The CD8-Derived Chemokine XCL1/Lymphotactin Is a Conformation-Dependent, Broad-Spectrum Inhibitor of HIV-1

Abstract

CD8+ T cells play a key role in the in vivo control of HIV-1 replication via their cytolytic activity as well as their ability to secrete non-lytic soluble suppressive factors. Although the chemokines that naturally bind CCR5 (CCL3/MIP-1α, CCL4/MIP- 1β, CCL5/RANTES) are major components of the CD8-derived anti-HIV activity, evidence indicates the existence of additional, still undefined, CD8-derived HIV-suppressive factors. Here, we report the characterization of a novel anti-HIV chemokine, XCL1/lymphotactin, a member of the C-chemokine family that is produced primarily by activated CD8+ T cells and behaves as a metamorphic protein, interconverting between two structurally distinct conformations (classic and alternative). We found that XCL1 inhibits a broad spectrum of HIV-1 isolates, irrespective of their coreceptor-usage phenotype. Experiments with stabilized variants of XCL1 demonstrated that HIV-1 inhibition requires access to the alternative, all-β conformation, which interacts with proteoglycans but does not bind/activate the specific XCR1 receptor, while the classic XCL1 conformation is inactive. HIV-1 inhibition by XCL1 was shown to occur at an early stage of infection, via blockade of viral attachment and entry into host cells. Analogous to the recently described anti-HIV effect of the CXC chemokine CXCL4/PF4, XCL1-mediated inhibition is associated with direct interaction of the chemokine with the HIV-1 envelope. These results may open new perspectives for understanding the mechanisms of HIV-1 control and reveal new molecular targets for the design of effective therapeutic and preventive strategies against HIV-1.

Cytokines Elevated in HIV Elite Controllers Reduce HIV ReplicationIn Vitroand Modulate HIV Restriction Factor Expression

“…These data are consistent with findings published after our experiments were performed, showing that an alternative all-␤ conformation of XCL1 forms a dimer, binds glycosaminoglycans, and suppresses HIV replication via blockade of attachment and entry into cells and that this form of the protein lacks chemotactic activity (35). The protein supplied by R&D Systems is tested to demonstrate chemotactic activity, so we conclude that the version of XCL1 used in our experiments was in the classic XCL1 conformation and would not be expected to suppress HIV replication.…”

“…Of note, soluble epidermal growth factor receptor (sEGFR) was also uniquely elevated in ECs compared to levels in the NEG group but was not studied further due to lack of available reagents. Of the five cytokines selected for further in vitro study, CCL14 (32), SDF-1 (33,34), and XCL1 (35) have been previously associated with control of HIV infection.…”

Structural Determinants for the Selective Anti-HIV-1 Activity of the All-β Alternative Conformer of XCL1

“…Using XCL1 variants stabilized in each of the two alternative conformations, we found that inhibition of HIV-1 requires access to the GAG-binding, alternative conformation, while the XCR1-binding (classic, chemokine-like) fold lacks anti-viral function. However, by enzymatic removal of GAGs on HIV-1 target cells, we demonstrated that interaction with cell surface GAGs is not required for the antiviral activity of XCL1, in agreement with the evidence that HIV-1 inhibition is mediated by direct interaction of the chemokine with gp120 (2). Altogether, these observations point to a role of electrostatic interactions, potentially related to those involved in GAG binding, in XCL1 interaction with gp120 (6)(7)(8).…”

“…To investigate the relationship between the antiviral and GAG-binding activities of XCL1, we generated a panel of mutants bearing individual alanine substitution of all basic amino acids in the structured core of XCL1 (amino acids 1 to 54), as well as three residues within the C-terminal tail (12). Since mutation of some basic residues was previously shown to shift the conformational equilibrium of XCL1 toward the monomeric chemokine conformation (13), which lacks HIV-inhibitory activity (2), mutagenesis was performed on the stabilized XCL1 variant W55D, in order to ensure that the functional impact of each substitution could be interpreted in the context of the all-␤ dimeric structure (2). All of the mutants were tested for antiviral activity against a reference CCR5-tropic (R5) HIV-1 isolate (BaL) in susceptible target cells (TZM-bl).…”

“…A major determinant of the pace of disease progression is the level of HIV-1 replication, which is regulated in vivo by an intricate network of bioactive molecules, including both soluble immune mediators and cell surface receptors. We recently reported that the C-chemokine XCL1/lymphotactin is a conformation-dependent broad-spectrum inhibitor of HIV-1 infection, which acts at the level of viral entry via an unconventional mechanism mediated by direct interaction with the external envelope glycoprotein, gp120 (2). XCL1 is a peculiar metamorphic chemokine that interconverts in solution between two distinct conformations: a monomeric chemokinelike fold (Ltn10), which binds and activates the cognate receptor, XCR1, and an alternatively folded, all-␤ conformation (Ltn40), which has a marked propensity to self-associate as a head-to-tail dimer and does not bind/activate XCR1 but rather interacts with cell surface glycosaminoglycans (GAGs) with high affinity (3,4).…”