The Potent Anti-HIV Activity of CXCL12γ Correlates with Efficient CXCR4 Binding and Internalization

Chemokines are small, structurally related chemoattractant cytokines characterized by conserved cysteine residues. Based on the positions of the first N-terminal cysteines, chemokines fall into four subfamilies. The CC and CXC subfamilies have been well characterized. The CC subfamily includes the following: regulated on activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory peptides 1 (MIP-1). The prototype of the CXC subfamily is interleukin-8 (IL-8)/CXCL8. The C chemokine (lymphotactine) and CX3C chemokine (fractalkine) subfamilies were recently identified (reviewed in reference 30). The physiological activities of chemokines are mediated by the selective recognition and activation of chemokine receptors belonging to the seven-membrane-domain G-protein-coupled receptor superfamily (GPCRs). In addition, chemokines also bind to glycosaminoglycans (GAGs) through distinct binding sites. Chemokine binding to GAGs on cells, particularly endothelial cells, results in chemotactic chemokine gradients that allow correct presentation of chemokines to leukocytes, therefore enabling target cells to cross the endothelial barrier and migrate into tissues (reviewed in reference 10). Stromal cell-derived factor 1 (SDF-1)/CXCL12 is a member of the CXC chemokine family and is a key regulator of B-cell lymphopoiesis, hematopoietic stem cell mobilization, and leukocyte migration (reviewed in reference 10). CXCL12 was originally thought to mediate these processes through the single receptor CXCR4 (9). However, later studies demonstrated that RDC-1/CXCR7 is also a receptor for CXCL12 (6, 11). CXCL12 has also been shown to block HIV-1 infection (5). There are two known human splice variants of CXCL12, referred to as CXCL12α and CXCL12β (27). The genomic structure of the CXCL12 gene revealed that human CXCL12α and CXCL12β are encoded by a single gene and arise by alternative splicing. The cDNAs corresponding to CXCL12α and CXCL12β encode proteins of 89 and 93 amino acids, respectively. A third splice variant, classified as CXCL12γ, has been identified in rats (14). The human equivalent of CXCL12γ was recently identified among other splice variants of CXCL12 (33). The novel human splice variants CXCL12γ, CXCL12ɛ, CXCL12δ, and CXCL12θ (also reported as CXCL12ϕ [33]) are expressed through alternative splicing events that result in different exons being added to the same first three exons. Therefore, all six splice variants of CXCL12 are identical in the first 88 amino acid residues from the amino terminus. It has been demonstrated that CXCL12α and -β are expressed in numerous tissues, with the highest expression levels in the liver, pancreas, and spleen (33). The mRNA encoding CXCL12γ was detectable in the adult human heart but hardly detectable in several other tissues. On the other hand, CXCL12δ, -ɛ, and -θ could be detected in several human adult and fetal tissues, with the pancreas expressing the highest levels (33). Recent studies have demonstrated the tissue expression of CXCL12γ in the adult heart (24). We previously demonstrated that CXCL12γ is the most potent anti-HIV-1 inhibitor, with the weakest chemotactic activity and no detectable enhancing activity for hematopoietic progenitor cell survival or replating capacity (2). The first three exons present in the CXCL12γ splice variant are identical to those found in CXCL12α and CXCL12β. The fourth exon, however, contains a large number of basic residues that result in at least four additional BBXB domains in addition to the conserved 24KHLK27 domain (33). It is not known whether the additional BBXB domains in the C terminus of CXCL12γ result in higher affinity for heparan sulfate proteoglycan (HSPG) and whether differences in HSPG binding could explain the observed anti-HIV-1 potency or the low chemotactic activity. The BBXB motif on RANTES has been suggested as the principal site for high-affinity binding to heparan sulfate. This binding controls receptor selectivity (22). It was previously demonstrated that a mutation of CXCL12α in the 24KHLK27 domain reduces the antiviral activity at least 50 percent without affecting the chemotactic activity (4, 29). It was proposed that chemokine binding to HSPG might concentrate the chemokine near the CXCR4 receptor or form a haptotactic chemokine gradient. In this study, we analyzed the mechanism of the potent antiviral activity of CXCL12γ. We examined the role of the additional BBXB domains of CXCL12γ in the observed biological activities of CXCL12γ. Mutations in CXCL12γ were introduced to knock out the BBXB domains either individually or in combination. We analyzed receptor internalization and binding affinities of the mutant chemokines for CXCR4 and HSPG. The results demonstrate that the potent anti-HIV activity of CXCL12γ is due to its efficient binding and internalization of CXCR4. The results provide important insight into the structure-function relationship of CXCL12γ and suggest that determinants other than the BBXB domains are involved in the observed biological activities of CXCL12γ.