Creation of an Improved HIV Latency Model to Test Latency Reversal Agents

HIV is a retrovirus that infects CD4+ T-cells by integrating into the human genome. During productive infection, the virus enters a latent state in a small subset of cells. This allows HIV to persist and remain undetected by the immune system and current antiretroviral therapies (ART). While ART is highly effective at controlling HIV, it is not a cure. If ART is discontinued, HIV can reactivate and restart the infectious cycle. One proposed cure is the “kick and kill” method, where latency reversal agents (LRAs) are used to reactivate the latent virus, allowing them to be detected and eradicated. Although current cell models exist to test LRA efficacy, these models are unrepresentative of HIV in vivo. We sought to create a new HIV latency model that addresses issues of current cell models, generating three cell lines that each contained both a unique constitutively active fluorescent protein and integrated HIV reporter. We hypothesized these could be pooled to assay LRAs simultaneously on three viruses with unique integration sites, whereby reactivation efficacy could be separated by integration location using the fluorescent protein. Fluorescent proteins were integrated into Jurkat cells using CRISPR, and cells were transduced with defective HIV, then maintained for a month to allow time for latency. We successfully generated two of three intended lines which responded to LRAs. However, constitutive overexpression of fluorescent proteins was toxic and limited the usefulness of this approach.