Dr. Michael Emerman, a professor in the Human Biology and Basic Sciences Divisions of Fred Hutch, has been hunting the secrets of HIV replication for decades. “There are nearly 40 million people living with HIV,” says Dr. Emerman. “We now have good drugs to keep viral loads below detectable levels and to prevent transmission. However, we do not have the means to “cure” people of their virus such that it would not come back if antiviral therapy were interrupted.” The reason? HIV can “hide” in a subset of cells after integrating into the host genome. Like undercover spies, these so-called HIV proviruses might be imperceptible while a person living with HIV is taking anti-retroviral therapy (ART) but are always spontaneously reactivating at low levels such that disruption of ART allows reactivate viruses to proliferate, wreaking havoc on the immune system. If doctors could specifically shock these latent viruses -into waking up while the person living with HIV was still taking their ART drugs, then other immune mechanisms might be able to clear the latent reservoir for good. Enter Emily Hsieh, a graduate student in the Emerman Lab, and her new tool to trick latent HIV into blowing its cover, published last month on bioRxiv as a pre-print.
“Many transcriptional and epigenetic mechanisms (including transcription initiation and elongation and epigenetic regulators, as highlighted below) have been described to regulate HIV-1 latency, but how these mechanisms interact is still an open question,” said Emily. “We were able to design a new type of CRISPR-based screen to study multiple mechanisms simultaneously (i.e. Latency Reversal Agent (LRA) treatment combined with gene knockout) and identify/validate a novel HIV-1 latency factor.” This “Latency HIV-CRISPR” approach builds on a well-established technique in the Emerman Lab that individually knocks out a large number of genes using CRISPR-Cas9 technology, then assesses the effects of those knockouts in bulk on HIV infection. Emily’s innovation was to flip this approach and look for genes that were involved in latency reactivation, as well as to pair the CRISPR-knockouts with LRA drugs. Dr. Molly OhAinle, a former postdoc who developed the original HIV-CRISPR screen in the Emerman Lab and now leads her own lab at UC Berkeley, expressed her enthusiasm for how Emily adapted the technique. “The biggest excitement [in my opinion] IMO came right out of the gates when Emily showed that [the HIV-CRISPR screening technique] could be adapted to study cell models of HIV latency,” Dr. OhAinle gushed. “It was not at all clear this would work. But the first data showing the Proof of Concept were compelling and a thesis project was born!”
The Emerman Lab combined the small molecule AZD5582, an LRA that activates a noncanonical-NFkB pathway (part of the immflamatory response), with their CRISPR screen. “We discovered that Inhibitor of Growth Family Member (ING3) knockout combined with AZD5582 treatment resulted in enhanced viral reactivation in the J-Lat cells [a type of in vitro T-lymphocyte-based HIV-1 latency model] and a primary CD4+ T cell model of HIV-1 latency,” explained Dr. Emerman. However, the team also had to show that their approach was specifically affecting transcription of the HIV-proviruses – hence, their collaboration with Dr. Derek Janssens of the Henikoff Lab. “We were able to successfully apply CUT&Tag technology in a new, HIV-1 latency context and use it to demonstrate that our screen finding had specificity and potency to the HIV-1 LTR instead of targeting all host promoters,” said Emily. Dr. Janssens emphasized, “when we examined the genome-wide transcriptional response of cells harboring an HIV-provirus to the combination therapies, we found the activation of the HIV-provirus was highly specific. This confirmed our hypothesis that the two pathways we identified through genetic screening really converged to regulate the HIV provirus in a way that is nearly unique in the genome.”
Overall, this project represented a powerful step forward in understanding, and potentially reversing, HIV-1 latency – but as always, more questions remain. “We are interested in understanding how all of the different transcriptional and epigenetic mechanisms interact together to establish and maintain HIV-1 latency,” Emily chimed in. “As a result, we are interested in performing the screen using different LRAs or even combinations of LRAs. Additionally, we are interested to understand if the molecular mechanisms of HIV-1 latency are similar or different in various cell types and primary cells, so we would also like to perform the screen with all of these different variations.” “I think a lot of questions remain,” agreed Dr. Janssens. “This was proof of concept – but can you use combination therapies clinically to reverse HIV latency? [Can] we expand the genetic screening strategy used in this study to identify additional combination therapies that may work even better? Why is the transcriptional mechanism we identified nearly unique to the HIV-provirus?” Dr. Emerman assures me that “others in the lab are testing additional combinations of LRAs in combination with both the epigenetics library and a transcription factor library to explore further leads for latency reversal,” so keep an eye on the Emerman Lab for the answers to these and other questions.
“This work was made possible by an incredible set of collaborators, most of whom are at the Hutch,” concluded Emily. “It has been a great privilege to have many helpful and creative discussions with these collaborators and many others at the Hutch and I am very grateful to be a part of this stimulating scientific community.” Dr. Janssens enthusiastically agreed, “This project was a fun collaboration between multiple labs at the Hutch. The enthusiasm of our collaborators was really inspiring and is part of what makes working at the Hutch so special.”
This work was funded by the National Institutes of Health, the University of Washington Viral Pathogenesis training grant, the National Science Foundation, the Hartwell Foundation, the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute of Allergy and Infectious Diseases, and the Howard Hughes Medical Institute.
Cancer Consortium members Dr. Michael Emerman, Dr. Steven Henikoff, and Dr. Patrick Paddison contributed to this work.
E Hsieh, DH Janssens, PJ Paddison, EP Browne, S Henikoff, M OhAinle, and M Emerman. 2022. A modular CRISPR screen identifies individual and combination pathways contributing to HIV-1 latency. bioRxiv. Online ahead of print.