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. 2016 Mar 29:13:22.
doi: 10.1186/s12977-016-0255-z.

CCR5 interaction with HIV-1 Env contributes to Env-induced depletion of CD4 T cells in vitro and in vivo

Li-Chung Tsao  1   2 , Haitao Guo  2 , Jerry Jeffrey  1 , James A Hoxie  3 , Lishan Su  4   5   6
Affiliations

CCR5 interaction with HIV-1 Env contributes to Env-induced depletion of CD4 T cells in vitro and in vivo

Li-Chung Tsao et al. Retrovirology. .

Abstract

Background: CD4 T cell depletion during HIV-1 infection is associated with AIDS disease progression, and the HIV-1 Env protein plays an important role in the process. Together with CXCR4, CCR5 is one of the two co-receptors that interact with Env during virus entry, but the role of CCR5 in Env-induced pathogenesis is not clearly defined. We have investigated CD4 T cell depletion mechanisms caused by the Env of a highly pathogenic CXCR4/CCR5 dual-tropic HIV-1 isolate R3A.

Results: We report here that R3A infection induced depletion of both infected and uninfected "bystander" CD4 T cells, and treatment with CCR5 antagonist TAK-779 inhibited R3A-induced bystander CD4 T cell depletion without affecting virus replication. To further define the role of Env-CCR5 interaction, we utilized an Env-mutant of R3A, termed R3A-5/6AA, which has lost CCR5 binding capability. Importantly, R3A-5/6AA replicated to the same level as wild type R3A by using CXCR4 for viral infection. We found the loss of CCR5 interaction resulted in a significant reduction of bystander CD4 T cells death during R3A-5/6AA infection, whereas stimulation of CCR5 with MIP1-β increased bystander pathogenesis induced by R3A-5/6AA. We confirmed our findings using a humanized mouse model, where we observed similarly reduced pathogenicity of the mutant R3A-5/6AA in various lymphoid organs in vivo.

Conclusion: We provide the first evidence that shows CCR5 interaction with a dual-tropic HIV-1 Env played a significant role in Env-induced depletion of CD4 T cells.

Keywords: Bystander CD4 T cells; CCR5; HIV-1 Env; HIV-1 pathogenesis; Humanized mice.

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Figures

Fig. 1
Fig. 1
A highly pathogenic HIV-1 isolate R3A induces depletion of both productively infected and bystander CD4 T cells. a HIV-R3A infection in PBMC leads to efficient depletion of CD4+T cells. PBMCs were infected with R3A virus, and stimulated with anti-CD3/CD28/CD2 activation beads at 3 h post infection and cultured in the presence of IL-2 (20 U/mL). FACS Plots show CD4 and CD8 staining of populations gated on live CD3(+) T cells in PBMC at 6 days post infection (dpi). HIV-1 fusion inhibitor T20 was used as negative control to prevent HIV-1 infection. b Graphical summary showing kinetics of CD4 T cells depletion by R3A from 3 to 10 dpi. CD4 T cells are identified as live CD8 (−) CD3(+) population. Data are presented as %CD4 T cell survival relative to mock infection (top) and total CD4 T cell numbers (bottom) over time. c FACS gating strategies are presented. CD4 T cells were identified as CD3(+) CD8(−) population. Uninfected bystander CD4 T cells were identified as p24(−) cells and infected CD4 T cells as p24(+) cells (right graphs). Cell viability of both bystander and infected populations was quantified by co-staining with a cell viability dye (left graphs). d Graphical summary of bystander p24(−) cell death at 6 days post infection by R3A. *p < 0.05
Fig. 2
Fig. 2
CCR5 antagonist TAK-779 protects CD4 T cell from R3A-induced bystander CD4 T cell depletion a PBMCs were treated with CCR5 antagonist TAK-779 (5 μM) before infection with R3A and maintained after infection. HIV-1 viral replication in CD4 T cells was measured by intracellular p24 staining or by extracellular HIV-1 reverse transcriptase levels. b CD4 T cell depletion by R3A in the presence of TAK-779 was measured by FACS analysis as described in Fig. 1b. %CD4 T cells over time relative to mock infected PBCMCs are presented. c Cell viability of bystander and infected CD4 T cells during R3A infection in the presence of TAK-779 was analyzed as described in Fig. 1c. d Graphical summary of viral induced bystander cell death from c, presented as cell death percentage of p24(−) CD4 T cells at 6 days post infection. Experimental results were repeated with PBMCs from 2 different blood donors. *p < 0.05
Fig. 3
Fig. 3
Ablation of CCR5 usage reduces dual-tropic HIV-1 pathogenesis in PBMC infection. a Intracellular p24 staining and FACS analysis was used to quantify viral replication in infected PBMCs. Infected (p24+) CD4 T cell numbers were calculated by multiplying the percentage of (p24+) cells with total CD4 T cell numbers at each time point (left). RT-qPCR analysis of HIV-1 genomic RNA levels was used to measure extracellular viral replication of R3A and R3A-5/6AA in infected PBMCs (right). b Representative FACS Plots are presented showing CD4 and CD8 populations gated on live CD3(+) T cells in PBMC at 6 days post infection. HIV-1 fusion inhibitor T20 treatment was used as negative control. c Kinetics of CD4 T cell depletion by R3A versus R3A-5/6AA was analyzed as described in Fig. 1b. CD4 T cell survival are measured as %CD4 T cells in infected PBMCs relative to mock infection (top), or live CD4 T cell numbers (bottom) over time. d p24(−) and p24(+) CD4 T cell death in R3A and R3A-5/6AA infected PBMCs are quantified at 6 days post infection, as described in Fig. 1C. FACS plots (left) and graphical summary (Right) from a representative experiment are presented. Experimental results were repeated with PBMCs from 3 different blood donors. *p < 0.05
Fig. 4
Fig. 4
CCR5 agonist MIP-1β enhances R3A-5/6AA pathogenesis to promote bystander CD4 T cell depletion PBMCs were infected with R3A-5/6AA virus and treated with recombinant MIP-1 β (200 ng/mL) at 3 h post infection. At 6 days post infection, viabilities of p24(−) and p24(+) CD4 T cell were measured as described in Fig. 1c. a Representative FACS plots and b graphical summary are presented. c %CD4 T cells survival at 6 days post infection was measured by gating on live CD8(−) CD3(+) cells. *p < 0.05
Fig. 5
Fig. 5
Ablation of CCR5 usage reduces pathogenesis of dual tropic HIV-1 in a humanized mouse model. a Humanized mice were infected with R3A or R3A-5/6AA as described in “Methods” section. Viral replication was assessed weekly by RT-qPCR measurement of HIV-1 genomic RNA in the blood. HIV-1 infection in CD4 T cells was analyzed by intracellular p24 staining of splenocytes isolated from infected mice at 3 weeks post infection (wpi). b Total PBMCs in blood, spleen and bone marrow from infected animals were harvested at 3wpi, and CD4 T cell depletion was analyzed by FACS staining as described in Fig. 1b. c p24(−) and p24(+) CD4 T cell viability in R3A versus R3A-5/6AA infection in the infected animal’s spleen and bone marrow were analyzed as described in Fig. 1c. *p < 0.05
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