Panning for gold in a dependent-sea of genes

From the Emerman Lab, Human Biology and Basic Sciences Divisions

Despite decades of research and several important breakthroughs, Human Immunodeficiency Virus (HIV) remains a prominent public health threat with no cure which has to this point claimed roughly 40 million lives. Like most small RNA viruses, HIV only contains a meager proportion of the genes it needs to survive; thus, the virus needs to hijack the biological machinery of its host cells to replicate and spread. Identifying which specific pieces of this host machinery are important for the virus—so-called ‘host dependency factors’—is crucial for understanding HIV replication and designing strategies for effecting HIV cures. The Emerman Lab in the Human Biology and Basic Science Divisions at Fred Hutch has studied the biology of HIV for over thirty years. In their recent work, published in mBio and led by Dr. Vanessa Montoya, the group has helped bring the study of HIV dependency factors into today’s high-throughput, CRISPR-fueled era of discovery.

At its heart, Montoya’s strategy to identify HIV host dependency factors centers on an innovative high-throughput technology recently developed by the Emerman Lab called HIV-CRISPR. The method leverages the genome-editing capabilities of CRISPR-Cas9 and the mechanics of HIV infection to systematically screen for host genes critical for HIV function. First, using a different genome-integrating lentivirus, the team generates a collection of T-cells (the primary cell type targeted by HIV in infected hosts), each of which expresses Cas9 and a guide RNA that directs Cas9 to delete a single gene from the host cell genome. Then, they expose this population of modified T-cells to HIV, but there’s a catch: they’ve modified the CRISPR vector so that, upon HIV infection, the guide RNA is incorporated into newly produced virions, which then burst from the infected cells. In a situation where a cell is missing a gene essential for any stage of the HIV life cycle, HIV won’t efficiently infect and replicate in that cell, so that specific guide RNA (corresponding to the essential gene) will not be packaged into newly formed virions and released. By using sequencing to detect guide RNA sequences which are underrepresented in the pool of released viruses, the team is thus able to infer that these guides target genes which are potential HIV host dependency factors.

A female scientists wades in a river containing DNA fragments
An artistic rendering of a scientist 'panning for gold' in a sea of genes, highlighting new discovery-based approaches made possible by CRISPR-based technologies. Image generted by the author using DALL-E 2.

With this powerful tool in hand, Montoya and colleagues implemented it in a line of T-cells which are naturally susceptible to HIV infection. After a suspenseful wait for the results to come back, they were delighted by what they found: not only had their screen returned a collection of previously known HIV dependency factors (giving the team confidence that their screen worked as they intended), but it had also identified many genes not previously thought to have a role in HIV biology. Using bioinformatic tools to summarize the cell processes represented by these top-scoring genes, Montoya found hits participating in nearly every stage of the HIV life cycle, from entry to viral RNA transcription and protein modification.

Not content with a single set of exciting discoveries, Dr. Montoya knew that—like all experiment biologists run—genome-wide screens aren’t perfect, and even ‘statistically significant’ hits may not check out on second look. So, to validate the results of the initial screen, she used the hits from the initial, genome-wide HIV-CRISPR screen to design a second, smaller guide RNA library comprising the top 500 hits, along with ample positive and negative controls. This approach also gave the team another powerful opportunity: since a smaller screen is less resource-intensive to run, they could use this smaller library to screen multiple HIV strains, resulting in a high-confidence list of both strain-specific and global bona fide HIV dependency factors.

Applying their smaller, targeted screen to five genetically distinct strains of HIV-1 virus, Montoya and colleagues were rewarded with good news—of the hits identified in their original, genome-wide screen, 88% were validated in subsequent targeted screens and infection experiments. Considering previously characterized HIV dependency factors, the team estimates that they’ve discovered a whopping 140 novel HIV dependency factors using their approach. This list includes genes involved in processes as diverse as protein degradation, transcription, and RNA splicing, among many others. With abundant replication and thoughtful controls, the study is a tour de force of reproducible science in today’s era of large-scale discovery-focused approaches. The team is following up with mechanistic studies on some of their hits but admits that there are simply too many novel dependency factors here for any one lab to investigate: they hope that these results can serve as an invaluable resource to the community of scientists seeking to better understand how HIV functions and use that knowledge to defeat this virus once and for all.

The spotlighted research was funded by the National Institutes of Health.

Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium member Dr. Michael Emerman contributed to this study.

Montoya, V. R., Ready, T. M., Felton, A., Fine, S. R., OhAinle, M., & Emerman, M. (2023). A Virus-Packageable CRISPR System Identifies Host Dependency Factors Co-Opted by Multiple HIV-1 Strains. MBio, 14(1), e00009-23.