Interferon (IFN) was discovered as an antiviral agent during studies on virus interference (180, 294). Isaacs and Lindenmann reported in 1957 that influenza virus-infected chick cells produced a secreted factor that mediated the transfer of a virus-resistant state active against both homologous and heterologous viruses (180). This seminal observation, along with similar findings described by Nagano and Kojima in 1958 (294), set the stage for subsequent studies that led to the elucidation of the IFN system in exquisite detail.

What is the IFN system? How do IFNs function to inhibit the multiplication of some, but not all, viruses? What strategies are used by viruses to counteract the antiviral actions of IFNs? Considerable progress has been made toward answering these and other questions about IFNs and their effects on the virus-host interaction. Furthermore, IFNs were approved as therapeutics and moved from the basic research laboratory to the clinic. Advances made while elucidating the IFN system contributed significantly to our understanding in multiple areas of mammalian cell biology and biochemistry, ranging from pathways of signal transduction to the biochemical mechanisms of transcriptional and translational control to the molecular basis of viral pathogenesis.

Several of the key features of the human IFN system are summarized in Fig. 1. The IFN system includes cells that synthesize IFN in response to an external stimulus such as viral infection and cells that respond to IFN by establishing an antiviral state (318, 351, 394). Animal viruses are inducers of IFN, and are also sensitive to the antiviral actions of IFNs. Some animal viruses also encode products that antagonize the IFN antiviral response. IFN proteins display autocrine as well as paracrine activities. The IFN response represents an early host defense, one that occurs prior to the onset of the immune response. IFNs possess a wide range of biological activities in addition to the characteristic antiviral activity by which they were discovered (36). This review will focus primarily on the antiviral activities of IFNs. However, IFN cytokines affect a number of other processes including those regulating cell growth, differentiation, and apoptosis, as well as the modulation of the immune response.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Schematic summary of the IFN system. Virion particles are illustrated as open hexagons, and IFN proteins are illustrated as open circles. The IFN-producing cell shown on the left depicts a cell induced to synthesize IFN in response to either virus infection (IFN- and IFN-) or antigen or mitogen stimulation (IFN-). The IFN-treated cell shown on the right depicts paracrine IFN action in a cell induced to synthesize IFN-regulated proteins that collectively constitute the antiviral response that is responsible for the inhibition of virus multiplication. IFN may also act in an autocrine manner on the IFN producer cell. The numbers refer to the chromosome (Ch) assignment of the human genes encoding the IFNs and their receptors. Adapted from reference 354 with permission of the publisher.

IFNs are a multigene family of inducible cytokines (40, 91, 340, 394, 443). They possess antiviral activity (318, 349, 394). Indeed, the biological activity of IFN is most commonly assayed by determining the antiviral activity in cell culture, although radioimmunoassays and enzyme-based immunoassays are also available for IFNs (318). IFNs are commonly grouped into two types (36, 110, 351). Type I IFNs are also known as viral IFNs and include IFN- (leukocyte), IFN-, (fibroblast), and IFN-. Type II IFN is also known as immune IFN (IFN-). The viral IFNs are induced by virus infection, whereas type II IFN is induced by mitogenic or antigenic stimuli. Most types of virally infected cells are capable of synthesizing IFN-/ in cell culture. By contrast, IFN- is synthesized only by certain cells of the immune system including natural killer (NK) cells, CD4 Th1 cells, and CD8 cytotoxic suppressor cells (10, 443). The natural IFN--producing cells appear to be precursor dendritic cells (114, 381). Purified CD4⁺CD11c type 2 dendritic cell precursors (pDC2s) from human blood produce up to 10³ times more IFN in cell culture than do other blood cells following microbial or viral challenge (381). IFN- genes can be divided into two groups: an immediate-early response gene (IFN-4), which is induced rapidly and without the need for ongoing protein synthesis, and, a set of IFN- genes, consisting of IFN-2, IFN-5, IFN-6, and IFN-8, that display delayed induction and are synthesized more slowly and require protein synthesis (256).

