Reversible and efficient activation of HIV-1 cell entry by a tyrosine-sulfated peptide dissects endocytic entry and inhibitor mechanisms.
HIV-1 membranes contain gp120-gp41 trimers. Binding of gp120 to CD4 and a coreceptor (CCR5 or CXCR4) reduces the constraint on metastable gp41, enabling a series of conformational changes that cause membrane fusion. An analytic difficulty occurs because these steps occur slowly and asynchronously within cohorts of adsorbed virions. We previously isolated HIV-1JRCSF variants that efficiently use CCR5 mutants severely damaged in the tyrosine-sulfated amino terminus or extracellular loop 2. Surprisingly, both independent adaptations included gp120 mutations S298N, F313L, and N403S, supporting other evidence that they function by weakening gp120's grip on gp41 rather than by altering gp120 binding to specific CCR5 sites. Although several natural HIV-1 isolates reportedly use CCR5(Δ18) (CCR5 with a deletion of 18 N-terminal amino acids, including the tyrosine-sulfated region) when the soluble tyrosine-sulfated peptide is present, we show that HIV-1JRCSF with the adaptive mutations [HIV-1JRCSF(Ad)] functions approximately 100 times more efficiently and that coreceptor activation is reversible, enabling synchronous efficient entry control under physiological conditions. This system revealed that three-stranded gp41 folding intermediates susceptible to the inhibitor enfuvirtide form slowly and asynchronously on cell surface virions but resolve rapidly, with virions generally forming only one target. Adsorbed virions asynchronously and transiently become competent for entry at 37°C but are inactivated if the CCR5 peptide is absent during their window of opportunity. This competency is conferred by endocytosis, which results in inactivation if the peptide is absent. For both wild-type and adapted HIV-1 isolates, early gp41 refolding steps obligatorily occur on cell surfaces, whereas the final step(s) is endosomal. This system powerfully dissects HIV-1 entry and inhibitor mechanisms.


OBJECTIVE
We present a powerful means to reversibly and efficiently activate or terminate HIV-1 entry by adding or removing a tyrosine-sulfated CCR5 peptide from the culture medium. This system uses stable cell clones and a variant of HIV-1JRCSF with three adaptive mutations. It enabled us to show that CCR5 coreceptor activation is rapidly reversible and to dissect aspects of entry that had previously been relatively intractable. Our analyses elucidate enfuvirtide (T-20) function and suggest that HIV-1 virions form only one nonredundant membrane fusion complex on cell surfaces. Additionally, we obtained novel and conclusive evidence that HIV-1 entry occurs in an assembly line manner, with some steps obligatorily occurring on cell surfaces and with final membrane fusion occurring in endosomes. Our results were confirmed for wild-type HIV-1. Thus, our paper provides major methodological and mechanistic insights about HIV-1 infection.
Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon, USA.
Emily J
EJ
Platt
Michelle M
MM
Gomes
David
D
Kabat
D000595:Amino Acid Sequence; D065100:CCR5 Receptor Antagonists; D002460:Cell Line; D004705:Endocytosis / Q000187:drug effects; D000077560:Enfuvirtide; D015700:HIV Envelope Protein gp41 / Q000494:pharmacology*; D023581:HIV Fusion Inhibitors / Q000494:pharmacology*; D015658:HIV Infections / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology / Q000821:virology*; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology*; D019713:Receptors, CCR5 / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry; D053586:Virus Internalization / Q000187:drug effects*
D065100:CCR5 Receptor Antagonists; D015700:HIV Envelope Protein gp41; D023581:HIV Fusion Inhibitors; D010446:Peptide Fragments; D010455:Peptides; D019713:Receptors, CCR5; D000077560:Enfuvirtide; C005662:tyrosine O-sulfate; D014443:Tyrosine