Peptides from Second Extracellular Loop of C-C Chemokine Receptor Type 5 (CCR5) Inhibit Diverse Strains of HIV-1

Laboratory of Bioorganic Chemistry, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2012; 287(18):15076-86. DOI: 10.1074/jbc.M111.332361
Source: PubMed


To initiate HIV entry, the HIV envelope protein gp120 must engage its primary receptor CD4 and a coreceptor CCR5 or CXCR4. In the absence of a high resolution structure of a gp120-coreceptor complex, biochemical studies of CCR5 have revealed the importance of its N terminus and second extracellular loop (ECL2) in binding gp120 and mediating viral entry. Using a panel of synthetic CCR5 ECL2-derived peptides, we show that the C-terminal portion of ECL2 (2C, comprising amino acids Cys-178 to Lys-191) inhibit HIV-1 entry of both CCR5- and CXCR4-using isolates at low micromolar concentrations. In functional viral assays, these peptides inhibited HIV-1 entry in a CD4-independent manner. Neutralization assays designed to measure the effects of CCR5 ECL2 peptides when combined with either with the small molecule CD4 mimetic NBD-556, soluble CD4, or the CCR5 N terminus showed additive inhibition for each, indicating that ECL2 binds gp120 at a site distinct from that of N terminus and acts independently of CD4. Using saturation transfer difference NMR, we determined the region of CCR5 ECL2 used for binding gp120, showed that it can bind to gp120 from both R5 and X4 isolates, and demonstrated that the peptide interacts with a CD4-gp120 complex in a similar manner as to gp120 alone. As the CCR5 N terminus-gp120 interactions are dependent on CD4 activation, our data suggest that gp120 has separate binding sites for the CCR5 N terminus and ECL2, the ECL2 binding site is present prior to CD4 engagement, and it is conserved across CCR5- and CXCR4-using strains. These peptides may serve as a starting point for the design of inhibitors with broad spectrum anti-HIV activity.

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    • "Moreover, specific antibodies against the ECL2 of the CCR5 were shown to block HIV entry in cells (38). Similarly, a peptide mimic of the C-terminal portion of CCR5 was shown to act as a NAM for HIV-1 entry (39). Not surprisingly, because ECLs can adopt different conformations in GPCR, either at basal state or upon receptor activation (46, 49), it can also be targeted to allosterically bias receptor signaling. "
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    • "C34 is derived from C-terminal heptad-repeat regions (CHR) of gp41 and known to inhibit HIV-1 Env-mediated membrane fusion by interfering with formation of the six-helix bundle [11], [26], [27], [28]. Another peptide 2N derived from the second extracellular loop of chemokine receptor type 5 (CCR5) was introduced as a negative control (HXB2-TM11D-2N), since it has no fusion inhibitory activity [29]. The expression and processing of tethered fusion protein was confirmed by immunoblotting analysis with anti-gp120 and anti-gp41 antibodies (data not shown). "
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    ABSTRACT: The envelope glycoprotein (Env) of human immunodeficiency virus type I (HIV-1) mediates membrane fusion. To analyze the mechanism of HIV-1 Env-mediated membrane fusion, it is desirable to determine the expression level of Env on the cell surface. However, the quantification of Env by immunological staining is often hampered by the diversity of HIV-1 Env and limited availability of universal antibodies that recognize different Envs with equal efficiency. To overcome this problem, here we linked a tag protein called HaloTag at the C-terminus of HIV-1 Env. To relocate HaloTag to the cell surface, we introduced a second membrane-spanning domain (MSD) between Env and HaloTag. The MSD of transmembrane protease serine 11D, a type II transmembrane protein, successfully relocated HaloTag to the cell surface. The surface level of Env can be estimated indirectly by staining HaloTag with a specific membrane-impermeable fluorescent ligand. This tagging did not compromise the fusogenicity of Env drastically. Furthermore, fusogenicity of Env was preserved even after the labeling with the ligands. We have also found that an additional foreign peptide or protein such as C34 or neutralizing single-chain variable fragment (scFv) can be linked to the C-terminus of the HaloTag protein. Using these constructs, we were able to determine the required length of C34 and critical residues of neutralizing scFv for blocking membrane fusion, respectively.
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    • "Experiments were performed in duplicate and are presented as average ± standard deviation. a small peptide corresponding to the C-terminal part of ECL2-R5 identified several gp120-interacting residues (underlined, 181- HFPYSQYQFW-190) corresponding to the second cluster pointed out in the present study (189-FYPNDLW-195) [84]. "
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