The large number of viral IFN genes in the human include 13 IFN- genes, 1 IFN- gene, and 1 IFN- gene (340). They all lack introns and are clustered on the short arm of chromosome 9 in the human and chromosome 4 in the mouse. The single IFN- gene possesses three introns and maps to the long arm of chromosome 12 in the human, and chromosome 10 in the mouse. Although some IFNs are modified posttranslationally by N- and O-glycosylation, the major human IFN- subspecies are not glycosylated. The IFN- gene products appear to function as monomers, whereas IFN- and IFN- appear to function as homodimers (10, 318, 351, 394). It is not known why there are so many IFN- genes. When the single IFN- gene is deleted from chromosome 4 by targeted disruption, the resultant mice are highly susceptible to viral infection (82). The IFN- subspecies do not compensate for the loss of IFN-, suggesting a unique role for IFN- that is essential for a fully effective antiviral response.

SIGNAL TRANSDUCTION AND ACTIVATION OF TRANSCRIPTION OF INTERFERON-INDUCIBLE GENES

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IFN Receptors

IFNs exert their actions through cognate cell surface receptors that are largely species specific (10, 165, 287, 318). The alpha, beta, and omega IFNs appear to have a common receptor consisting of two subunits, IFNAR-1 and IFNAR-2. Both IFNAR-1 and IFNAR-2 map to chromosome 21 in the human, and chromosome 16 in the mouse. There is a single form of the IFNAR-1 subunit. However, alternative processing of the IFNAR-2 gene transcript produces long (2c), short (2b), and soluble (2a) forms of the encoded subunit (287, 327). IFN- binds to a receptor distinct from that used by IFN-/. Two kinds of subunits also constitute the IFN- receptor complex. The IFN- ligand-binding IFNGR-1 subunit and the accessory IFNGR-2 subunit map to chromosomes 6 and 21 in the human and chromosomes 10 and 16 in the mouse, respectively (10). IFN signaling involves an IFN-mediated heterodimerization of the cell surface receptor subunits, IFNAR-1 and IFNAR-2 with IFN-/ and IFNGR-1 and IFNGR-2 with IFN- (10, 12, 287, 379a, 394).

Pathogenesis studies with knockout mice in which the IFN-/ receptor function has been eliminated by targeted gene disruption illustrate the central importance of the IFN response in virus-host interactions (198). IFN-/ receptor-null mice are unable to establish an antiviral state, demonstrating that IFN-/ appears to be of particular importance in the host response to viral pathogens. IFN-/ receptor-deficient mutant animals are highly susceptible to infection by an array of different viruses exemplified by members of the Poxviridae (vaccinia virus), Arenaviridae (lymphocytic choriomeningitis virus), Rhabdoviridae (vesicular stomatitis virus [VSV]), and Togaviridae (Sindbis virus, Semliki Forest virus, and Venezuelan equine encephalitis virus) despite the presence of an otherwise intact immune system and a normal resistance to the microbial pathogen Listeria monocytogenes (128, 146, 177, 290, 345, 416). By contrast, IFN- receptor-deficient mice in which either the IFNGR-1 or IFNGR-2 gene has been disrupted are greatly impaired in their ability to resist a variety of microbial pathogens and some viral pathogens including vaccinia virus and herpes simplex virus (10, 48, 176, 244, 290). Thus, the IFNAR and IFNGR gene knockout studies established that the viral IFN and immune IFN systems are functionally nonredundant. Interestingly, for IFN antiviral responses involving IFN- and its receptor, IFNGR knockout mutant mice lacking the receptor are more susceptible to both HSV-1 and vaccinia virus challenge than are mutant mice lacking the gene for the IFN- ligand (48). Although animals with IFNGR gene disruptions show no overt defects in embryonic development, they have severe immune system defects (10, 47, 48).

IFN- plays an important role in both innate and adaptive immunity (36, 42). It stimulates innate cell-mediated immunity through NK cells; it stimulates specific cytotoxic immunity based on the recognition of cell surface-bound viral antigens expressed in association with major histocompatibility complex (MHC) proteins; and it activates macrophages (10, 42). These immune responses play an important role in the antiviral and antimicrobial actions of IFN-. Th cells can be divided into three classes based on the pattern of cytokines produced following activation by antigens and mitogens. In addition to IFN-, Th1 cells produce interleukin-2 (IL-2) and tumor necrosis factor beta (TNF-), Th2 cells produce IL-4 and IL-5, and Tr cells produce IL-10 (56, 260). Cellular immunity mediated by Th1 cells and humoral immunity mediated by Th2 cells are modulated by IFN-, which affects the differentiation of naive T cells into either Th1 or Th2 cells (36). IL-12 and IL-18 are IFN--inducing cytokines; IL-12 induction of IFN- is dependent on caspase-1 processing of the IL-18 precursor protein (106, 435).

