The interferon-stimulated genes: targets of direct signaling by interferons, double-stranded RNA, and viruses.

The interferon system plays a profound role in determining the outcome of viral infection in mammals. Viruses induce the synthesis of interferon, which, in turn, blocks virus replication by inducing the expression of antiviral proteins encoded by interferon-stimulated genes. It is not widely appreciated that without the participation of interferon, many of the same genes can also be induced by a variety of virus-related agents, such as double-stranded RNA and viral proteins. In this chapter, we discuss different signaling pathways, activated by these agents, that lead to the induction of partially overlapping sets of genes, including the interferon-stimulated genes. We also review the biochemical and cellular properties of the protein products of a selected number of these genes including ISG56, ISG54, and ISG15.

Apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis.

IFNs are a family of cytokines with pleiotropic biological effects mediated by scores of responsive genes. IFNs were the first human proteins to be effective in cancer therapy and were among the first recombinant DNA products to be used clinically. Both quality and quantity of life has been improved in response to IFNs in various malignancies. Despite its beneficial effects, unraveling the mechanisms of the anti-tumor effects of IFN has proven to be a complex task. IFNs may mediate anti-tumor effects either indirectly by modulating immunomodulatory and anti-angiogenic responses or by directly affecting proliferation or cellular differentiation of tumor cells. Both direct or indirect effects of IFNs result from induction of a subset of genes, called IFN stimulated genes (ISGs). In addition to the ISGs implicated in anti-viral, anti-angiogenic, immunomodulatory and cell cycle inhibitory effects, oligonucleotide microarray studies have identified ISGs with apoptotic functions. These include TNF-alpha related apoptosis inducing ligand (TRAIL/Apo2L), Fas/FasL, XIAP associated factor-1 (XAF-1), caspase-4, caspase-8, dsRNA activated protein kinase (PKR), 2'5'A oligoadenylate synthetase (OAS), death activating protein kinases (DAP kinase), phospholipid scramblase, galectin 9, IFN regulatory factors (IRFs), promyelocytic leukemia gene (PML) and regulators of IFN induced death (RIDs). In vitro IFN-alpha, IFN-beta and IFN-gamma induced apoptosis in multiple cell lines of varied histologies. This review will emphasize possible mechanisms and the role of ISGs involved in mediating apoptotic function of IFNs.

Organizations and promoter analyses of the human and the mouse genes for PACT, the protein-activator of the interferon-induced protein kinase, PKR.

PACT is an activator of the protein kinase, PKR. Here we report the isolation and the characterization of the mouse Pact gene. It contains eight exons ranging in size from 79 to 630 bp spanning a region of 18 kb with the largest and smallest introns being 3700 and 500, respectively. The human PACT gene, as analyzed from sequence available in the GenBank database, has a very similar organization. The 5' flanking regions of both mouse and human PACT genes are devoid of TATA boxes but are rich in GC boxes. Although there are putative binding sites of numerous transcription factors on both promoters, their organizations and identities are different. For examining promoter activities, about 2 kb of DNA 5' to the transcription start sites of both genes was cloned upstream of a reporter luciferase gene. Transient transfection assays demonstrated that both promoters are strong. Deletion analyses revealed that most of the positive cis-elements lie within 400 bp upstream of the transcription start sites of both mouse and human PACT genes.

Viruses and interferons.

The interferon system is the first line of defense against viral infection in mammals. This system is designed to block the spread of virus infection in the body, sometimes at the expense of accelerating the death of the infected cells. As expected of potent cytokines, in addition to their antiviral effects, interferons have profound effects on many aspects of cell physiology. All these actions of interferons are mediated by hundreds of interferon-induced proteins that are usually not synthesized in resting cells. Interferons induce their synthesis by activating the Jak-STAT pathways, a paradigm of cell signaling used by many cytokines and growth factors. Surprisingly, some of the same genes can also be induced directly by viruses and double-stranded RNA, a common viral by-product. Some of the interferon-induced proteins have novel biochemical properties and some are inactive as such but can be activated by double-stranded RNA produced during virus infection. Finally, almost all viruses have evolved mechanisms to evade the interferon system by partially blocking interferon synthesis or interferon action. Thus, in nature interferons and viruses maintain an equilibrium that allows regulated viral replication.

