HIV type I envelope determinants for use of the CCR2b, CCR3, STRL33, and APJ coreceptors

Abstract

The envelope (Env) proteins of primate lentiviruses interact sequentially with CD4 and a coreceptor to infect cells. Changes in coreceptor use strongly influence viral tropism and pathogenesis. We followed the evolution of coreceptor use in pig-tailed macaques that developed severe CD4 T-cell loss during the derivation of a pathogenic simian HIV (SHIV) that contained the tat, rev, vpu, and env genes of the HXBc2 strain of HIV-1 in a genetic background of SIVmac239. The Env from the parental virus as well as one derived from the first macaque to develop AIDS exclusively used CXCR4 as a coreceptor, indicating that CXCR4 can function as a coreceptor in macaques even though it is rarely used by simian immunodeficiency viruses. One Env (Pnb5), obtained from a macrophage-tropic virus isolated from the cerebral spinal fluid, did not use CCR5 or CXCR4. Instead, it used CCR2b and to a lesser extent CCR3, STRL33, and APJ to infect cells. Chimeras between Pnb5 and the parental X4 Env indicated that the V3 loop is the major determinant of CXCR4 use, with other regions of Env influencing the efficiency with which this coreceptor was used. In contrast, the Pnb5 V1/2 and V3 regions in combination were both necessary and sufficient to confer full use of CCR2b, CCR3, STRL33, and APJ to the parental X4 Env protein. These results are consistent with a single, conserved binding site in Env that interacts with multiple coreceptors in conjunction with the V1/2 and V3 loops, and suggest that the V1/2 region plays a more important role in governing the use of CCR2b, CCR3, STRL33, and APJ than for CXCR4.

Figure 1

Coreceptor use profiles of SHIV Env proteins. Viral pseudotypes generated by cotransfection of env and NL-luc-E⁻R⁻ constructs into 293T cells were used to infect feline CCCS target cells expressing CD4 and indicated coreceptor. Infection of target cells leads to integration of the reporter virus genome and long terminal repeat-mediated production of luciferase, which was quantified in cell lysates 3 days after infection. Values were normalized to the level of infection of SHIV-4 with CXCR4 for each experiment. Experiments were performed ≥3 times by using at least two independently derived virus stocks. Error bars represent SEMs. HIV-1 JRFL is shown as a control for CCR5 expression.

Figure 2

The Pnb5 Env protein mediates infection of macaque macrophages. A recombinant virus containing the Pnb5 gp120 in a SHIV-4 background was constructed as described in Materials and Methods and used to inoculate rhesus macaque macrophage cultures. All cultures were inoculated with 1,000 TCID₅₀ of each virus for 2 hr, washed three times to remove the virus inoculum, and maintained in macrophage differentiation medium for the course of the infection (44). Culture medium was harvested at the time points indicated and assayed for the presence of p27 antigen by using antigen capture assays.

Figure 3

Chimeric Env proteins. The gp120 diagram at the top indicates the restriction sites used to generate Env chimeras between SHIV-4 and Pnb5 and their positions relative to the variable regions of HIV-1 Env. Each arrow represents an amino acid difference between the SHIV-4 and Pnb5 Env proteins. The identity of these changes is listed in Table 1. Each chimera was tested for coreceptor use by pseudotype infection (Fig. 4) and for expression by Western blot (data not shown).

Figure 4

Analysis of chimeric Env proteins. Pseudotype reporter viruses for each Env chimera shown in Fig. 3 was tested for infection against CCCS cells expressing CD4 and the indicated coreceptor. Values represent the average of ≥3 experiments using at least two independent virus stocks, and data are normalized to the level of infection of SHIV-4 with CXCR4 in each experiment. Error bars indicate SEMs.

Figure 5

Env determinants for coreceptor use. The ribbon diagram shows gp120 in purple and CD4 in white (56). The V1/V2 and V3 loops are lacking in the structure because of their truncation in the crystallized protein, but the points at which each emanate are shown in yellow. The Env determinants for coreceptor use in this paper reside in or very near to the V1/V2 and V3 loops. Conserved residues in gp120 shown by Rizzuto et al. (56) to play important roles in CCR5 binding are shown in light blue and reside in the bridging sheet, an antiparallel, four-stranded β-sheet structure that links the inner and outer domains of gp120 (56).