While IFN- possesses unique immunoregulatory activities that are especially important in the innate host response to microbial infections, it also plays a role in mediating protection against viral infection, especially long-term control of viral infections (48, 176, 244, 290). Double-knockout mice lacking both the IFN- receptor and the IFN-/ receptor are especially sensitive to viral infection (416). Finally, while studies of mice made deficient in components of the IFN receptors by targeted gene disruption have firmly established the importance of the IFN system in the host antiviral response to a wide range of different RNA and DNA viruses (48, 128, 146, 176, 290, 416), the role of IFNs as inhibitors of the replication do vary with the animal system examined (7, 354, 368). This is illustrated by some members of the Reoviridae, including rotaviruses and orthoreoviruses. Studies of mutant mice deficient in either the IFN-/ receptor or IFN- suggest that neither IFN-/ nor IFN- responses play a major role in the clearance of primary rotavirus infection or affect the duration of rotavirus-induced disease in suckling mice (7). However, treatment of calves with recombinant IFN gave substantial protection from bovine rotavirus-induced diarrhea (368). Differences in IFN-inducing capacity between virus strains may be an important parameter contributing to the host response to viral infection by members of the Reoviridae as well as by other viruses (354). For example, reovirus-induced acute myocarditis in mice correlates with viral RNA synthesis rather than with the generation of infectious virus in cardiac myocytes; it has been speculated that the IFN-inducing capacity of reoviruses may determine in part their potential for causing virus-induced acute myocarditis (376). IFN- production can also be induced by reovirus infection, but the capacity of the serotype 1 Lang strain to induce IFN- in peripheral lymph node lymphocytes is dependent on the mouse strain; lymphocytes from C3H mice produce significantly higher levels of IFN- than do those from BALB/c, C57BL/6, and B10.D2 mice (253). In mouse fibroblasts in culture, serotype 3 is a better inducer of IFN-/ than is serotype 1 (374); curiously, serotype 3 virus also is more cytopathic in culture than is serotype 1 virus (291).

IFN-mediated signaling and transcriptional activation of cellular gene expression are best understood in the context of JAK-STAT pathway proteins (75, 232, 362, 363, 394). The principal components of the pathway are summarized in Fig. 2. The signal transducer and activator of transcription (STAT) family of proteins are latent cytoplasmic transcription factors that become tyrosine phosphorylated by the Janus family of tyrosine kinase (JAK) enzymes in response to cytokine stimulation. There are seven known members of the STAT protein family, Stat-1, Stat-2, Stat-3, Stat-4, Stat-5a, Stat-5b, and Stat-6, and four members of the JAK family, Jak-1, Jak-2, Jak-3, and Tyk-2. Different members of the JAK and STAT families have distinct functions in cytokine signaling. Receptor-associated JAKs are activated following binding of IFNs to their cognate multi-subunit transmembrane receptor. Of the known JAKs and STATs, the Jak-1, Jak-2, and Tyk-2 kinases and the Stat-1 and Stat-2 transcription factors play central roles in mediating IFN-dependent biological responses, including induction of the antiviral state (74, 169, 231, 394).

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Schematic of IFN signaling by the Jak-Stat pathway. The signaling process is initiated by binding of the IFN ligand to its cognate receptor subunits. IFN- and IFN- are depicted by the diamond, and IFN- is depicted by the sphere. IFN binding leads to activation of overlapping pairs of Jak and Stat transcription factors by tyrosine phosphorylation. The Jak-1 and Tyk-2 kinases are activated by IFN-/, which leads to the phosphorylation and dimerization of the Stat-1 (p91) and Stat-2 (p113) proteins and subsequent translocation, along with IRF-9 (p48), to the nucleus. The complex of these three proteins, known as IFN-stimulated gene factor 3 (ISGF-3), activates the transcription of IFN-/-inducible genes through the ISRE. The Jak-1 and Jak-2 kinases are activated by IFN-, which leads to the phosphorylation and homodimerization of the Stat-1 protein and subsequent translocation to the nucleus. The Stat-1 dimer complex, known as GAF for gamma activation factor, activates the transcription of IFN- inducible genes through the GAS enhancer element.

