Interferon-Stimulated Genes and Their Protein Products: What and How?

I nterferons (IFNs) are an essential part of vertebral inhibition of virus replication, immune surveillance, and suppression of malignant cells. Cellular actions of IFN-stimulated genes (ISGs), of which several hundreds are transcriptionally regulated, have helped elucidate mechanisms virus inhibition and resistance, roles in host resistance to tumor emergence, and influences on cell homeostasis and communications. Studies of the modes of action of IFNs and ISGs have resulted in fundamental discoveries relevant to translational control, regulation of RNA stability and editing, and protein transport and turnover.

Thirty years ago emerging technologies for molecular biology stimulated interest in not only cloning and expressing IFNs but also for identification of any gene products transcriptionally induced by IFNs. From these latter type of studies discovered were 2 genes of novel function, 2-5A synthetase and protein kinase R (Stark and others 1998). Equally striking was the diversity of functionally important genes identified as regulated with no known direct influence on antiviral activities—such as metallothioneins and major histocompatability complex (MHC) components (Friedman and others 1984). This early work stimulated additional interest in IFNs by investigators in the biochemistry, molecular biology, and immunology research communities. 2-A synthetase and PKR also became an archetype for findings that followed—on both identification of new cellular mechanisms and potently stimulated genes of initially little-defined function.

Because antiviral and cellular actions of these gene and their protein products are now in the process of being functionally further elucidated but have been comprehensively summarized to only limited extents, we have selected some of the most potently induced for review in this issue. Some are clearly part of critical pathways of antiviral actions—the 2′,5′-oligoadenylate-synthetase-directed ribonuclease L proteins, protein kinase R, the ISG15 ubiquitin-like functional pathway, and the Mx GTPase (Chakrabarti and others 2010; Haller and Kochs 2010; Kristiansen and others 2010; Pindel and Sadler 2010; Zhang and Zhang 2010). Adenosine deminases are involved in nucleotide editing of both viral and cellular RNA (George and others 2010). BST-2 (tetherin, HM1.24), initially identified as an antigen on B cells and myeloma, markedly restricts enveloped virus release (Andrew and Strebel 2010). Viperin (cig5) influences lipid rafts, lipid-related virus replication, and protein secretion (Fitzgerald 2010). These ISGs thus individually degrade various viral RNAs, block viral transcription, inhibit translation, and/or modify protein assembly steps for viral replication and release.

Possibly because they have been less associated with the fundamental antiviral definition of IFNs, several other genes that are potently induced have not always been widely recognized as transcriptionally activated ISGs. The unphosphorylated products of STAT1 and STAT2 may well have important cellular or antiviral roles (Cheon and others 2010). Like STATs, members of the IFN regulatory factors (IRF) family have important roles in regulating expression and amplifying effects of ISGs (Tamura and others 2008). IFIT proteins may be induced not only through IRF or STAT pathways but also directly as activation products of toll-like receptors (TLRs) (Fensterl and Sen 2010). Many of the activation products downstream of TLRs are themselves ISGs (Khoo and others 2010; Onoguchi and others 2010). STATs, IRFs, TLR signaling, and IFITs exemplify the interconnected and amplifying nature of ISGs and the IFN-signaling cascade. Other ISG products, not included in this issue, may well prove important proteins in host antiviral resistance. For example, nucleoporins are being identified that have critical roles in both RNA and DNA virus replication (Park and others 2008). Phospholipid scramblase 1, a protein implicated in Ca²⁺-dependent reorganization of plasma membrane phospholipids, may not only alter the plasma membrane or bind DNA but may also increase other ISGs (Dong and others 2004).

Because of the growing number, many other genes induced with similar potency either in various cell types in vitro and/or in patients receiving IFNs have not been reviewed in depth in this issue (Hertzog and others 2011). For example, while of great interest for their role in antiviral and antitumor host responses, ISG products such as those of the MHC complex, the chemokine family, or proapoptotic proteins such as TRAIL have been comprehensively reviewed in other journals. Type I IFNs or IFN-γ can upregulate MHC class I-dependent antigen presentation; other ISGs, such as legumain, IFI30, and transporters for antigen processing, are involved in antigen processing. Chemokines, potently induced by IFNs, accumulate in activated cells of innate and adaptive immunity; these include CCL8 (monocyte chemotactic protein 2), CXCL9 (monokine induced by IFN-γ), CXCL10 (IP-10: IFN-induced peptide), and CXCL11 (IFN-inducible T cell alpha-chemoattractant). Some ISGs may require the transcription factor (promyleocytic zinc finger factor, PLZF) together with histone deacetlyase I and an ISG promyelocytic leukemia protein, for induction (Xu and others 2009; Geoffroy and Chelbi-Alix 2010). Although PLZF is itself not an ISG product, several critical antiviral ISGs are not induced in PLZF-deficient mice and as a consequence these mice are not protected by IFNs against virus infection.

