Identification of the platelet-derived chemokine CXCL4/PF-4 as a broad-spectrum HIV-1 inhibitor

Abstract

The natural history of HIV-1 infection is highly variable in different individuals, spanning from a rapidly progressive course to a long-term asymptomatic infection. A major determinant of the pace of disease progression is the in vivo level of HIV-1 replication, which is regulated by a complex network of cytokines and chemokines expressed by immune and inflammatory cells. The chemokine system is critically involved in the control of HIV-1 replication by virtue of the role played by specific chemokine receptors, most notably CCR5 and CXCR4, as cell-surface coreceptors for HIV-1 entry; hence, the chemokines that naturally bind such coreceptors act as endogenous inhibitors of HIV-1. Here, we show that the CXC chemokine CXCL4 (PF-4), the most abundant protein contained within the α-granules of platelets, is a broad-spectrum inhibitor of HIV-1 infection. Unlike other known HIV-suppressive chemokines, CXCL4 inhibits infection by the majority of primary HIV-1 isolates regardless of their coreceptor-usage phenotype or genetic subtype. Consistent with the lack of viral phenotype specificity, blockade of HIV-1 infection occurs at the level of virus attachment and entry via a unique mechanism that involves direct interaction of CXCL4 with the major viral envelope glycoprotein, gp120. The binding site for CXCL4 was mapped to a region of the gp120 outer domain proximal to the CD4-binding site. The identification of a platelet-derived chemokine as an endogenous antiviral factor may have relevance for the pathogenesis and treatment of HIV-1 infection.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Inhibition of HIV-1 replication by recombinant human CXCL4. (A) Dose-dependent inhibition of a prototypic X4 HIV-1 strain (IIIB) in the immortalized human CD4+ T-cell lines MT-2, PM1, and Sup-T1. (B) Dose-dependent inhibition of two HIV-1 variants with different coreceptor-usage phenotype [HIV-1 IIIB (X4) and HIV-1 BaL (R5)] in primary human CD4+ T cells. (C) Dose-dependent inhibition of a prototypic macrophage-tropic HIV-1 strain (BaL, R5) in primary human macrophage cultures. Although CXCL4 is predominantly a tetramer in solution, molar values were calculated on the basis of the molecular weight of the monomer (∼7.8 Kd); thus, the data represent the most conservative estimate of the antiviral potency of CXCL4. Virus replication was assessed by measuring the amount of extracellular p24 Gag protein by enzyme immunoassay. To reduce interexperimental variability, the data were normalized with respect to the level of virus replication detected in control cultures (not treated with CXCL4). All of the data represent mean values (±SD) from at least three independent experiments, each performed in duplicate.

Fig. 2.

CXCL4 inhibits the early steps of the HIV-1 replication cycle. (A) Time dependence of CXCL4 sensitivity as evaluated using the MAGI assay. Enumeration of blue cells after 48 h denotes the completion of a single infectious cycle of HIV-1. To reduce interexperimental variability, the data were normalized with respect to the number of blue cells detected in control cultures (C) not treated with CXCL4. CXCL4 was added at different times relative to infection with HIV-1 IIIB or HIV-1 BaL, and the test was developed after 48 h of infection. Results shown are from a representative experiment performed in duplicate wells. (B) Effect of CXCL4 on HIV-1 attachment. The attachment assay was performed using activated primary human CD4+ T cells incubated with the infectious stock of a CXCL4-sensitive primary HIV-1 isolate (92HT599; R5X4). Virus attachment was measured as the total amount of cell-associated HIV-1 p24 Gag protein after subtraction of background p24 levels (measured in cells incubated with the virus at 4 °C and then treated with trypsin to remove cell surface-bound HIV-1 virions). (C) Effect of CXCL4 on HIV-1 entry. For the entry assay, at the end of the incubation period the cells were treated with trypsin to remove residual extracellular virions bound to the external surface of the cells. Virus entry was measured as the amount of trypsin-resistant, intracellular p24 protein. The recombinant chemokines and the anti-CD4 mAb (RPA-T4) were used at 5 μg/mL; peptide T20 was used at 50 μg/mL. Data represent mean values (±SD) from at least three independent experiments. *P < 0.05 and **P < 0.001 vs. untreated control by paired two-tailed Student t test.

Fig. 3.

CXCL4 directly interacts with the major HIV-1 envelope glycoprotein, gp120. (A) Capture of HIV-1 virions (strain IIIB; X4) by CXCL4-armed immunomagnetic beads. Polyclonal goat anti-CXCL4 IgG and irrelevant goat IgG were used at 20 μg/mL; anti-CXCL4 mAbs (nos. 7951 and 7952) at 5 μg/mL. Data represent mean values (±SD) from at least three independent experiments. **P < 0.001 by paired two-tailed Student t test. (B) Coimmunoprecipitation of the HIV-1 envelope glycoproteins gp120 and gp160 by CXCL4. Assays were performed using the PM1 clone (28) persistently infected with HIV-1 IIIB (X4), which expresses on its surface both gp120 and its uncleaved precursor, gp160. Lane 1, gp120 and gp160 precipitated with CXCL4 and anti-CXCL4 rabbit antiserum; lane 2, anti-CXCL4 rabbit antiserum alone; lane 3, CCL3 with anti-CCL3 rabbit antiserum; lane 4, anti-CCL3 rabbit antiserum alone; lane 5, positive control (direct immunoprecipitation with the anti-gp120 mAb 2G12). (C) Inhibition of HIV-1 virion capture by polyclonal antisera, soluble CD4 (sCD4) and human mAbs directed against HIV-1 gp120. Polyclonal goat antisera against recombinant gp120 outer-domain fragments (PB1 and PB1 Sub 7) were used at 1:50 dilution; irrelevant goat IgG at 100 μg/mL; 4-domain sCD4 at 10 μg/mL; human anti-gp120 mAbs at 10 μg/mL. Data represent the mean values (±SD) from at least four independent experiments performed for each inhibitor. *P < 0.05 and **P < 0.01 vs. untreated control by paired two-tailed Student t test.