Overlapping subsets of JAKs are involved in signaling by the two types of IFNs. Jak-1 and Tyk-2 kinases function in IFN-/ signaling, and the Jak-1 and Jak-2 kinases function in IFN- signaling (10, 287). Tyk-2 interacts with the IFNAR-1 receptor subunit, and Jak-1 interacts with the IFNAR-2 subunit of the IFN-/ receptor. Jak-1 also interacts with the IFNGR-1 receptor subunit, and Jak-2 interacts with the IFNGR-2 subunit of the IFN- receptor. Activation of the receptor-associated JAKs leads to the subsequent phosphorylation of latent cytoplasmic STAT transcription factors. For IFN-/, the phosphorylated forms of Stat-1/ and Stat-2, along with an additional non-STAT protein, p48 (also known as IRF-9), translocate to the nucleus and constitute a complex known as ISGF-3. The ISGF-3 trimeric complex binds to a cis-acting DNA element, designated ISRE, found in IFN-/-inducible genes. For IFN-, the phosphorylated Stat-1 factor homodimerizes, translocates to the nucleus, and binds to a different cis-acting element, designated the gamma-activated sequence (GAS), that is commonly found in IFN--inducible genes (11, 15, 232).

Targeted disruption of the Jak-1, Jak-2, Stat-1, and Stat-2 genes in mice have firmly established the obligatory physiological roles of the encoded Jak-1 and Jak-2 kinases and the Stat-1 transcription factor in the IFN response (103, 169, 278, 307, 327, 341, 362). Disruption of the mouse Stat-1 gene revealed an unexpected physiologic specificity. Stat-1 null mice show a greatly reduced responsiveness to both IFN- and IFN- and show a compromised innate immunity to viral disease (103, 278). Stat-1-deficient mice are extremely susceptible to viral pathogens. However, if reared in a pathogen-free environment, mice lacking Stat-1 are fertile and show no overt developmental abnormalities. Consistent with the biochemical evidence, Stat-2 null mice are defective primarily in their response to IFN-/ (327, 362). The Jak-1 gene disruption results in mice that are runted at birth and typically die within 24 h; mutant mouse embryo fibroblasts (MEF) lacking the Jak-1 kinase are unresponsive to either IFN- or IFN- (341). The Jak-2 gene disruption is embryonic lethal on day 12 to 13; mutant MEF lacking the Jak-2 kinase are not responsive to IFN- but remain responsive to IFN- (307).

The IFN regulatory factor (IRF) family of transcriptional regulators, like the STATs, are important regulatory factors in the IFN response (299). The IRF-1 protein was first identified as a regulator of the IFN-/ gene promoter, as well as the IFN-stimulated response element (ISRE) found in the promoters of some IFN-/-regulated genes (363). The IRF family of factors includes nine known members: IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF-6, IRF-7, IRF-8, and IRF-9 (p48, ISGF-3). These factors are homologous to each other in the N-terminal region, corresponding to their conserved DNA-binding domain. The IRFs and STATs can function in conjunction with each other to establish the signal transduction and gene regulation events that lead to the induced expression of the proteins that collectively constitute the antiviral state. IRF-9 was initially identified as a component of the trimeric ISGF-3 complex, along with Stat-1 and Stat-2. It is the only component of the ISGF-3 complex for which a nuclear localization signal has been identified (222), and it is the DNA sequence recognition subunit of ISGF-3 (185). The Stat-2 protein appears to form a cytoplasmic complex with IRF-9 that is retained in the cytoplasm in the absence of IFN treatment. The Stat protein subunits are localized in the cytoplasm in untreated cells but rapidly translocate to the nucleus following IFN treatment. IRF-9, on the other hand, is found in both the nucleus and cytoplasm of untreated and IFN-treated cells. Preassociation of IRF-9 with Stat-2 in the cytoplasm has the potential to poise the complex for activation by dimerization with Stat-1, as a rapid response to signaling by IFNs-/. IRF-9 also appears to play an ISGF-3-independent role in responses mediated by IFN- (195). IRF-9 and IRF-1 play essential but nonredundant roles in the IFN response. IRF-1 binds directly to the ISRE found in the promoters of some IFN-/-regulated genes (299) and plays an important role in the antiviral actions of IFN (146, 265, 337).