Another important action of ISGs relates to direct influences on malignant transformation. Some products of ISGs, such as those of the IFITM family and the p200 (HIN) family, may mediate tumor suppressor actions (Gariglio and others 2010; Siegrist and others 2010). The promyelocytic leukemia protein sensitizes cells to apoptosis by death ligands through suppression of NF-κB survival pathways (Geoffroy and Chelbi-Alix 2010). ISG12 (IFI27) also has proapoptotic effects, but a related product of the FAM gene family, G1P3 (ISG6-16), inhibits apoptosis (Cheriyath and others 2010). TRAIL and fas are critical for extrinsic apoptotic cascade activation. Neutralization of TRAIL can block IFN-induced cell apoptosis, demonstrating that it was essential, although not necessarily sufficient, to mediate cell death (Chawla-Sarkar and others 2003). XAF1, one of several ISGs that are promoter hypermethylated in malignant cells, can augment proapoptotic effects of TRAIL (Chawla-Sarkar and others 2003). Human polynucleotide phosphorylase can not only degrade miRNA but also induce growth arrest (Das and others 2010). The literature has grown recently, so products of these latter 2 ISGs, XAF1 and human polynucleotide phosphorylase (and others not included herein), could merit comprehensive future review.

In malignant cells resistant to DNA damaging and cytotoxic therapies, ISG products such as IFI27, IFIT1, IFI35, MX1, guanylate-binding protein 2 (GBP2), and ISG15 can be potently and basally expressed (Weichselbaum and others 2008; Luszczek and others 2010). In contrast to sensitive cells in which DNA damage resulted, in radiation-resistant breast carcinoma cells, the ISG expression pattern was a predictor for adverse clinical outcome to radiation and chemotherapy (Weichselbaum and others 2008)—an apparent paradox but possibly reflecting an evolutionary cell survival mechanism. Similar may be the observation that IFNs resulted in activation of hematopoietic stem cells from a dormant state (Essers and others 2009). This unexpected expression of ISGs may contribute to enhanced mechanistic understandings of antitumor effects of IFNs and new therapeutic approaches to overcoming malignant cell resistance.

Inhibition of angiogenesis has emerged as another action of IFNs. ISG products such as the GBPs, ISG20, and CXCL10 may have important roles in this effect. GBPs, though less well characterized for antiviral activity, influence cytoskeletal organization and may have antiangiogenic effects and antiviral effects (Vestal and Jeyaratnam 2010). ISG20, an RNase with single-stranded exonuclease activity, is highly expressed in endothelial cells and thus may have antiangiogenic in addition to antiviral effects (Espert and others 2003; Taylor and others 2008). In contrast to most IFN-regulated gene products, VEGF-protein expression was in treated patients (Yurkovetsky and others 2007). Although of uncertain mechanism, an antiangiogenic chemokine, CXCL5, was markedly inhibited in treated patients (Borden and others 2010). Although decreases in ISGs have not been reproducibly identified in expression arrays, the reduction in VEGF after the addition of IFN-γ in vitro occurred at the translational level (Ray and others 2009).

Many ISGs, indeed most of those reviewed herein, have been identified as RNA and protein products in serum and peripheral blood cells of patients receiving IFNs (see, eg, Yurkovetsky and others 2007; Zimmerer and others 2008; Rani and others 2009; Borden and others 2010). These have included STAT1, IFI27, G1P3, MX1, 2-5AS, GBP1, XAF1, IRF7, ISG15, TRAIL, viperin, BST2, IFIT proteins, CXCL10, CCL8, and IFITM2—in addition to others. Studies have usually been for identification of pharmacodynamic effects of different IFNs. Although target disease tissues have infrequently been biopsied singly or serially, no individual ISG has yet been definitively correlated with therapeutic antiviral, antitumor, or inhibitory effects on multiple sclerosis.

The 25th anniversary of the approval of IFNs-α2 by the U.S. Food and Drug Administration for clinical use is 2011. As the first recombinant DNA product previously unavailable as a pharmaceutical, U.S. Food and Drug Administration approval was a milestone. By improving outcomes for patients worldwide, clinical use of IFNs has been one of the major advances from the biotechnology industry over the past 3 decades. The protein products of ISGs are the critical mediators providing fundamental cellular defense mechanisms against viral infections and cancer. They are thus critically important to the health of animals and humans.

Because all biologic effects of IFNs are mediated through the action of ISGs, understanding the functions of these genes, both individually and collectively, may lead to more efficacious cancer and antiviral therapeutics. For example, IFN-regulated proteins, such as OAS, RNase L, and PKR, exist in either latent inactive or active states; drugs that might act as a molecular switches could have potent antitumor and/or antiviral effects. Insight into the actions of ISGs, the gene products regulated by IFNs, will remain critical to improved clinical application of modulators of the TLR and IFN signaling cascades.