A comprehensive view of regulation of gene expression by double-stranded RNA-mediated cell signaling.

Double-stranded (ds) RNA, a common component of virus-infected cells, is a potent inducer of the type I interferon and other cellular genes. For identifying the full repertoire of human dsRNA-regulated genes, a cDNA microarray hybridization screening was conducted using mRNA from dsRNA-treated GRE cells. Because these cells lack all type I interferon genes, the possibility of gene induction by autocrine actions of interferon was eliminated. Our screen identified 175 dsRNA-stimulated genes (DSG) and 95 dsRNA-repressed genes. A subset of the DSGs was also induced by different inflammatory cytokines and viruses demonstrating interconnections among disparate signaling pathways. Functionally, the DSGs encode proteins involved in signaling, apoptosis, RNA synthesis, protein synthesis and processing, cell metabolism, transport, and structure. Induction of such a diverse family of genes by dsRNA has major implications in host-virus interactions and in the use of RNA(i) technology for functional ablation of specific genes.

A specific isozyme of 2'-5' oligoadenylate synthetase is a dual function proapoptotic protein of the Bcl-2 family.

2-5(A) synthetases are a family of interferon-induced enzymes that polymerize ATP into 2'-5' linked oligoadenylates that activate RNase L and cause mRNA degradation. Because they all can synthesize 2-5(A), the reason for the existence of so many synthetase isozymes is unclear. Here we report that the 9-2 isozyme of 2-5(A) synthetase has an additional activity: it promotes apoptosis in mammalian cells. The proapoptotic activity of 9-2 was isozyme-specific and enzyme activity-independent. The 9-2-expressing cells exhibited many properties of cells undergoing apoptosis, such as DNA fragmentation, caspase activation, and poly ADP-ribose polymerase and lamin B cleavage. The isozyme-specific carboxyl-terminal tail of the 9-2 protein was shown, by molecular modeling, to contain a Bcl-2 homology 3 (BH3) domain, suggesting that it may be able to interact with members of the Bcl-2 family that contain BH1 and BH2 domains. Co-immunoprecipitate assays and confocal microscopy showed that 9-2 can indeed interact with the anti-apoptotic proteins Bcl-2 and Bclx(L) in vivo and in vitro. Mutations in the BH3 domain that eliminated the 9-2-Bcl-2 amd 9-2-Bclx(L) interactions also eliminated the apoptotic activity of 9-2. Thus, we have identified an interferon-induced dual function protein of the Bcl-2 family that can synthesize 2-5(A) and promote cellular apoptosis independently. Moreover, the cellular abundance of this protein is regulated by alternative splicing; the other isozymes encoded by the same gene are not proapoptotic.

Modular structure of PACT: distinct domains for binding and activating PKR.

PACT is a 35-kDa human protein that can directly bind and activate the latent protein kinase, PKR. Here we report that PKR activation by PACT causes cellular apoptosis in addition to PKR autophosphorylation and translation inhibition. We analyzed the structure-function relationship of PACT by measuring its ability to bind and activate PKR in vitro and in vivo. Our studies revealed that among three domains of PACT, the presence of either domain 1 or domain 2 was sufficient for high-affinity binding of PACT to PKR. On the other hand, domain 3, consisting of 66 residues, was absolutely required for PKR activation in vitro and in vivo. When fused to maltose-binding protein, domain 3 was also sufficient for efficiently activating PKR in vitro. However, it bound poorly to PKR at the physiological salt concentration and consequently could not activate it properly in vivo. As anticipated, activation of PKR by domain 3 in vivo could be restored by attaching it to a heterologous PKR-binding domain. These results demonstrated that the structure of PACT is modular: it is composed of a distinct PKR-activation domain and two mutually redundant PKR-interacting domains.