Most, but not all, of the activities of IFN- are mediated through the Stat-1 protein target of the JAK-STAT signaling pathway. IFN- treatment leads to the tyrosine phosphorylation of the IFNGR-1 receptor subunit on Tyr-440 and the subsequent interaction and phosphoryation of Stat-1 at Tyr-701. Dimerization of Stat-1 through reciprocal SH2 interactions is followed by nuclear translocation and then DNA binding at GAS elements to activate transcription of IFN--inducible genes (169, 232, 362). Serine phosphorylation of the transcriptional activation domain of Stat-1 at Ser-727, positioned within the C-terminal region of Stat-1, enhances transcription by recruitment of p300/CBP, a ubiquitously expressed global transcriptional coactivator with histone acetyltransferase (HAT) activity (397, 446), and also BRCA1 (306). The BRCA1 tumor suppressor acts in concert with Stat-1 to differentially activate the transcription of a subset of IFN--regulated target genes involved in growth control, including the cyclin-dependent kinase inhibitor p21^WAF1. The possible roles of these interactions in the IFN-mediated inhibition of virus multiplication, by affecting responses associated with cell growth and proliferation, are unknown.

STAT factors in addition to Stat-1 and Stat-2, namely, Stat-3, Stat-4, Stat-5, and Stat-6, have also been observed to be activated by IFNs (107, 274, 332). Furthermore, although IFN-mediated signaling and transcriptional activation is presently best understood in the context of the JAK-STAT signal transduction pathway, additional pathways of signal transduction possibly involving the RNA-dependent protein kinase (PKR) (212), mitogen-activated protein kinase (78, 141), and phosphatidylinositol 3-kinase (319) may also be operative under some circumstances. Both IFN- and IFN- can regulate the expression of IFN-responsive genes by a Stat1- and PKR-independent alternative signaling mechanism that requires both the IFN- receptor and Jak-1 kinase (140a, 330a). Surprisingly, some genes are activated by IFN- only when Stat1 is absent, a finding that may be of special significance in those viral infections where Stat1 function is antagonized (330a). Thus, the possibility of an elaborate network that conceivably allows for considerable cross talk between separate pathways involved in IFN signal transduction and the actions of IFNs must be considered.

Two cis-acting DNA elements are the known targets of the Stat and IRF transcription factors: ISRE and GAS. ISRE and GAS are largely responsible for the IFN-regulated promoter activity seen for IFN-inducible genes. The ISRE drives the expression of genes inducible by IFN-/ and has the consensus sequence AGTTTCNNTTTCNPy. It is the binding site for the IFN-stimulated gene factor ISGF-3 and for some of the IRFs (74, 231, 394). Indeed, the crystal structure of an IRF-DNA complex reveals the DNA recognition sequence AANNGAAA and cooperative binding of IRF-2 to a tandem repeat of the GAAA core sequence (118). A novel 15-bp element, designated KCS (for "kinase-conserved sequence"), also is required both for basal and IFN-inducible activity of the promoter for the RNA-dependent protein kinase PKR. The KCS element, GGGAAGGCGGAGTCC, is exactly conserved in sequence and position between the human and mouse Pkr promoters and is the binding site for a protein complex including the Sp1 factor (207, 210).

The GAS element was initially identified as the IFN--responsive DNA element. However, in addition to Stat-1 homodimers that bind DNA at the GAS site, a number of other Stat proteins activated by various cytokines have been shown to bind GAS-like elements as either homo- or heterodimers, including Stat-3, Stat-4, Stat-5, and Stat-6 (74, 169, 231, 394). GAS-like elements have a palindromic core sequence, TTNNNNNAA (10, 362, 394).

