subunits anchor the complex in the viral membrane and mediate the fusion of viral and cellular membranes. Env undergoes several conformational changes that culminate in membrane fusion. The gp120 subunit binds the CD4 receptor, resulting in creation and/or exposure of the binding site for a coreceptor, usually CCR5 or CXCR4 (6, 7). Two α-helical leucine zipper-like motifs, heptad repeat 1 (HR1) and heptad repeat 2 (HR2), located in the extracellular part of gp41, play a major role in the following conformational changes. Binding of the receptors to gp120 induces formation of the pre-hairpin intermediate of gp41 in which HR1 is exposed and the N-terminal fusion peptide is inserted into the target cell membrane (1, 8–12). Subsequently, three HR1 and three HR2 domains assemble into a highly stable six-helix bundle structure that juxtaposes the viral and cellular membranes for the membrane merger. Other viruses with class I viral fusion proteins use similar HR1-HR2-mediated membrane fusion for target cell entry.Peptides based on the HR domains of class I viral fusion proteins have proven to be efficient inhibitors of virus entry for a broad range of viruses (13–17). The HIV-1 fusion inhibitor T20 (enfuvirtide (Fuzeon)) has been approved for clinical use. T20 mimics HR2 and can bind to HR1, thereby preventing the formation of the six-helix bundle (Fig. 1) (18–21). T1249 is a second-generation fusion inhibitor with improved antiviral potency compared with the first-generation peptide T20 (22–25). Recently, a series of more potent third-generation fusion inhibitors were designed (26, 27). These include T2635, which has an improved helical structure that increases stability and activity against both wild type (WT) HIV-1 and fusion inhibitor resistant variants.Open in a separate windowFIGURE 1.Schematic of the gp41 ectodomain. HR1 and HR2 are represented as cylinders, and position 38 in HR1 is indicated. Residues Gln-142, Asn-145, Glu-146, and Leu-149, which interact with residue 38, are underlined in the HR2 sequence. HR2-based peptide fusion inhibitors are shown underneath. Mutations introduced in T1249^mut and T2635^mut are bold and underlined. Numbering is based on the sequence of HXB2 gp41.Both the in vitro and in vivo selection of resistance has been described for T20 (28–33) and T1249 (23, 34–36). Resistance is often caused by mutations in the HR1 binding site of the fusion inhibitor. In particular, substitutions at positions 36 (G36D/M/S), 38 (V38A/W/M/E), and 43 (N43D/K) of gp41 can cause resistance. Strikingly, substitutions at position 38 can cause resistance to both T20 and T1249, but distinct amino acid substitutions are required. At position 38 only charged amino acids (V38E/R/K) cause resistance to T1249 (35). Surprisingly, none of the known T20 and T1249 resistance mutations at position 38 affect the susceptibility to the third generation inhibitor T2635.We hypothesized that the use of HIV-1 as a model system could provide a more detailed understanding of resistance to fusion inhibitors. We, therefore, analyzed the effect of all 20 amino acids at resistance hotspot 38 on Env function, viral fitness, biochemical properties of gp41, and resistance to the fusion inhibitors. From the results we can propose four resistance mechanisms that differ in the way the drug-target interaction is affected at the molecular level. Furthermore, we can deduce general principles on the mechanisms of resistance against fusion inhibitors and the requirements for effective antiviral drugs.