Single-Cell Characterization of Viral Translation-Competent Reservoirs in HIV-Infected Individuals

Cell Host Microbe. 2016 Sep 14;20(3):368-380. doi: 10.1016/j.chom.2016.07.015. Epub 2016 Aug 18.


HIV cure efforts are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in patients receiving antiretroviral therapy. We combine fluorescent in situ RNA hybridization with detection of HIV protein and flow cytometry, enabling detection of 0.5-1 gag-pol mRNA(+)/Gag protein(+)-infected cells per million. In the peripheral blood of untreated persons, active HIV replication correlated with viremia and occurred in CD4 T cells expressing T follicular helper cell markers and inhibitory co-receptors. In virally suppressed subjects, the approach identified latently infected cells capable of producing HIV mRNA and protein after stimulation with PMA/ionomycin and latency-reversing agents (LRAs). While ingenol-induced reactivation mirrored the effector and central/transitional memory CD4 T cell contribution to the pool of integrated HIV DNA, bryostatin-induced reactivation occurred predominantly in cells expressing effector memory markers. This indicates that CD4 T cell differentiation status differentially affects LRA effectiveness.

MeSH terms

  • CD4-Positive T-Lymphocytes / virology*
  • Cells, Cultured
  • Flow Cytometry
  • HIV Infections / drug therapy*
  • HIV Infections / virology*
  • Humans
  • In Situ Hybridization, Fluorescence
  • RNA, Messenger / analysis*
  • Single-Cell Analysis
  • Sustained Virologic Response*
  • gag Gene Products, Human Immunodeficiency Virus / analysis*
  • pol Gene Products, Human Immunodeficiency Virus / analysis*


  • RNA, Messenger
  • gag Gene Products, Human Immunodeficiency Virus
  • pol Gene Products, Human Immunodeficiency Virus