Replication-Competent Variants of Human Immunodeficiency Virus Type 2 Lacking the V3 Loop Exhibit Resistance to Chemokine Receptor Antagonists

Abstract

Entry of human immunodeficiency virus type 1 (HIV-1) and HIV-2 requires interactions between the envelope glycoprotein (Env) on the virus and CD4 and a chemokine receptor, either CCR5 or CXCR4, on the cell surface. The V3 loop of the HIV gp120 glycoprotein plays a critical role in this process, determining tropism for CCR5-or CXCR4-expressing cells, but details of how V3 interacts with these receptors have not been defined. Using an iterative process of deletion mutagenesis and in vitro adaptation of infectious viruses, variants of HIV-2 were derived that could replicate without V3, either with or without a deletion of the V1/V2 variable loops. The generation of these functional but markedly minimized Envs required adaptive changes on the gp120 core and gp41 transmembrane glycoprotein. V3-deleted Envs exhibited tropism for both CCR5-and CXCR4-expressing cells, suggesting that domains on the gp120 core were mediating interactions with determinants shared by both coreceptors. Remarkably, HIV-2 Envs with V3 deletions became resistant to smallmolecule inhibitors of CCR5 and CXCR4, suggesting that these drugs inhibit wild-type viruses by disrupting a specific V3 interaction with the coreceptor. This study represents a proof of concept that HIV Envs lacking V3 alone or in combination with V1/V2 that retain functional domains required for viral entry can be derived. Such minimized Envs may be useful in understanding Env function, screening for new inhibitors of gp120 core interactions with chemokine receptors, and designing novel immunogens for vaccines.During viral entry, the human immunodeficiency virus (HIV) envelope glycoprotein (Env) mediates complex and highly coordinated steps that include binding of gp120 to CD4, a subsequent interaction with a chemokine receptor (either CCR5 or CXCR4), and the release of the transmembrane protein (TM) to interact and ultimately fuse with the target cell membrane (11,41). These events continue to occur in the face of strong host humoral immune responses owing to a number of structural attributes of Env, particularly its ability to tolerate extensive genetic variation (40, 66). The sites for this variation are located predominantly on gp120 variable loops, V1/V2, V3, and V4, which face outward on the trimeric gp120/TM oligomer (3,18,28,30,71). Variation is greatest in the V1/V2 and V4 loops, while for V3 variation is most prominent among isolates that utilize CXCR4 (16,18,22,28,63). In addition, the V1/V2 and V3 loops may protect critical domains on the gp120 core that include, respectively, the recessed CD4 binding site and the bridging sheet, a four-stranded antiparallel beta sheet, formed from amino acids in the V1/V2 stem and the C4 domain, that likely binds to the chemokine receptor amino terminus (15,29,47,48,57,65). The V3 loop also plays a key role in interacting with chemokine receptors and determines tropism for CCR5-or CXCR4-expressing cells (8,9,12,18,20,23,39,55,69). The recently solved V3 structure on a CD4-bound gp120 core shows that its base is contiguous with th...

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).…”

“…How might a virus utilize the drug-bound form of CXCR4? 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).…”

Structural Comparison of HIV-1 Envelope Spikes with and without the V1/V2 Loop

“…On the other hand, the observations that soluble recombinant ⌬V1/V2 mutant constructs can be less stable than WT constructs and replication-competent ⌬V1/V2 mutant spikes are less stable than virion-associated WT Env spikes point to at least some direct or indirect stabilizing effect imparted by the V1/V2 loop (6). Interestingly, under selective pressure, HIV-1, HIV-2, and SIV ⌬V1/V2 mutants can revert to replication competence and trimer stability through mutations in other regions of gp120 and even in gp41, implying that enhancement in other contact regions can compensate for the loss of V1/V2 loop structural interactions (6,31,45,72,87). It would be interesting to determine if reversion to enhanced fusion competence in such situations is concomitant with the reestablishment of a more native structure and/or reduced gp120 flexibility.…”

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].…”