Effects of Partial Deletions within the Human Immunodeficiency Virus Type 1 V3 Loop on Coreceptor Tropism and Sensitivity to Entry Inhibitors

Abstract

The human immunodeficiency virus type 1 (HIV-1) V3 loop is critical for coreceptor binding and principally determines tropism for the CCR5 and CXCR4 coreceptors. The recent crystallographic resolution of V3 shows that its base is closely associated with the conserved coreceptor binding site on the gp120 core, whereas more distal regions protrude toward the cell surface, likely mediating interactions with coreceptor extracellular loops. However, these V3-coreceptor interactions and the structural basis for CCR5 or CXCR4 specificity are poorly understood. Using the dual-tropic virus HIV-1 R3A , which uses both CCR5 and CXCR4, we sought to identify subdomains within V3 that selectively mediate R5 or X4 tropism. An extensive panel of V3 mutants was evaluated for effects on tropism and sensitivity to coreceptor antagonists. Mutations on either side of the V3 base (residues 3 to 8 and 26 to 33) ablated R5 tropism and made the resulting X4-tropic Envs more sensitive to the CXCR4 inhibitor AMD3100. When mutations were introduced within the V3 stem, only a deletion of residues 9 to 12 on the N-terminal side ablated X4 tropism. Remarkably, this R5-tropic ⌬9-12 mutant was completely resistant to several small-molecule inhibitors of CCR5. Envs with mutations in the V3 crown (residues 13 to 20) remained dual tropic. Similar observations were made for a second dual-tropic isolate, HIV-1 89.6 . These findings suggest that V3 subdomains can be identified that differentially affect R5 and X4 tropism and modulate sensitivity to CCR5 and CXCR4 inhibitors. These studies provide a novel approach for probing V3-coreceptor interactions and mechanisms by which these interactions can be inhibited.Human immunodeficiency virus (HIV) entry requires a coordinated interaction between envelope glycoprotein (Env) trimers on the virion surface with CD4 and a chemokine receptor, typically CCR5 (2,11,18,20,22) or CXCR4 (24), on the target cell. Whereas binding of gp120 to CD4 is required for the initial conformational changes that facilitate coreceptor interactions (9, 35), binding to CCR5 or CXCR4 is required to release gp41 to interact with the cell membrane and to form the six-helix bundle that provides the energy for membrane fusion (8,19,60). The gp120-coreceptor interactions that are required for these events likely involve (i) the bridging sheet (a four-stranded ␤-sheet on the gp120 core) and the base of the V3 loop with the coreceptor N terminus and (ii) more distal regions of V3 with coreceptor extracellular loops (ECLs) (14,15,29,33,48,49,56).The V3 loop is the primary determinant for R5 or X4 tropism (28). However, the mechanism and structural basis that underlie the specificity of V3-coreceptor interactions are poorly understood. During HIV infection, viruses that utilize CCR5 are characteristically transmitted (16,38,51,53,58), whereas viruses that utilize CXCR4 can evolve during the progression to disease (13, 47). The evolution of X4 tropism in vivo and in vitro has been associated with an increase in the net positive charge in V3 ...

Baseline Resistance of Primary Human Immunodeficiency Virus Type 1 Strains to the CXCR4 Inhibitor AMD3100

“…The ability of HIV strains to use the AMD3100-bound form of CXCR4 has not previously been described, to the best of our knowledge, though utilization of drug-bound CCR5 has been described for several virus strains derived by passage in the presence of increasing concentrations of CCR5 antagonists (29,37,44,57) and by viruses into which V3 loop deletions have been introduced (30,34,42). Taken together, these studies indicate that mutations conferring resistance to CCR5 antagonists often, but not always, involve mutations in the V3 loop, and when introduced into other Env backgrounds, these mutations have not conferred the drug-resistant phenotype (37).…”

“…A possible model is suggested by studies of how HIV-1 Env interacts with CCR5 and CXCR4 in general, as well as insights gleaned from a virus strain that can efficiently utilize drugbound forms of CCR5 due to partial deletion of its V3 loop (30,34,42). There is good evidence that the distal portion of the V3 loop interacts with the extracellular loops of CCR5 or CXCR4, while the base of the V3 loop and bridging sheet bind to the amino-terminal domain of the coreceptor, with sulfated tyrosine residues playing a particularly important role in the case of CCR5 (13,25,41,42). Removal of 15 residues from the center of the V3 loop from the R5X4 virus strain R3A renders the virus completely resistant to CCR5 antagonists through a mechanism that most likely involves efficient utilization of the drug-bound coreceptor (30).…”

Enhanced Exposure of the CD4-Binding Site to Neutralizing Antibodies by Structural Design of a Membrane-Anchored Human Immunodeficiency Virus Type 1 gp120 Domain

“…gp145⌬CFI⌬V12(9,9) was derived from the HIV-1 R3A TA1 envelope (15,23) with cytoplasmic, cleavage-site, fusion peptide, interhelical-domain, and V1V2 deletions, as previously described (7,34). The outer domain construct OD(9,9)(hCD4-TM) was made by PCR of amino acids 252 to 482 ( 252 PVVST……WRSE 482 ) (numbering based on HXBc2) from R3A-gp145⌬CFI⌬V12 (9,9).…”

“…An HIV-1 clade B R5 and X4 dual-tropic virus, R3A, was selected as a prototype (20). Laboratory-adapted virus strain R3A TA1 contains a truncated V1/V2 and a truncated V3 (named 9,9), maintains CCR5 tropism, and is highly sensitive to b12 neutralization (15,23). We used available atomiclevel structures to model an R3A gp120 core and to design truncations of flexible, potentially immunodominant structures, which emanate from OD, including the ␤20-␤21 hairpin and the V3 loop.…”

Chemokine Receptors as Therapeutic Targets in HIV Infection

“…Accordingly, substantial deletions in V3 from both HIV-1 and HIV-2 confer complete resistance to coreceptor antagonists, presumably by disrupting the interaction between V3 and ECL2 [146][147][148][149][150]. Any adverse effect the V3 sequence changes have at the CCR5-binding stage may be compensated for by increases in the affinity of resistant viruses for CD4 and/or in the kinetics of virus entry [147,151].…”