Characterization of chemokine receptor utilization of viruses in the latent reservoir for human immunodeficiency virus type 1

Abstract

Latently infected resting CD4(+) T cells provide a long-term reservoir for human immunodeficiency virus type 1 (HIV-1) and are likely to represent the major barrier to virus eradication in patients on combination antiretroviral therapy. The mechanisms by which viruses enter the latent reservoir and the nature of the chemokine receptors involved have not been determined. To evaluate the phenotype of the virus in this compartment with respect to chemokine receptor utilization, full-length HIV-1 env genes were cloned from latently infected cells and assayed functionally. We demonstrate that the majority of the viruses in the latent reservoir utilize CCR5 during entry, although utilization of several other receptors, including CXCR4, was observed. No alternative coreceptors were shown to be involved in a systematic fashion. Although R5 viruses are present in the latent reservoir, CCR5 was not expressed at high levels on resting CD4(+) T cells. To understand the mechanism by which R5 viruses enter latent reservoir, the ability of an R5 virus, HIV-1 Ba-L, to infect highly purified resting CD4(+) T lymphocytes from uninfected donors was evaluated. Entry of Ba-L could be observed when virus was applied at a multiplicity approaching 1. However, infection was limited to a subset of cells expressing low levels of CCR5 and markers of immunologic memory. Naive cells could not be infected by an R5 virus even when challenged with a large inoculum. Direct cell fractionation studies showed that latent virus is present predominantly in resting memory cells but also at lower levels in resting naive cells. Taken together, these findings provide support for the hypothesis that the direct infection of naive T cells is not the major mechanism by which the latent infection of resting T cells is established.

Figures

FIG. 1

CCR5 expression on resting CD4+ T cells at various stages of purification. Highly purified resting CD4+ T cells were isolated from the peripheral blood of HIV-negative donors by removing unwanted cell lineages and activated CD4+ T cells through sequential bead depletion and cell sorting procedures. HLA-DR was used as a marker of activation. The purity of resting CD4+ T cells was invariably greater than 99%. Cells at multiple points of the procedure were stained for CCR5 expression using a FITC conjugate of the anti-CCR5 monoclonal 2D7.

FIG. 2

Levels of CCR5 on highly purified resting T lymphocytes are sufficient to mediate infection by the R5 virus Ba-L. The ability of highly purified resting cells to be infected with the R5 virus HIV-1 Ba-L and the X4 virus HIV-1 IIIb was determined. Cells were infected at an MOI of 0.1 (A) or 1.0 (B). After incubation for the indicated time at 37°C, cells were lysed, and entry was monitored by PCR assay for early (LTR) and late (LTR-Gag) products of reverse transcription as described (46). In control lanes, virus was heat-inactivated (HI) before infection. The sensitivity of the PCR in each experiment was assayed using a series of ACH-2 dilutions as a target for the PCR described above. A fragment of the β-globin gene was amplified to normalize for the number of cells used in each PCR experiment. Infection experiments were performed at least twice on cells from four different donors.

FIG. 3

Purification of naive and memory resting CD4+ T cell. PBMC from HIV-1-negative donors were subjected to magnetic depletion using monoclonal antibodies to CD8, CD19, CD16, CD14, CD69, CD25, HLA-DR, and either CD45 RA or RO. Cells were sorted for those expressing CD4 while lacking expression of either HLA-DR or the relevant CD45 isoform. Sorted populations contained less than 1.0% contaminants expressing HLA-DR or the inappropriate CD45 isoform.

FIG. 4

Infection of resting cells by R5 viruses is restricted to those expressing markers of immunologic memory. Resting CD4+ lymphocytes depleted of either naive or memory subsets were obtained and infected with HIV-1 Ba-L at a multiplicity of 1.0. Infection was monitored over the course of 3 days. Infection was monitored through the detection of the products of reverse transcription as described using M661 and M667 (46). The sensitivity of the PCR in each experiment was assayed using a series of ACH-2 dilutions as a target for the PCR against the LTR-Gag junction. Amplification of β-globin was performed to demonstrate the presence of an equal mass of DNA in each sample. This experiment was performed on samples from two donors with identical results.