HIV CRISPR Screen Provides Novel Dependency Factors

A screening strategy from the Emerman Lab at Fred Hutchinson Cancer Research Center in Seattle has shown capability to identify specific genes that possibly play novel biological roles in HIV infection.

Presentation of the CRISPR screen for HIV dependency factors, showcased at the Conference on Retroviruses and Opportunistic Infections (CROI) 2021 virtual sessions this week, show its potential for helping advance understanding of the HIV-1 life cycle by interpreting dependency factors that could inform therapy and even curative strategies.

Presented by Vanessa R. Montoya, a researcher PhD student from Fred Hutchinson, the HIV-CRISPR screen was designed by herself and colleagues at Emerman Lab to help identify genes that encode the host factors known to be associated with supported HIV infection.

Each HIV life cycle begins with host cellular proteins being hijacked by the virus to set and increase infection of the patient. Though many dependency factors have already been identified, investigators lack a complete interpretation of all involved and in what capacity.

The virus-packagable screen uses a lentiviral vector with a repaired long terminal repeat, making it able package into new virions.

“Packaging of these guideRNAs into nascent virions serves as barcodes to identify which gene targets are required for successful HIV infection,” Montoya and colleagues wrote.

The team conducted a pair of independent HIV-CRISPR screens to comprehensively observed cellular factors at the genome-wide level. They did so via the Toronto Knockout version 3 (TKOv3) library, as well as the targeted human epigenomic/ epigenetic (HuEpi) library, in Jurkat T lymphocyte cells.

The results of the approximate top 400 hits were curated to make new libraries that could interrogate HIV dependency factors across multiple strains of the virus.

Montoya and colleagues identified multiple already known HIV dependency factors across multiple parts of the virus cycle to serve as positive controls, including the HIV receptor and co-receptor, LEDGF/p75, NFkB, and genes encoding components of the Mediator complex.

They also noted numerous genes not previously observed to play a role in HIV biology associated with protein degradation and epigenetic factors, including MEN1, KMT2A, and KMT2D.

As such, they confirmed the screening approach with HIV-CRISPR is sensitive enough to identify host factors for HIV that are involved at all parts of the life cycle. The outcome provides “ample opportunity” to investigate novel gene roles in HIV replication.

“Initial validation experiments confirm the involvement of top hits of the screen in HIV replication,” they wrote.

In her presentation at CROI, Montoya said her toolkit to identify HIV dependency factors and the established library of results from screening has allowed for identification of both clade-specific and strain-specific HIV dependency factors.

“And so far, validation studies indicate that I have found some novel dependency factors,” she concluded.