Genetic and Phenotypic Analyses of Human Immunodeficiency Virus Type 1 Escape from a Small-Molecule CCR5 Inhibitor

Abstract

, Proc. Natl. Acad. Sci. USA 99:395-400, 2002). The escape mutant, CC101.19, continued to use CCR5 for entry, but it was at least 20,000-fold more resistant to AD101 than the parental virus, CC1/85. We have now cloned the env genes from the the parental and escape mutant isolates and made chimeric infectious molecular clones that fully recapitulate the phenotypes of the corresponding isolates. Sequence analysis of the evolution of the escape mutants suggested that the most relevant changes were likely to be in the V3 loop of the gp120 glycoprotein. We therefore made a series of mutant viruses and found that full AD101 resistance was conferred by four amino acid changes in V3. Each change individually caused partial resistance when they were introduced into the V3 loop of a CC1/85 clone, but their impact was dependent on the gp120 context in which they were made. We assume that these amino acid changes alter how the HIV-1 Env complex interacts with CCR5. Perhaps unexpectedly, given the complete dependence of the escape mutant on CCR5 for entry, monomeric gp120 proteins expressed from clones of the fully resistant isolate failed to bind to CCR5 on the surface of L1.2-CCR5 cells under conditions where gp120 proteins from the parental virus and a partially AD101-resistant virus bound strongly. Hence, the full impact of the V3 substitutions may only be apparent at the level of the native Env complex.Several members of a new class of inhibitors based on blocking human immunodeficiency virus type 1 (HIV-1) entry into target cells are now in, or approaching, human clinical trials (8,52,77,80,85,90,98). These various compounds antagonize different stages in the multistep pathway by which HIV-1 fuses with susceptible cells. For example, the CD4-immunoglobulin G2 protein (CD4-IgG2; PRO 542) attaches to the viral envelope glycoprotein gp120 to prevent it from interacting with the CD4 receptor (3, 109). The T-20 and T-1249 peptides act later, by inhibiting conformational changes in the viral gp41 glycoprotein that are necessary for membrane fusion to be initiated (8,40,72,115). All these inhibitors cause plasma viremia reductions in HIV-1-infected people (8,49,53,62,66,71,80).A step in the entry process intermediate between gp120-CD4 attachment and gp41 conformational changes involves a coreceptor for gp120 (21,31,37,85,98). Thus, after gp120 has bound to CD4, it changes conformation to enable it to bind to a coreceptor from the G-protein-coupled receptor superfamily (21,31,37,85,98,107,116). The most physiologically relevant coreceptors are the chemokine receptors CCR5 or CXCR4, the former used by HIV-1 strains that usually dominate early in infection and the latter used by viruses that sometimes emerge several years later or that are detectable only transiently (21,31,85,99,122). The presence of viruses able to use CXCR4 (X4 strains) is associated with an accelerated disease course, due in part to the loss of naive CD4 ϩ T cells that express CXCR4 but not CCR5 (32,44,69). Viruses using CCR5 (R5 strains) target memory CD4 ϩ...

HIV-1 Resistance to CCR5 Antagonists Associated with Highly Efficient Use of CCR5 and Altered Tropism on Primary CD4 + T Cells

“…Addressing the implications of resistance should make it possible to determine if virologic failure in the face of CCR5 antagonist therapy will affect viral tropism and disease progression in any meaningful way. Passaging HIV-1 in the presence of increasing concentrations of a CCR5 antagonist in vitro can result in the development of mutations in Env that enable it to recognize the drugbound conformation of CCR5 (22,33,51,56). A similar resistance pathway can occur in vivo or, as we have shown, can even occur in the absence of drug selection (48,53).…”

“…A similar resistance pathway can occur in vivo or, as we have shown, can even occur in the absence of drug selection (48,53). This resistance pathway often, but not always, results in a virus that is resistant to multiple CCR5 antagonists (22,33,39,53,56). The CCR5 antagonists that have been used clinically all seem to bind to a hydrophobic region in the transmembrane helices of CCR5 and induce conformational alterations in the extracellular domains of the coreceptor that inhibit recognition by gp120 (13,31,32,46,52).…”

“…Viral determinants critical for CCR5 antagonist resistance are often localized to the V3 loop, consistent with the role of this domain in mediating coreceptor interactions (22,33,53,56). However, while V3 loop mutations appear to be a common though not universal theme underlying resistance to CCR5 antagonists, the specific changes can vary and are often context dependent in that mutations important for CCR5 antagonist resistance often fail to impart complete resistance when introduced into a heterologous background (22,37).…”

“…Because all Env clones isolated from this patient were resistant to complete suppression by aplaviroc, we performed a protein BLAST search on the Env clone demonstrating the greatest resistance to aplaviroc (clone pre5.2) and identified JRFL as a closely related, commonly studied HIV strain that is sensitive to inhibition by aplaviroc and other CCR5 antagonists. Since mutations in the V3 loop are commonly associated with resistance to CCR5 antagonists (22,33,53,56), we constructed chimeric viruses in which the V3 loops of pre5.2 and JRFL were exchanged. Pseudotype infection assays were performed on NP2/CD4/CCR5 cells in the presence or absence of 10 M aplaviroc, a level empirically determined to be 3 log units higher than required to saturate CCR5 receptors on this cell line.…”

“…A second mechanism of HIV resistance to CCR5 antagonists is the use of drug-bound CCR5 as a coreceptor for entry. Resistant viruses that utilize drug-bound CCR5 have been identified following in vitro passaging with multiple CCR5 antagonists (1,2,22,33,36,51,56). Recently, we identified a panel of viral Envs able to use aplaviroc (APL)-bound CCR5 that were isolated from a patient (21,48).…”