Development of an HIV reporter virus that identifies latently infected CD4⁺ T cells

There is no cure for HIV infection, as the virus establishes a latent reservoir, which escapes highly active antiretroviral treatments. One major obstacle is the difficulty identifying cells that harbor latent proviruses. We devised a single-round viral vector that carries a series of versatile reporter molecules that are expressed in an LTR-dependent or LTR-independent manner and make it possible to accurately distinguish productive from latent infection. Using primary human CD4⁺ T cells, we show that transcriptionally silent proviruses are found in more than 50% of infected cells. The latently infected cells harbor proviruses but lack evidence for multiple spliced transcripts. LTR-silent integrations occurred to variable degrees in all CD4⁺ T subsets examined, with CD4⁺ T_EM and CD4⁺ T_REG displaying the highest frequency of latent infections. This viral vector permits the interrogation of HIV latency at single-cell resolution, revealing mechanisms of latency establishment and allowing the characterization of effective latency-reversing agents.