Investigating the role of HIV-1 Nef during reactivation of latent viral reservoirs

A major barrier to development of a cure for HIV-1 is the ability of the virus to establish latent infection that reactivates to cause disease if antiretroviral therapy is discontinued. The HIV-1 protein Nef displays multiple in vitro and in vivo functions, including the ability to modulate T cell signaling events that may alter the activation status of infected cells. Nef is critical for viral pathogenesis, but its role during latency remains unclear. To investigate this, I generated a novel panel of latent T cell clones (C-Lat) harbouring a single integrated copy of HIV-1 encoding functional or defective nef genes. By assessing the location and genomic features of the proviral DNA integration sites in these clones, I observed that the presence of functional Nef variants was associated with a broader repertoire of inducible latent T cell lineages. By characterizing the reactivation phenotypes of these clones following stimulation with latency reversing agents (LRAs), I observed higher early and late viral protein expression in C-Lat clones encoding functional nef compared to those encoding defective nef. I confirmed these observations by disrupting the functional nef gene in C-Lat clones using CRISPR/Cas9 strategies. Variable viral reactivation phenotypes were observed in Nef knock-out (NefKO) clones following stimulation with LRAs, but the efficiency of early and late viral protein expression was consistently lower in NefKO clones compared to their corresponding parental isolates. My research highlights the ability of Nef to modulate HIV-1 reactivation from latency. Results indicate that Nef may play an important role in determining the breadth and diversity of inducible viral reservoirs following infection. If so, a better mechanistic understanding of Nef’s impact may uncover new strategies to enhance viral reactivation from latency that are clinically beneficial.