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

Abstract

HIV-1 replication is regulated in vivo by a complex network of cytokines and chemokines. XCL1/lymphotactin, a unique metamorphic chemokine, was recently identified as a broad-spectrum endogenous HIV-1 inhibitor that blocks viral entry via direct interaction with the gp120 envelope glycoprotein. HIV-1 inhibition by XCL1 requires access to the alternative all-␤ conformation, which interacts with glycosaminoglycans (GAGs) but not with the specific XCL1 receptor, XCR1. To investigate the structural determinants of the HIV-inhibitory function of XCL1, we performed a detailed structure-function analysis of a stabilized all-␤ variant, XCL1 W55D. Individual alanine substitutions of two basic residues within the 40s' loop, K42 and R43, abrogated the ability of XCL1 to bind to the viral envelope and block HIV-1 infection; moreover, a loss of HIV-inhibitory function, albeit less marked, was seen upon individual mutation of three additional basic residues: R18, R35, and K46. In contrast, mutation of K42 to arginine did not cause any loss of function, suggesting that the interaction with gp120 is primarily electrostatic in nature. Strikingly, four of these five residues cluster to form a large (ϳ350 Å 2 ) positively charged surface in the all-␤ XCL1 conformation, whereas they are dissociated in the classic chemokine fold, which is inactive against HIV-1, providing a structural basis for the selective antiviral activity of the alternatively folded XCL1. Furthermore, we observed that changes to the N-terminal domain, which is proximal to the cluster of putative HIV-1 gp120-interacting residues, also affect the antiviral activity of XCL1. Interestingly, the complement of residues involved in HIV-1 blockade is partially overlapping, but distinct from those involved in the GAG-binding function of XCL1. These data identify key structural determinants of anti-HIV activity in XCL1, providing new templates for the development of HIV-1 entry inhibitors. IMPORTANCEThe host immune system controls HIV-1 infection through a wide array of inhibitory responses, including the induction of cytotoxic effector cells and the secretion of noncytolytic soluble antiviral factors such as cytokines and chemokines. We recently identified XCL1/lymphotactin, a chemokine primarily produced by CD8 ؉ T cells, as a novel endogenous factor with broad anti-HIV activity. Strikingly, only one of the two conformations that XCL1 can adopt in solution, the alternative all-␤ fold, mediates antiviral activity. At variance with the classic HIV-inhibitory chemokines such as CCL5/RANTES, XCL1 acts via direct interaction with the external viral envelope glycoprotein, gp120. Here, we identify the interactive surface of XCL1 that is implicated in binding to the HIV-1 envelope and HIV-1 inhibition, providing a structural basis to explain why only the all-␤ XCL1 conformer is effective against HIV-1. Our findings may be useful in guiding the rational design of new inhibitors of HIV-1 entry.T he natural history of HIV-1 infection is highly heterogeneous in different i...

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

“…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. In fact, the R&D Systems XCL1 was tested recently and was shown to have only modest suppressive activity compared to that of other preparations (53). If XCL1 were to be utilized for anti-HIV therapy, it would need to be formulated in the ␤-dimer form, and a variant with a disulfide bond that stabilizes the all-␤ conformation has been engineered (54).…”

Structure-function guided modeling of chemokine-GPCR specificity for the chemokine XCL1 and its receptor XCR1

“…Several studies demonstrate that the metamorphic behavior of XCL1 plays an important role in its biological activity. For example, the alternative XCL1 dimer conformation inhibits HIV infection in peripheral blood mononuclear cells, but the monomeric chemokine fold does not (31)(32)(33).…”

Examination of Glycosaminoglycan Binding Sites on the XCL1 Dimer

“…12 Mutational analysis indicated that the anti-HIV-1 activity of XCL1 depends on residues that also participate in GAG binding. 13 A link between XCL1–GAG binding and antiviral potency may reveal the evolutionary advantage conferred by the metamorphic XCL1 native state.…”