A doubly fluorescent HIV-1 reporter shows that the majority of integrated HIV-1 is latent shortly after infection.

HIV-1 latency poses a major barrier to viral eradication. Canonically, latency is thought to arise from progressive epigenetic silencing of active infections. However, little is known about when and how long terminal repeat (LTR)-silent infections arise since the majority of the current latency models cannot differentiate between initial (LTR-silent) and secondary (progressive silencing) latency. In this study, we constructed and characterized a novel, double-labeled HIV-1 vector (Red-Green-HIV-1 [RGH]) that allows for detection of infected cells independently of LTR activity. Infection of Jurkat T cells and other cell lines with RGH suggests that the majority of integrated proviruses were LTR-silent early postinfection. Furthermore, the LTR-silent infections were transcriptionally competent, as the proviruses could be reactivated by a variety of T cell signaling agonists. Moreover, we used the double-labeled vector system to compare LTRs from seven different subtypes with respect to LTR silencing and reactivation. These experiments indicated that subtype D and F LTRs were more sensitive to silencing, whereas the subtype AE LTR was largely insensitive. Lastly, infection of activated human primary CD4(+) T cells yielded LTR-silent as well as productive infections. Taken together, our data, generated using the newly developed RGH vector as a sensitive tool to analyze HIV-1 latency on a single-cell level, show that the majority of HIV-1 infections are latent early postinfection.