Stable exposure of the coreceptor-binding site in a CD4-independent HIV-1 envelope protein

Abstract

We recently derived a CD4-independent virus from HIV-1/IIIB, termed IIIBx, which interacts directly with the chemokine receptor CXCR4 to infect cells. To address the underlying mechanism, a cloned Env from the IIIBx swarm (8x) was used to produce soluble gp120. 8x gp120 bound directly to cells expressing only CXCR4, whereas binding of IIIB gp120 required soluble CD4. Using an optical biosensor, we found that CD4-induced (CD4i) epitopes recognized by mAbs 17b and 48d were more exposed on 8x than on IIIB gp120. The ability of 8x gp120 to bind directly to CXCR4 and to react with mAbs 17b and 48d in the absence of CD4 indicated that this gp120 exists in a partially triggered but stable state in which the conserved coreceptor-binding site in gp120, which overlaps with the 17b epitope, is exposed. Substitution of the 8x V3 loop with that from the R5 virus strain BaL resulted in an Env (8x-V3BaL) that mediated CD4-independent CCR5-dependent virus infection and a gp120 that bound to CCR5 in the absence of CD4. Thus, in a partially triggered Env protein, the V3 loop can change the specificity of coreceptor use but does not alter CD4 independence, indicating that these properties are dissociable. Finally, IIIBx was more sensitive to neutralization by HIV-positive human sera, a variety of anti-IIIB gp120 rabbit sera, and CD4i mAbs than was IIIB. The sensitivity of this virus to neutralization and the stable exposure of a highly conserved region of gp120 suggest new strategies for the development of antibodies and small molecule inhibitors to this functionally important domain.

Figure 1

CD4-independent cell–cell fusion and virus infection. (a) QT6 effector cells expressing the indicated Env and T7 polymerase were mixed with QT6 target cells expressing chemokine receptor, CD4, and the luciferase gene under control of the T7 promoter. Luciferase is produced in this assay only if Env mediates fusion between effector and target cells. Results for each Env are expressed in relative light units and are normalized to the amount of fusion obtained with 8x Env effector and CXCR4/CD4 target cells. Results from a typical experiment are shown. (b) Luciferase reporter viruses bearing the indicated Env proteins were used to infect 293T cells expressing CCR5, CD4, or both receptors, and the amount of luciferase activity determined 2 days after infection. Results for each Env are expressed in relative light units and are normalized to the results obtained with virions bearing the 8x-V3BaL Env and CCR5/CD4 target cells. A representative experiment is shown.

Figure 2

Cell-surface gp120 binding. Radioiodinated gp120s were incubated with 293T cells transiently transfected with coreceptor or CD4 plasmids. sCD4 was added to the binding reaction where indicated. The amount of specific radioactivity bound to the cells is presented and is normalized for each gp120 such that binding to CD4 represents 100%. Each value represents the average of at least three independent experiments and error bars represent SEMs.

Figure 3

Overlap between the coreceptor-binding site and the 17b epitope. A space-filling model of gp120 bound to sCD4 is depicted. Residues shown by Rizzuto et al.(17) to decrease CCR5 binding by >50% when mutated while reducing CD4 binding by <50% are colored red (17), contact residues for 17b are shown in light blue (16), and residues involved in both CCR5 and 17b binding are shown in purple. Although 8x contains numerous mutations in gp120 that contribute to the CD4-independent phenotype [C.C.L., T.L.H., J. Romano, B. S. Haggarty, T. J. Matthews, R.W.D. and J.A.H (unpublished work)], only one mutation in 8x affects a contact site for 17b (I423V, shown in green). The stems of the V1/V2 and V3 loops are colored orange.

Figure 4

Sensorgrams for gp120 binding to the CD4i mAb 17b. mAb 17b was attached to the sensor surface, after which the indicated gp120 molecule (at equal concentrations), with or without prior incubation with saturating levels of sCD4, was applied to the flow cell. A 300-sec association was followed by a wash with running buffer for an additional 300 sec, during which dissociation was measured. Kinetic constants derived from linear transformations of the data are presented in Table 1.