Interestingly, the ISRE is not limited to IFN-inducible cellular genes. The Q promoter (Qp) of Epstein-Barr virus (EBV) and the enhancer-1 region of hepatitis B virus (HBV) have an ISRE-like element, vISRE, that is the target of IRF factors (297, 447). Qp is used for the transcription of EBV nuclear antigen 1 (EBNA1) during the highly restricted type I latent infection but is inactive in type III latency. Constitutive activation of EBV EBNA1 gene transcription is mediated in part by IRF-1 activation of Qp. A different IRF factor, IRF-7, binds to the vISRE-like sequence of Qp and represses transcriptional activation by both IFN and IRF-1. Expression of IRF-7 is high in type III latency cells but almost undetectable in type I latency, corresponding to the activity of the Q promoter of EBV in these latency states (447). The EBV latency promoter is positively regulated by Stat factors, and Zta interference with signaling leads to a loss of promoter activity (64). IFN- treatment suppresses the activity of the hepatitis B virus enhancer-1, which possesses an ISRE-like element that is bound by both IRF-1 and IRF-9. Mutation of the HBV vISRE reduces the suppressive effect of IFN-, whereas overexpression of IRF-9 enhances the inhibition of enhancer-1 activity in human hepatoma HuH7 cells (297).

Virus infection activates the transcription of a large number of cellular genes (58, 198, 451). The activation may occur either directly through activation of cellular transcription factors such as IRF-3 or indirectly through prior induction of IFN-/. IRF-3, a key transcriptional activator affected by viral infection, is a subunit of the double-stranded RNA (dsRNA)-activated transcription factor complex (DRAF) (167, 425). It is constitutively expressed in many cells and tissues. It is directly activated by dsRNA or by virus infection and subsequently plays a role in the transcriptional activation of the IFN- and IFN- promoters and IFN-/-responsive genes (299, 361, 440). Activation of IRF-3 involves protein serine/threonine phosphorylation, which results in its cytoplasmic-to-nuclear translocation, stimulation of DNA binding, and association with p300/CBP coactivator, leading to increased transcriptional activation (167, 213, 371). Along with IRF-3, IRF-7 probably plays important roles in the regulation of IFN- synthesis in response to virus infection (424). IRF-7 is also a critical determinant for the induction of IFN- genes in infected cells and functions in part by a positive feedback induction loop mechanism (256, 438).

Results of analyses of genetically altered cell lines suggest that signal transduction by dsRNA is mediated by ISREs without activation of ISGF-3. The IFN signaling pathway components Jak-1, Tyk-2, IRF-9, and Stat-2, necessary for ISGF-3 activation, are not absolutely required for dsRNA-mediated signal transduction (15). Interestingly, virus infection mediates the induction of the IFN-inducible P56 protein in mutant P2.1 cells even though both dsRNA and IFN signaling are nonfunctional in these cells (155). The biochemical nature of the pathway components implicated in direct induction of IFN-inducible genes by virus in the P2.1 cells in the absense of functional dsRNA and IFN signaling pathways have not yet been defined.

Negative regulators of JAK-STAT-mediated signaling and gene activation have been identified. Among these are both viral and cellular proteins which down-regulate JAK-STAT-mediated signals. The family of suppressors of cytokine signaling (SOCS)/STAT-induced STAT inhibitors (SSI)/ cytokine-inducible SH2 protein (CIS) represent cellular proteins that function as negative-feedback regulators of JAK-STAT signaling (295, 395). On the basis of two characteristic conserved domains, a central SH2 domain and the so-called SOCS box at the carboxyl end, there are eight known members (295, 437). SOCS-1 is induced by numerous cytokines including IFN-, binds to the kinase domain of all four members of the JAK family of kinases, and inhibits signaling by suppression of Jak activation (104, 295). Consequently, SOCS-1 inhibits the tyrosine phosphorylation and nuclear translocation of Stat-1 in response to both IFN- and IFN-. SOCS-1, but not SOCS-2, inhibits the antiviral and antiproliferative activities of IFNs in cell lines stably expressing the SOCS proteins (388). Targeted gene disruption of SOCS-1 suggests that the most important physiological function of this signaling suppressor is the negative regulation of IFN- signaling and Stat-1 function (6, 437).

Two members of the protein inhibitor of activated Stat (PIAS) family of cellular proteins, PIAS-1 and PIAS-3, are also negative regulators of STAT signaling. However, the mechanism is different from that of SOCS, which inactivates the Jak kinases. The PIAS-1 and PIAS-3 proteins directly associate with Stat-1 and Stat-3, respectively, in response to treatment with IFNs or IL-6 (380). PIAS-1 specifically interacts with the Stat-1 dimer, but not monomer, to block the DNA-binding activity and thus the Stat-1-mediated gene activation (234, 380).

