Cooperation of Multiple CCR5 Coreceptors Is Required for Infections by Human Immunodeficiency Virus Type 1

Abstract

In addition to the primary cell surface receptor CD4, CCR5 or another coreceptor is necessary for infections by human immunodeficiency virus type 1 (HIV-1), yet the mechanisms of coreceptor function and their stoichiometries in the infection pathway remain substantially unknown. To address these issues, we studied the effects of CCR5 concentrations on HIV-1 infections using wild-type CCR5 and two attenuated mutant CCR5s, one with the mutation Y14N at a critical tyrosine sulfation site in the amino terminus and one with the mutation G163R in extracellular loop 2. The Y14N mutation converted a YYT sequence at positions 14 to 16 to an NYT consensus site for N-linked glycosylation, and the mutant protein was shown to be glycosylated at that position. The relationships between HIV-1 infectivity values and CCR5 concentrations took the form of sigmoidal (S-shaped) curves, which were dramatically altered in different ways by these mutations. Both mutations shifted the curves by factors of approximately 30-to 150-fold along the CCR5 concentration axis, consistent with evidence that they reduce affinities of virus for the coreceptor. In addition, the Y14N mutation specifically reduced the maximum efficiencies of infection that could be obtained at saturating CCR5 concentrations. The sigmoidal curves for all R5 HIV-1 isolates were quantitatively consistent with a simple mathematical model, implying that CCR5s reversibly associate with cell surface HIV-1 in a concentration-dependent manner, that approximately four to six CCR5s assemble around the virus to form a complex needed for infection, and that both mutations inhibit assembly of this complex but only the Y14N mutation also significantly reduces its ability to successfully mediate HIV-1 infections. Although several alternative models would be compatible with our data, a common feature of these alternatives is the cooperation of multiple CCR5s in the HIV-1 infection pathway. This cooperativity will need to be considered in future studies to address in detail the mechanism of CCR5-mediated HIV-1 membrane fusion.

A biosensor assay for studying ligand-membrane receptor interactions: Binding of antibodies and HIV-1 Env to chemokine receptors

“…In addition, this shows that at least two different proteins can be incorporated into a given virus particle; it also shows that because membrane fusion is a cooperative process requiring multiple receptor binding events (31), multiple copies of each can be incorporated. In the case of HIV-1, it is estimated that six CCR5 molecules are needed to support membrane fusion (32) and that multiple CD4 molecules are also needed (33). Our results support these conclusions; the 7TM and type I membrane proteins studied here retained their native conformations as judged by their abilities to bind a variety of conformationally sensitive ligands.…”

Cooperative subunit interactions within the oligomeric envelope glycoprotein of HIV-1: Functional complementation of specific defects in gp120 and gp41

“…Numerous experimental variables may contribute to complementation efficiency, including the surface densities of each of the participating proteins, the binding affinities between gp120 and each receptor, the efficiencies of subunit interactions, etc. The findings of constitutive interactions between CD4 and coreceptors may have significance in this regard (14,31), as may the recent proposal that cooperative interactions involving multiple CCR5 molecules are required for the HIV-1 infection pathway (32). Also of note is that certain complementation effects were not uniformly noted; for example, LAV-BS complemented SF162 wild type for fusion with CXCR4-expressing targets cells (Fig.…”

Reduction of CCR5 with low-dose rapamycin enhances the antiviral activity of vicriviroc against both sensitive and drug-resistant HIV-1

“…It is thought that HIV requires the engagement of several (about four to six) CCR5 molecules to form a fusion pore (31). We postulate that RAPA reduction of CCR5 density could prevent R5 HIV virions from engaging a minimum number of CCR5s required for effective fusion.…”