Restriction factors are host intrinsic antiviral proteins that are often expressed in response to pathogens, triggered by the interferon (IFN)-mediated response. They are involved in inhibiting viruses throughout a range of different steps of viral infection and replication. Identifying the restriction factors involved in the host defense against human immunodeficiency virus (HIV) is essential for understanding virus infection strategies and the adaptation of HIV to humans. Techniques already exist to determine these restriction factors, but they are either unreliable or time consuming. Researchers from the Emerman lab in the Human Biology Division developed a new method for the identification of HIV-targeting restriction factors, published in the journal Elife in December 2018.
“We developed a novel technology that uses high throughput CRISPR screening specific to HIV research that allows for comprehensive discovery of host factors important for HIV infection,” said Dr. Molly OhAinle. In their CRISPR/Cas9 genome editing system, the guide or sgRNA (the nucleotide template allowing the targeted editing of the genome) and the Cas9 protein (a nuclease that is directed by the sgRNA template to edit the genome) can be packaged in the budding HIV virions when the cells are infected. Target cells are engineered with a library of CRISPR/Cas9 lentiviral vectors that knock out the expression of any of the 1905 Interferon Stimulated Genes (ISGs). Genome-edited cells are stimulated with interferon (the trigger for restriction factor expression) and then infected with different strains of HIV that are sensitive to IFN-mediated inhibition. Control cells (without genome editing) are more resistant to HIV infection in the presence of IFN. The virus that infects cells in which the gene for a restriction factor involved in the resistance to HIV infection has been edited will package the sgRNA-Cas9 DNA fragment from the cell genome to a greater extent than cells in which the restriction factors are intact. By sequencing the sgRNA fragment in the viruses released from infected cells, researchers identified restriction factors that are important for the resistance against HIV infection including MxB, IFITM1, Tetherin and TRIM5alpha. To validate these screening hits, they tested the ability of HIV to infect cells with MxB, IFITM1, Tetherin or TRIM5alpha knockouts and confirmed that the infection rate was increased in absence of these restriction factors, thereby validating their method and results. Dr. OhAinle explains: “It is the first proof-of-concept that this type of approach will work.”
Interestingly, the authors also identified sgRNA sequences in virions with a decreased infection rate compared to control, meaning that these sgRNA are targeting genes that the virus hijacks to enter the cell. The proteins encoded by these genes are called dependency factors. They found that SIGLEC1/CD169 and SEC62 are involved in the virus entry whereas TLR2-mediated facilitation of HIV infection is independent of viral entry, which shows that their screening method can also give mechanistic insights.
As Dr. OhAinle highlights, “This technology has the potential to transform our understanding of how host genes affect HIV replication. By harnessing the power of gene editing technology, together with tricking the virus itself into doing most of the work, we have developed a new method to find host genes important for HIV infection. Our findings suggest that there are strain-specific differences in sensitivity to antiviral factors and that we can find these using our HIV-CRISPR approach.”
Although no easy therapeutic target was identified in this version of the screen, the Emerman lab is already working on “variations of this screen […] that will be more targeted for therapeutic uses.” Meanwhile, they hope their work should help the field to “better understand the host factors HIV requires for infection, how our cells fight back against infection and how HIV has evolved to evade or antagonize our antiviral defense,” says Dr. OhAinle.
This work was supported by the National Institutes of Health, the Center for Aids Research (CFAR) and the Belgian American Educational Foundation.
Fred Hutch faculty members Drs Michael Emerman and Julie Overbaugh contributed to this research.
OhAinle M, Helms L, Vermeire J, Roesch F, Humes D, Basom R, Delrow JJ, Overbaugh J, Emerman M. 2018. A virus-packageable CRISPR screen identifies host factors mediating interferon inhibition of HIV. Elife, 7:e39823. DOI: https://doi.org/10.7554/eLife.39823