A new pathway of translational regulation mediated by eukaryotic initiation factor 3.

We report a new pathway of translation regulation that may operate in interferon-treated or virus-infected mammalian cells. This pathway is activated by P56, a protein whose synthesis is strongly induced by interferons or double-stranded RNA. Using a yeast two-hybrid screen, we identified the P48 subunit of the mammalian translation initiation factor eIF-3 as a protein that interacts with P56. The P56-P48 interaction was confirmed in human cells by co-immunoprecipitation assays and confocal microscopy. Gel filtration assays revealed that P56 binds to the large eIF-3 complex that contains P48. Purified recombinant P56 inhibited in vitro translation of reporter mRNAs in a dose-dependent fashion, and that inhibition was reversed by the addition of purified eIF-3. In vivo, expression of transfected P56 or induction of the endogenous P56 by interferon caused an inhibition of overall cellular protein synthesis and the synthesis of a transfected reporter protein. As expected, a P56 mutant that does not interact with P48 and eIF-3 failed to inhibit protein synthesis in vitro and in vivo.

Different subcellular localizations for the related interferon-induced GTPases, MuGBP-1 and MuGBP-2: implications for different functions?

The guanylate-binding proteins (GBPs) are a family of 65-67-kDa proteins induced by both type I and type II interferons (IFN). Members of the GBP family of GTPases are among the most abundant IFN-gamma-induced proteins. GBPs contain an unusual GTP binding site, which is consistent with GBP hydrolysis of GTP to both GDP and GMP. In addition, six of the eight known GBPs have a carboxy-terminal CaaX motif for the addition of isoprenyl lipids. Despite their abundance, however, little is known about the biologic function or cellular location of GBPs. We report here on studies to localize both a newly identified murine GBP (MuGBP-2) and its closely related family member, MuGBP-1. In both IFN-treated macrophages and fibroblasts, MuGBP-2 is found in both a granular distribution throughout the cytoplasm and localized to vesicle populations of heterogeneous sizes. The localization of MuGBP-2 to vesicles is dependent on its isoprenylation. Despite a high degree of sequence identity and the presence of an identical CaaX sequence, MuGBP-1 has a very homogeneous cytoplasmic distribution and fails to localize to intracellular vesicles. The different intracellular distribution of these two closely related family members suggests differential function(s).

Novel functions of interferon-induced proteins.

Interferons are important cytokines which regulate antiviral, cell growth, immune modulatory and anti-tumor functions. These pleiotropic effects of interferons are brought about by a large number of cellular proteins, the interferon-inducible proteins. Investigation of the biochemical and cellular activities of some of these proteins have revealed new pathways of regulation of cellular RNA and protein metabolism, growth and differentiation, apoptosis and signal transduction. In this article we discuss recent findings on the novel activities of a selected number of interferon-induced proteins.

Physiological non-equivalence of the two isoforms of angiotensin-converting enzyme.

The structurally related somatic and germinal isoforms of angiotensin-converting enzyme (ACE) contain the same catalytic active center and are encoded by the same gene, whose disruption causes renal atrophy, hypotension, and male sterility. The reason for the evolutionary conservation of both isozymes is an enigma, because, in vitro, they have very similar enzymatic properties. Despite the common enzymatic properties, discrete expression of both isoforms is maintained in alternate cell types. We have previously shown that sperm-specific expression of transgenic germinal ACE in Ace -/- male mice restores fertility without curing their other abnormalities (Ramaraj, P., Kessler, S. P., Colmenares, C. & Sen, G. C. (1998) J. Clin. Invest. 102, 371-378). In this report we tested the biological equivalence of somatic ACE and germinal ACE utilizing an in vivo isozymic substitution approach. Here we report that restoration of male fertility was not achieved by the transgenic expression of enzymatically active, somatic ACE in the sperm of Ace -/- mice. Therefore, the requisite physiological functions of the two tissue-specific isozymes of ACE are not interchangeable.

