The role of the N-terminal segment of CCR5 in HIV-1 Env-mediated membrane fusion and the mechanism of virus adaptation to CCR5 lacking this segment.
BACKGROUND
HIV-1 envelope glycoprotein (Env) induces membrane fusion as a result of sequential binding to CD4 and chemokine receptors (CCR5 or CXCR4). The critical determinants of CCR5 coreceptor function are the N-terminal domain (Nt) and the second extracellular loop. However, mutations in gp120 adapt HIV-1 to grow on cells expressing the N-terminally truncated CCR5(Delta 18) (Platt et al., J. Virol. 2005, 79: 4357-68).


RESULTS
We have functionally characterized the adapted Env (designated Env(NYP)) using a quantitative cell-cell fusion assay. The rate of fusion with target cells expressing wild-type CCR5 and the resistance to fusion inhibitors was virtually identical for wild-type Env and Env(NYP), implying that the coreceptor affinity had not increased as a result of adaptation. In contrast, Env(NYP)-induced fusion with cells expressing CCR5(Delta 18) occurred at a slower rate and was extremely sensitive to the CCR5 binding inhibitor, Sch-C. Resistance to Sch-C drastically increased after pre-incubation of Env(NYP)- and CCR5(Delta 18)-expressing cells at a temperature that was not permissive to fusion. This indicates that ternary Env(NYP)-CD4-CCR5(Delta 18) complexes accumulate at sub-threshold temperature and that low-affinity interactions with the truncated coreceptor are sufficient for triggering conformational changes in the gp41 of Env(NYP) but not in wild-type Env. We also demonstrated that the ability of CCR5(Delta 18) to support fusion and infection mediated by wild-type Env can be partially reconstituted in the presence of a synthetic sulfated peptide corresponding to the CCR5 Nt. Pre-incubation of wild-type Env- and CCR5(Delta 18)-expressing cells with the sulfated peptide at sub-threshold temperature markedly increased the efficiency of fusion.


CONCLUSIONS
We propose that, upon binding the Nt region of CCR5, wild-type Env acquires the ability to productively engage the extracellular loop(s) of CCR5 - an event that triggers gp41 refolding and membrane merger. The adaptive mutations in Env(NYP) enable it to more readily release its hold on gp41, even when it interacts weakly with a severely damaged coreceptor in the absence of the sulfopeptide.
Institute of Human Virology, University of Maryland School of Medicine,725 W, Lombard St,, Baltimore, MD 21201, USA. melikian@umbi.umd.edu
Gregory B
GB
Melikyan
Emily J
EJ
Platt
David
D
Kabat
D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000494:pharmacology; D065100:CCR5 Receptor Antagonists; D003497:Cyclic N-Oxides / Q000494:pharmacology; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D023581:HIV Fusion Inhibitors / Q000494:pharmacology; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008561:Membrane Fusion / Q000235:genetics; D009154:Mutation; D010091:Oximes; D010880:Piperidines / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000494:pharmacology; D019713:Receptors, CCR5 / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013696:Temperature; D053586:Virus Internalization
D019380:Anti-HIV Agents; D065100:CCR5 Receptor Antagonists; D003497:Cyclic N-Oxides; D015699:HIV Envelope Protein gp120; D023581:HIV Fusion Inhibitors; D010091:Oximes; D010880:Piperidines; D011725:Pyridines; D019713:Receptors, CCR5; C438583:Ancriviroc