Soluble CD4 and CD4-Mimetic Compounds Inhibit HIV-1 Infection by Induction of a Short-Lived Activated State

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Abstract
Binding to the CD4 receptor induces conformational changes in the human immunodeficiency virus (HIV-1) gp120 exterior envelope glycoprotein. These changes allow gp120 to bind the coreceptor, either CCR5 or CXCR4, and prime the gp41 transmembrane envelope glycoprotein to mediate virus–cell membrane fusion and virus entry. Soluble forms of CD4 (sCD4) and small-molecule CD4 mimics (here exemplified by JRC-II-191) also induce these conformational changes in the HIV-1 envelope glycoproteins, but typically inhibit HIV-1 entry into CD4-expressing cells. To investigate the mechanism of inhibition, we monitored at high temporal resolution inhibitor-induced changes in the conformation and functional competence of the HIV-1 envelope glycoproteins that immediately follow engagement of the soluble CD4 mimics. Both sCD4 and JRC-II-191 efficiently activated the envelope glycoproteins to mediate infection of cells lacking CD4, in a manner dependent on coreceptor affinity and density. This activated state, however, was transient and was followed by spontaneous and apparently irreversible changes of conformation and by loss of functional competence. The longevity of the activated intermediate depended on temperature and the particular HIV-1 strain, but was indistinguishable for sCD4 and JRC-II-191; by contrast, the activated intermediate induced by cell-surface CD4 was relatively long-lived. The inactivating effects of these activation-based inhibitors predominantly affected cell-free virus, whereas virus that was prebound to the target cell surface was mainly activated, infecting the cells even at high concentrations of the CD4 analogue. These results demonstrate the ability of soluble CD4 mimics to inactivate HIV-1 by prematurely triggering active but transient intermediate states of the envelope glycoproteins. This novel strategy for inhibition may be generally applicable to high–potential-energy viral entry machines that are normally activated by receptor binding. Human immunodeficiency virus type 1 (HIV-1) is the cause of the global AIDS epidemic. HIV-1 gains entry into its target cells by fusing with the cell membrane, a process that begins with the interaction of the viral envelope glycoproteins with cell-surface receptors. HIV-1 uses two receptors on the target cell: CD4 and CCR5/CXCR4. Binding of the virus to the primary receptor, CD4, primes the viral envelope glycoproteins to mediate the fusion of the viral membrane and the membrane of the target cell. Soluble forms of the CD4 receptor and small molecules that mimic the effects of CD4 can inhibit virus infection; however, how this inhibition occurs is still unknown. In this report, we show that soluble mimics of CD4 inhibit HIV-1 infection by prematurely triggering the viral envelope glycoproteins. The unstable activated state of the virus lasts only a few minutes, after which the virus loses the ability to infect cells. This novel strategy for inhibition may be generally applicable to other viruses besides HIV-1, some of which are also activated by binding to their receptors.