Induction of the human protein P56 by interferon, double-stranded RNA, or virus infection.

P56 is the most abundant protein induced by interferon (IFN) treatment of human cells. To facilitate studies on its induction pattern and cellular functions, we expressed recombinant P56 as a hexahistidine-tagged protein in Escherichia coli and purified it to apparent homogeneity using affinity chromatography. A polyclonal antibody raised against this recombinant protein was used to show that P56 is primarily a cytoplasmic protein. Cellular expression of P56 by transfection did not inhibit the replication of vesicular stomatitis virus and encephalomyocarditis virus. P56 synthesis was rapidly induced by IFN-beta, and the protein had a half-life of 6 h. IFN-gamma or poly(A)(+) could not induce the protein, but poly(I)-poly(C) or an 85-bp synthetic double-stranded RNA efficiently induced it. Similarly, infection of GRE cells, which are devoid of type I IFN genes, by vesicular stomatitis virus, encephalomyocarditis virus, or Sendai virus caused P56 induction. Surprisingly, Sendai virus could also induce P56 in the mutant cell line P2.1, which cannot respond to either IFN-alpha/beta or double-stranded RNA. Induction of P56 in the P2.1 cells and the parental U4C cells by virus infection was preceded by activation of IRF-3 as judged by its translocation to the nucleus from the cytoplasm.

Characterization of the interaction between the interferon-induced protein P56 and the Int6 protein encoded by a locus of insertion of the mouse mammary tumor virus.

For determining cellular functions of the interferon-inducible human cytoplasmic protein P56, we undertook a Saccharomyces cerevisiae two-hybrid screen that identified Int6 as a P56-interacting protein. That the interaction also occurs in human cells was confirmed by coimmunoprecipitation and the observed cytoplasmic displacement of nuclear Int6 upon coexpression of P56. Because Int6 has been claimed to be both a cytoplasmic and a nuclear protein, we investigated the structural basis of this discrepancy. By mutational analyses, we showed that the Int6 protein contains a bipartite nuclear localization signal and a nuclear export signal at the far end of the amino terminus. The 20 amino-terminal residues of Int6, when they were attached to a different nuclear protein, were sufficient to translocate that protein to the cytoplasm. Within this region, replacement of any of the three leucine residues with alanine destroyed the function of the export signal. The specific domain of P56 that is required for its interaction with Int6 was mapped using the yeast two-hybrid assay and a mammalian coimmunoprecipitation assay. Both assays demonstrated that the C-terminal region of P56 containing three specific tetratricopeptide motifs is required for this interaction. In contrast, removal of an internal domain of P56 enhanced the interaction, as quantified by the two-hybrid assay.

Cell growth regulatory and antiviral effects of the P69 isozyme of 2-5 (A) synthetase.

Among the many interferon-induced proteins that carry out multiple cellular functions of interferons is the family of enzymes called 2'-5' oligoadenylate synthetases. We examined the anticellular and antiviral activities of a specific member of that family, the P69 isozyme. P69 was expressed in human cells by transfection and shown to be localized primarily in the endoplasmic reticulum. For further studies, permanent cell lines expressing different levels of P69 or an enzymatically inactive mutant were isolated. Constitutive P69 expression caused inhibition of replication of encephalomyocarditis virus but not of vesicular stomatitis virus, Sendai virus, or reovirus. Increasing levels of P69 expression also caused increasing perturbations in cell growth properties. There was increasing accumulations of the P69-expressing cells in the G1 phase of the cell cycle; cell-doubling time was increased by P69 expression; and there were many multinucleated cells in the P69-expressing line, indicating a defect in cytokinesis.

The nature of the catalytic domain of 2'-5'-oligoadenylate synthetases.

