V3 Loop Truncations in HIV-1 Envelope Impart Resistance to Coreceptor Inhibitors and Enhanced Sensitivity to Neutralizing Antibodies

Abstract

The V1/V2 region and the V3 loop of the human immunodeficiency virus type I (HIV-1) envelope (Env) protein are targets for neutralizing antibodies and also play an important functional role, with the V3 loop largely determining whether a virus uses CCR5 (R5), CXCR4 (X4), or either coreceptor (R5X4) to infect cells. While the sequence of V3 is variable, its length is highly conserved. Structural studies indicate that V3 length may be important for interactions with the extracellular loops of the coreceptor. Consistent with this view, genetic truncation of the V3 loop is typically associated with loss of Env function. We removed approximately one-half of the V3 loop from three different HIV-1 strains, and found that only the Env protein from the R5X4 strain R3A retained some fusion activity. Loss of V1/V2 (ΔV1/V2) was well tolerated by this virus. Passaging of virus with the truncated V3 loop resulted in the derivation of a virus strain that replicated with wild-type kinetics. This virus, termed TA1, retained the V3 loop truncation and acquired several adaptive changes in gp120 and gp41. TA1 could use CCR5 but not CXCR4 to infect cells, and was extremely sensitive to neutralization by HIV-1 positive human sera, and by antibodies to the CD4 binding site and to CD4-induced epitopes in the bridging sheet region of gp120. In addition, TA1 was completely resistant to CCR5 inhibitors, and was more dependent upon the N-terminal domain of CCR5, a region of the receptor that is thought to contact the bridging sheet of gp120 and the base of the V3 loop, and whose conformation may not be greatly affected by CCR5 inhibitors. These studies suggest that the V3 loop protects HIV from neutralization by antibodies prevalent in infected humans, that CCR5 inhibitors likely act by disrupting interactions between the V3 loop and the coreceptor, and that altered use of CCR5 by HIV-1 associated with increased sensitivity to changes in the N-terminal domain can be linked to high levels of resistance to these antiviral compounds.

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

“…In contrast, nearly all changes within the ECL regions of CCR5 did not inhibit infection, indicating that these residues are not essential for the use of drug-bound receptor by the pre5.2 Env. These findings are consistent with recently described CCR5 antagonist-resistant Envs that are dependent on the NЈ terminus of CCR5 for entry (5,25,36). Similar results were found using maraviroc to block infection, suggesting that pre5.2 relies upon the NЈ terminus of CCR5 for entry in the presence of CCR5 antagonists (data not shown).…”

Rapid Degranulation of NK Cells following Activation by HIV-Specific Antibodies

“…The V3 loop of the Env protein has long been recognized as a major target of Env-specific Abs (19). By using gp140 protein devoid of the V3-loop and mapping the specificity of Ab epitopes using overlapping peptides, we demonstrated, not surprisingly, that some patients preferentially target the V3 loop.…”

HIV Type 1 from a Patient with Baseline Resistance to CCR5 Antagonists Uses Drug-Bound Receptor for Entry

“…[45][46][47] Likewise, in vitro-derived CCR5 antagonist-resistant viruses have varied cross-resistance profiles, with some viruses demonstrating broad cross-resistance and others showing very narrow cross-resistance, again revealing variability in CCR5 interactions. 23,24 We have characterized a virus with a partially deleted V3 loop that is resistant to all CCR5 antagonists tested, and that appears to interact primarily with the N-terminal domain of CCR5, 28 a region of the receptor that may not be greatly impacted by this class of entry inhibitors that bind to the transmembrane domains immediately contiguous with the extracellular loops. The viruses derived from the patient studied here were broadly cross-resistant to other CCR5 antagonists, although they remained sensitive to enfuvirtide, which targets a separate stage of the entry process.…”

“…28 For the fusion assay, 29 ''target'' QT6 cells were transiently cotransfected with CD4, CXCR4, or CCR5, and a luciferase reporter plasmid under the control of a T7 promoter (pGEM2 T7-luc, Promega). ''Effector'' QT6 cells, transfected with env expression plasmids, were infected with a recombinant vaccinia virus expressing T7 polymerase (vTF1.1).…”