Use of inhibitors to evaluate coreceptor usage by simian and simian/human immunodeficiency viruses and human immunodeficiency virus type 2 in primary cells

Abstract

We have used coreceptor-targeted inhibitors to investigate which coreceptors are used by human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency viruses (SIV), and human immunodeficiency virus type 2 (HIV-2) to enter peripheral blood mononuclear cells (PBMC). The inhibitors are TAK-779, which is specific for CCR5 and CCR2, aminooxypentane-RANTES, which blocks entry via CCR5 and CCR3, and AMD3100, which targets CXCR4. We found that for all the HIV-1 isolates and all but one of the HIV-2 isolates tested, the only relevant coreceptors were CCR5 and CXCR4. However, one HIV-2 isolate replicated in human PBMC even in the presence of TAK-779 and AMD3100, suggesting that it might use an undefined, alternative coreceptor that is expressed in the cells of some individuals. SIV(mac)239 and SIV(mac)251 (from macaques) were also able to use an alternative coreceptor to enter PBMC from some, but not all, human and macaque donors. The replication in human PBMC of SIV(rcm) (from a red-capped mangabey), a virus which uses CCR2 but not CCR5 for entry, was blocked by TAK-779, suggesting that CCR2 is indeed the paramount coreceptor for this virus in primary cells.

FIG. 1

Testing of the specificity of coreceptor-targeted inhibitors. The replication of the test viruses in GHOST-CD4 cells expressing the coreceptor indicated in the presence and absence of AMD3100 (1.2 μM), AOP-RANTES (AOP-R) (120 nM), TAK-779 (10 μM), or SDF-1α (500 nM) was evaluated. The extent to which replication was inhibited by each agent was recorded.

FIG. 2

Effects of coreceptor-targeted inhibitors on HIV-1 replication in human PBMC. The replication of the HIV-1 isolates P6-v3 and M6-v3 (a) and 5073 and 5160 (b) in human PBMC in the presence and absence of AOP-RANTES (AOP-R) (40 nM [left bar], 4 nM [middle bar], and 0.4 nM [right bar]), TAK-779 (3.3 μM, 330 nM, and 33 nM), or AMD3100 (400 nM, 40 nM, and 4 nM) or with combinations of AMD3100 and either AOP-RANTES or TAK-779 was evaluated. When combinations were used, the concentration of each agent was the same as when the agents were used alone. The extent to which replication was inhibited by each agent or combination was recorded. The coreceptors that can be used by each isolate in GHOST-CD4 cells are indicated below the isolate designation in parentheses.

FIG. 3

Effects of coreceptor-targeted inhibitors on SHIV replication in PBMC. The experimental design was like that described in the legend to Fig. 2. The SHIV isolates evaluated were 89.6PD in macaque and human PBMC (a) and KU-2 and SF33A in human PBMC (b).

FIG. 4

Effects of coreceptor-targeted inhibitors on HIV-2 replication in human PBMC. The experimental design was like that described in the legend to Fig. 2. The HIV-2 isolates evaluated were 310342 and 7312A (a) and 77618 and 7924A (b).

FIG. 5

Effects of coreceptor-targeted inhibitors on HIV-2 isolate 7924A in PBMC from different donors. The replication of HIV-2 7924A in PBMC from four different human donors in the presence of AMD3100 at 40 μM, 4 μM, 400 nM, and 40 nM (a) and SDF-1α at 400 nM, 40 nM, 4 nM, and 0.4 nM (b) was evaluated. HIV-1 NL4-3 was also tested with SDF-1α. In each case, replication was measured after 7 and 10 days. IC₅₀s of AMD3100 were calculated and are shown in panel a. The data shown were obtained on day 10, but values from day 7 were similar.

FIG. 6

Effects of coreceptor-targeted inhibitors on SIV replication in PBMC. The experimental design was like that described in the legend to Fig. 2. The SIV isolates evaluated were SIV_mac251 and SIV_mac239 in macaque PBMC (a), SIV_mac251/1390 and SIV_mac239/5501 in macaque PBMC (b), and SIV_rcm in human PBMC (c). MCP-1 and MCP-3 were used at 400, 40, and 4 nM (left to right); TAK-779 was used at 3.3 μM, 330 nM, and 33 nM.

FIG. 6

Effects of coreceptor-targeted inhibitors on SIV replication in PBMC. The experimental design was like that described in the legend to Fig. 2. The SIV isolates evaluated were SIV_mac251 and SIV_mac239 in macaque PBMC (a), SIV_mac251/1390 and SIV_mac239/5501 in macaque PBMC (b), and SIV_rcm in human PBMC (c). MCP-1 and MCP-3 were used at 400, 40, and 4 nM (left to right); TAK-779 was used at 3.3 μM, 330 nM, and 33 nM.

FIG. 6

Effects of coreceptor-targeted inhibitors on SIV replication in PBMC. The experimental design was like that described in the legend to Fig. 2. The SIV isolates evaluated were SIV_mac251 and SIV_mac239 in macaque PBMC (a), SIV_mac251/1390 and SIV_mac239/5501 in macaque PBMC (b), and SIV_rcm in human PBMC (c). MCP-1 and MCP-3 were used at 400, 40, and 4 nM (left to right); TAK-779 was used at 3.3 μM, 330 nM, and 33 nM.

FIG. 7

Donor-dependent variation in the effects of coreceptor-targeted inhibitors in PBMC. (a) SIV_mac239 replication in PBMC from four different macaques was evaluated in the presence of TAK-779 at 33 μM, 3.3 μM, 330 nM, and 33 nM. SIV_mac251 was similarly evaluated with cells from two donors. (b) HIV-1 P6-v3 replication in PBMC from four different human donors was evaluated in the presence of TAK-779 at 3.3 μM, 330 nM, 33 nM, and 3 nM. In each case, replication was measured after 7 and 10 days, and IC₅₀s of the inhibitor were calculated. The data shown were obtained on day 10, but values from day 7 were similar.