In 1957, the term “interferon” (IFN) was coined by Isaacs & Lindenmann to describe a substance, likely produced by cells, that interferes with viral replication. IFN was indeed later shown to be a small protein, produced and secreted by host cells as a protective mechanism following viral infection. Type I IFNs can activate the innate immune system by turning on a transcriptional program that enhances the expression of Interferon Stimulated Genes (ISG), which are highly effective at resisting and controlling pathogens. Notably, the InterFeron Induced Transmembrane (IFITM) proteins are ISGs located at the cell surface and endosomal compartments, and exert their antiviral activity by restricting the cellular entry of a variety of viruses including the human immunodeficiency virus (HIV).
In turn, viruses often make proteins to counteract the infected host’s antiviral activity in an effort to promote their replication in infected cells. Vpx is one example of an accessory protein released by lentiviruses to promote degradation of the host antiviral protein SAMHD1, which functions to deplete dNTPs from the cell and restrict viral replication. A previous study published in 2013 showed that IFN treatment of human monocytic THP1 cells prevents degradation of SAMHD1 in the presence of Vpx, suggesting that other proteins activated by IFN may have a protective effect on SAMHD1. To explain how IFN protects SAMHD1 from degradation by Vpx, members of the Emerman laboratory in the Human Biology division designed a flow cytometry-based CRISPR knockout screen to identify ISGs involved in this process. In their study published in the journal Retrovirology, they identified the IFITM proteins to be the main regulators of this phenotype, and highlight the importance in the pathways viruses use for viral entry as a potential method of HIV-1 evolution to escape restriction factors.
Merging the high-throughput qualities of flow cytometry and next-generation sequencing technologies, the CRISPR knock-out screen consisted of single-guide RNAs (sgRNAs) targeting 1906 human ISGs assembled into a Cas9-encoded lentiviral backbone, transduced into THP1 cells and cultured for gene knockout to occur. The cells were then treated with IFN, exposed to Vpx, and sorted by flow cytometry for cells with low levels of SAMHD1. The rationale behind this was the following: sgRNAs that target genes necessary for SAMHD1 protection would be enriched in the population of cells with low levels of SAMHD1. The authors found that IFITM 2 and 3, but not IFITM1, play a major role in protecting SAMHD1.
To investigate which stage of viral entry is affected by IFN and IFITMs, the authors compared the entry block on HIV-1 pseudotyped with different envelopes. The VSV-G protein can replace the existing envelope proteins of viruses to produce pseudotyped virus particles with new cell tropisms. In this study, the authors used a beta-lactamase assay to measure viral entry. Compared to the wild-type HIV-1 envelope, VSV-G is inhibited to a greater degree, suggesting that HIV-1 may escape IFN induced blocks to viral entry via different cellular pathways for membrane fusion, particularly since IFITMs are expressed on the cell surface.
In summary, using a flow cytometry-based CRISPR knockout screen, IFITMs were identified as the most predominant ISGs responsible for protecting SAMHD1 from degradation by blocking viral entry via inhibition of VSV-G but not HIV-1 envelope viral fusion. IFITMs are known to restrict a number of enveloped viruses such as Influenza, Zika, Ebola, and several others suggesting that the effects of IFITMs on VSV-G-mediated entry/fusion described in this study would be a generalized phenomenon in the life cycle of other viruses. Lead author Dr. Ferdinand Roesch further explained how their study revealed other biological implications: “It is particularly interesting to us that IFITM3 has a very strong effect on a lot of viruses using the endocytic pathway for entry, but only moderately inhibits HIV-1. We interpret this as an indication that HIV fuses predominantly at the plasma membrane, and not in endosomes.”
CRISPR screens often generate vast amounts of information. Dr. Roesch explained that he and his colleagues are continuously mining this valuable resource: “We are currently investigating other genes of interest that we discovered in this screen. In particular, by comparing this data with results from other screens in the lab focusing on wild type HIV, we are identifying genes that may affect viral replication in an envelope-dependent fashion.”
Roesch F, OhAinle M, Emerman E. 2018. A CRISPR screen for factors regulating SAMHD1 degradation identifies IFITMs as potent inhibitors of lentiviral particle delivery. Retrovirology. 15:26
Funding was provided by the National Institutes of Health, and the Co-operative Center for Excellence in Hematology (CCEH) at the Fred Hutch.