2'-5'-Oligoadenylate (2-5(A)) synthetases are a family of interferon-induced enzymes that are activated by double-stranded RNA. To understand why, unlike other DNA and RNA polymerases, they catalyze 2'-5' instead of 3'-5' phosphodiester bond formation, we used molecular modeling to compare the structure of the catalytic domain of DNA polymerase beta (pol beta) to that of a region of the P69 isozyme of 2-5(A) synthetase. Although the primary sequence identity is low, like pol beta, P69 can assume an alphabetabetaalphabetabetabeta structure in this region. Moreover, mutation of the three Asp residues of P69, which correspond to the three catalytic site Asp residues of pol beta, inactivated the enzyme without affecting its substrate and activator binding capacity, providing further credence to the concept that this region is the catalytic domain of P69. This domain is highly conserved among all 2-5(A) synthetase isozymes. Biochemical and mutational studies demonstrated that dimerization of the P69 protein is required for its enzyme activity. However, a dimer containing a wild type subunit and an inactive catalytic domain mutant subunit was also active. The rate of catalysis of the heterodimer was half of that of the wild type homodimer, although the two proteins bound double-stranded RNA and ATP equally well.

DRBP76, a double-stranded RNA-binding nuclear protein, is phosphorylated by the interferon-induced protein kinase, PKR.

The interferon-induced double-stranded RNA-activated protein kinase PKR is the prototype of a class of double-stranded (dsRNA)-binding proteins (DRBPs) which share a dsRNA-binding motif conserved from Drosophila to humans. Here we report the purification of DRBP76, a new human member of this class of proteins. Sequence from the amino terminus of DRBP76 matched that of the M phase-specific protein, MPP4. DRBP76 was also cloned by the yeast two-hybrid screening of a cDNA library using a mutant PKR as bait. Analysis of the cDNA sequence revealed that it is the full-length version of MPP4, has a bipartite nuclear localization signal, two motifs that can mediate interactions with both dsRNA and PKR, five epitopes for potential M phase-specific phosphorylation, two potential sites for phosphorylation by cyclin-dependent kinases, a RG2 motif present in many RNA-binding proteins and predicts a protein of 76 kDa. DsRNA and PKR interactions of DRBP76 were confirmed by analysis of in vitro translated and purified native proteins. Cellular expression of an epitope-tagged DRBP76 demonstrated its nuclear localization, and its co-immunoprecipitation with PKR demonstrated that the two proteins interact in vivo. Finally, purified DRBP76 was shown to be a substrate of PKR in vitro, indicating that this protein's cellular activities may be regulated by PKR-mediated phosphorylation.

Enzymatic characteristics of recombinant medium isozyme of 2'-5' oligoadenylate synthetase.

P69 is an isozyme of the medium size class of human 2'-5' oligoadenylate synthetases. In this study, recombinant P69 was expressed and used for enzymological and structural investigations. Bacterially expressed P69 was inactive whereas the same protein expressed in insect cells was highly active. Whether this difference could be due to differential post-translational modifications of the protein was investigated. Mutations of appropriate residues showed that myristoylation of the protein was not necessary for enzyme activity. In contrast, inhibition of glycosylation of P69, by tunicamycin treatment of the insect cells, produced an enzymatically inactive protein. Recombinant P69 produced in insect cells was purified by affinity chromatography. It was a dimeric glycoprotein, very stable and completely dependent on double stranded (ds) RNA for activity. The enzyme catalyzed the non-processive synthesis of 2'-5'-linked oligoadenylate products containing up to 30 residues. 2'-O-Methylated dsRNA was incapable of activating P69 and a 25-base pair dsRNA was as effective as larger dsRNA. This expression system will be useful for large scale production of P69 and its mutants for structural studies.

Transcriptional induction of the p69 isoform of 2',5'-oligoadenylate synthetase by interferon-beta and interferon-gamma involves three regulatory elements and interferon-stimulated gene factor 3.

