Novel pathway for induction of latent virus from resting CD4(+) T cells in the simian immunodeficiency virus/macaque model of human immunodeficiency virus type 1 latency

Although combination therapy allows the suppression of human immunodeficiency virus type 1 (HIV-1) viremia to undetectable levels, eradication has not been achieved because the virus persists in cellular reservoirs, particularly the latent reservoir in resting CD4(+) T lymphocytes. We previously established a simian immunodeficiency virus (SIV)/macaque model to study latency. We describe here a novel mechanism for the induction of SIV from latently infected resting CD4(+) T cells. Several human cell lines including CEMx174 and Epstein-Barr virus-transformed human B-lymphoblastoid cell lines mediated contact-dependent activation of resting macaque T cells and induction of latent SIV. Antibody-blocking assays showed that interactions between the costimulatory molecule CD2 and its ligand CD58 were involved, whereas soluble factors and interactions between T-cell receptors and major histocompatibility complex class II were not. Combinations of specific antibodies to CD2 also induced T-cell activation and virus induction in human resting CD4(+) T cells carrying latent HIV-1. This is the first demonstration that costimulatory signals can induce latent virus without the coengagement of the T-cell receptor, and this study might provide insights into potential pathways to target latent HIV-1.     

Human immunodeficiency virus type 1 can establish latent infection in resting CD4+ T cells in the absence of activating stimuli

Resting CD4(+) T cells are the best-defined reservoir of latent human immunodeficiency virus type 1 (HIV-1) infection, but how the reservoir is formed is unclear. Understanding how the reservoir of latently infected cells forms is critical because it is a major barrier to curing HIV infection. The system described here may provide an in vitro model of latent HIV-1 infection in resting CD4(+) T cells. We demonstrated that HIV-1 integrates into the genomes of in vitro-inoculated resting CD4(+) T cells that have not received activating stimuli and have not entered cell cycle stage G(1b). A percentage of the resting CD4(+) T cells that contain integrated DNA produce virus upon stimulation, i.e., are latently infected. Our results show that latent HIV-1 infection occurs in unstimulated resting CD4(+) T cells and suggest a new route for HIV-1 reservoir formation.     

Disulfiram reactivates latent HIV-1 in a Bcl-2-transduced primary CD4+ T cell model without inducing global T cell activation

Highly active antiretroviral therapy (HAART) can reduce plasma HIV-1 levels to below the detection limit. However, due to the latent reservoir in resting CD4(+) cells, HAART is not curative. Elimination of this reservoir is critical to curing HIV-1 infection. Agents that reactivate latent HIV-1 through nonspecific T cell activation are toxic. Here we demonstrate in a primary CD4(+) T cell model that the FDA-approved drug disulfiram reactivates latent HIV-1 without global T cell activation. The extent to which disulfiram reactivates latent HIV-1 in patient cells is unclear, but the drug alone or in combination may be useful in future eradication strategies.     

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

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.     

Human immunodeficiency virus type 1 (HIV-1) antigen secretion by latently infected resting CD4+ T lymphocytes from HIV-1-infected individuals

In resting CD4(+) T lymphocytes harboring human immunodeficiency virus type 1 (HIV-1), replication-competent virus persists in patients responding to highly active antiretroviral therapy (HAART). This small latent reservoir represents between 10(3) and 10(7) cells per patient. However, the efficiency of HIV-1 DNA-positive resting CD4(+) T cells in converting to HIV-1-antigen-secreting cells (HIV-1-Ag-SCs) after in vitro CD4(+)-T-cell polyclonal stimulation has not been satisfactorily evaluated. By using an HIV-1-antigen enzyme-linked immunospot assay, 8 HIV-1-Ag-SCs per 10(6) CD4(+) resting T cells were quantified in 25 patients with a plasma viral load of <20 copies/ml, whereas 379 were enumerated in 10 viremic patients. In parallel, 369 and 1,238 copies of HIV-1 DNA per 10(6) CD4(+) T cells were enumerated in the two groups of patients, respectively. Only a minority of latently HIV-1 DNA-infected CD4(+) T cells could be stimulated in vitro to become HIV-1-Ag-SCs, particularly in aviremic patients. The difference between the number of HIV-1 immunospots in viremic versus aviremic patients could be explained by HIV-1 unintegrated viral DNA that gave additional HIV-1-Ag-SCs after in vitro CD4(+)-T-cell polyclonal stimulation. The ELISPOT approach to targeting the HIV-1-Ag-SCs could be a useful method for identifying latently HIV-1-infected CD4(+) T cells carrying replication-competent HIV-1 in patients responding to HAART.