Dual-color HIV reporters trace a population of latently infected cells and enable their purification

Abstract

SUMMARY HIV latency constitutes the main barrier for clearing HIV infection from patients. Our inability to recognize and isolate latently infected cells hinders the study of latent HIV. We engineered two HIV-based viral reporters expressing different fluorescent markers: one HIV promoter-dependent marker for productive HIV infection, and a second marker under a constitutive promoter independent of HIV promoter activity. Infection of cells with these viruses allows the identification and separation of latently-infected cells from uninfected and productively infected cells. These reporters are sufficiently sensitive and robust for high-throughput screening to identify drugs that reactivate latent HIV. These reporters can be used in primary CD4 T lymphocytes and reveal a rare population of latently infected cells responsive to physiological stimuli. In summary, our HIV-1 reporters enable visualization and purification of latent cell populations and open up new perspectives for studies of latent HIV infection.

HIV Latency Is Established Directly and Early in Both Resting and Activated Primary CD4 T Cells

“…A small percentage of the uninfected cell population became infected, which most likely reflects pre-integration latency [18]. More interestingly, 98.8% of the productively infected cell population continued to express GFP throughout the 11 days, with 1.18% of the cells no longer expressing GFP.…”

“…In the uninfected cell population, stimulation produced a small amount of reactivatable provirus which, again, is likely due to pre-integration latency [18]. Interestingly, stimulation of the productively infected cell population did not lead to reactivation of the ~1% of cells that no longer expressed GFP.…”

Eradicating HIV-1 infection: seeking to clear a persistent pathogen

“…Latency may primarily be established in activated CD4 + T cells that are infected as they transition to the resting memory state 11 . However, recent studies using primary CD4 + T cells that are infected with dual-labelled HIV-1 reporter viruses suggest that a small fraction of transcriptionally silent infection occurs directly in activated CD4 + T cells that have not yet transitioned to a resting state 12 . Whether this phenomenon occurs in vivo remains to be determined.…”

COMMD1/Murr1 Reinforces HIV-1 Latent Infection through IκB-α Stabilization

“…Although clinical studies have indicated the existence of HIV-1 latency in myeloid cells and ex vivo analysis also indicated the potential of myeloid cells as a latent-HIV-1 reservoir, the establishment of an optimal model of HIV-1 latency reflecting HIV/AIDS patients is required (60)(61)(62). Recently, a latent HIV-1 infection model was established by using isolated CD4 T cells from peripheral blood mononuclear cells (63,64). However, there is no report of a model for the analysis of latent HIV-1 infection in primary myeloid cells.…”

“…That could make it difficult to analyze latently HIV-1-infected myeloid cells in HIV/AIDS patients. Therefore, to establish a latently HIV-1-infected primary myeloid cell model, we plan to use the double-labeled HIV-1 that encodes LTR-dependent and independent fluorescent proteins recently established by two groups (63,64). This approach might resolve the lack of a primary model in the HIV-1 latency field of study.…”