The 2',5'-oligoadenylate synthetases are key enzymes that mediate antiviral actions of interferon (IFN). The mRNAs for the intermediate isoforms (p69) of human 2',5'-oligoadenylate synthetase are rapidly induced 10- to 20-fold in HT1080 glioma cells by IFN-beta and induced 3-fold at 24 h by IFN-gamma. Induction is mediated by three regulatory elements, an IFN-stimulated response element and two identical sites resembling interferon response factor binding sites that are located within 300 bp of the transcriptional start site. Maximal induction requires all three elements, yet mutation in the most distal IRF-1-like site diminishes transcription only slightly. Mutation in the ISRE substantially decreases constitutive expression but does not abrogate the response to IFNs. Simultaneous mutation in all three elements abolishes responsiveness to both IFN-beta and IFN-gamma. Both constitutive and IFN-beta-induced expression from the p69 promoter is blocked in mutant cell lines deficient in components of the transcription factor, interferon-stimulated gene factor 3, suggesting that it is the primary factor controlling IFN-beta induced expression of this gene.

Requirement of PKR dimerization mediated by specific hydrophobic residues for its activation by double-stranded RNA and its antigrowth effects in yeast.

The roles of protein dimerization and double-stranded RNA (dsRNA) binding in the biochemical and cellular activities of PKR, the dsRNA-dependent protein kinase, were investigated. We have previously shown that both properties of the protein are mediated by the same domain. Here we show that dimerization is mediated by hydrophobic residues present on one side of an amphipathic alpha-helical structure within this domain. Appropriate substitution mutations of residues on that side produced mutants with increased or decreased dimerization activities. Using these mutants, we demonstrated that dimerization is not essential for dsRNA binding. However, enhancing dimerization artificially, by providing an extraneous dimerization domain, increased dsRNA binding of both wild-type and mutant proteins. In vitro, the dimerization-defective mutants could not be activated by dsRNA but were activated normally by heparin. In Saccharomyces cerevisiae, unlike wild-type PKR, these mutants could not inhibit cell growth and the dsRNA-binding domain of the dimerization-defective mutants could not prevent the antigrowth effect of wild-type PKR. These results demonstrate the biological importance of the dimerization properties of PKR.

Production, purification, and characterization of recombinant 2', 5'-oligoadenylate synthetases.

2',5'-Oligoadenylate [2-5(A)] synthetases are a family of interferon-induced enzymes that polymerize ATP into 2'-5'-linked oligoadenylates in the presence of double-stranded RNA (dsRNA), their cofactor. The 2-5(A) molecules, in turn, activate the latent ribonuclease RNase L by promoting its dimerization. The 2-5(A) synthetase pathway has been implicated in interferon's antiviral and anticellular activities. In addition to their interesting cellular properties, these enzymes are also enzymologically interesting because they are the only known template and primer independent nucleotide (DNA or RNA)polymerases that synthesize 2'-5'-linked oligonucleotides. Moreover, their mode of activation by dsRNA remains unknown. In the past, biochemical and structure-function studies have been hampered by the lack of a convenient system for expressing recombinant 2-5(A) synthetases. These proteins are toxic to mammalian cells, probably because of RNase L activation, and proteins produced in bacteria do not have full enzymatic activity. To circumvent these problems, we have developed a baculovirus-insect cell system for high-yield expression of the small and medium isozymes. Here, methods are described for the production, purification, and characterization of the mouse small (9-2) (S. K. Ghosh, J. Kusari, S. K. Bandyopadhyay, H. Samanta, R. Kumar, and G. C. Sen, 1991, J. Biol. Chem. 266, 15293-15299) and human medium (P69) (I. Marie and A. G. Hovanessian, 1992, J. Biol. Chem. 267, 9933-9939) 2-5(A) synthetase isozymes and their mutants using the insect cell system. We also report methods for studying 2-5(A) synthetase-dsRNA interactions and protein-protein interactions among the subunits of the two isozymes.