Negative regulation of JAK-STAT signal transduction is also achieved by dephosphorylation catalyzed by the protein tyrosine phosphatase SHP-1. SHP-1 suppresses the signal transduction process of a variety of cytokines, including IFN- (77), by directly interacting with JAKs and catalyzing their dephosphorylation. A dominant negative form of SHP-2 phosphatase suppresses ISGF-3-dependent transcription (79), suggesting that SHP-2 phosphatase, in contrast to SHP-1, plays a role in IFN-/-induced gene expression.

Both DNA and RNA viruses encode proteins that impair the activity of the JAK-STAT signaling pathway (198). Multiple mechanisms appear to be involved. Among these is mimicry. Several examples exist in which viruses encode products that mimic cellular components of the IFN signal transduction pathway. This molecular mimicry can lead to an antagonism of the IFN signaling process and subsequent impairment of the development of an antiviral state. Poxviruses, for example, encode soluble IFN receptor homologues (vIFN-Rc). These vIFN-Rc homologues are secreted from poxvirus-infected cells and bind IFNs, thereby preventing them from acting through their natural receptors to elicit an antiviral response (385). M-T7, the first poxvirus receptor homologue identified, was found in myxoma virus-infected cells and acts as a decoy to inhibit the biological activity of rabbit IFN-. The vIFN-Rc M-T7 gene product is a critical virulence factor for poxvirus pathogenesis. Other poxviruses also encode soluble IFN-Rc homologues, including vaccinia virus, where the B8R gene encodes the IFN- receptor homologue. A vIFN-/Rc protein is secreted by vaccinia virus and several additional orthopoxviruses. The vIFN-/ receptor homologue, the B18R gene product in the Western Reserve strain and the B19R product in the Copenhagen strain, binds several different IFN- subspecies as well as IFN- and blocks IFN-/ signaling activity (5, 71).

Three additional DNA viruses that affect IFN signaling are adenovirus, papillomavirus, and human herpesvirus 8 (HHV-8). The adenovirus E1A protein blocks IFN-mediated signaling at a point upstream of the activation of ISGF-3. The DNA binding activity of ISGF-3 is inhibited by E1A (230). Overexpression of the IRF-9 subunit of ISGF-3 restores IFN responses and the transcription of ISRE-driven genes in adenovirus-infected cells (230). Sendai virus (SeV), a paramyxovirus that replicates in the cytoplasm of the host, circumvents the IFN-induced antiviral response by interfering with the transcriptional activation of IFN-inducible cellular genes. The C proteins of SeV play an essential role (130); infections with two different C gene mutants of SeV eliminate the ability to prevent the establishment of an antiviral state against vesicular stomatitis virus (131). Impairment of the IFN-induced antiviral response appears to be a key determinant of SeV pathogenicity (93, 131).

Human papillomavirus (HPV) E6 oncoprotein binds selectively to IRF-3 but only very poorly to other cellular IRFs including IRF-2 and IRF-9. Association of E6 with IRF-3 inhibits transactivation, thereby providing HPV with a mechanism to circumvent the antiviral response (343). Adenovirus E1A protein also inhibits IRF-3-mediated transcriptional activation by a mechanism dependent on the ability of E1A to bind p300. CBP/p300 and PCAF histone acetyltransferase (HAT) enzymes are coactivators for several transcription factors including Stat-1; the viral E1A protein represses HAT activity and inhibits p300-dependent transcription and nucleosomal histone modifications by PCAF (57).

HHV-8, a gammaherpesvirus associated with Kaposi's sarcoma, synthesizes an IRF homologue (vIRF) that functions as a repressor of transcriptional activation induced by IFN-/ and IFN- (127, 167). The HHV-8-encoded vIRF protein also represses IRF-1-mediated transcriptional activation. HHV8 vIRF probably plays an important role in HHV-8 pathogenesis and neoplastic transformation by antagonizing IFN- and IRF-mediated transcriptional control. Expression of vIRF antisense in HHV-8-infected cells increases IFN-mediated transcriptional activation and downregulates the expression of HHV-8 genes. Two other herpesviruses, varicella-zoster virus (VZV) and cytomegalovirus (CMV), also disrupt the function of the JAK-STAT signal transduction pathway (1, 283). VZV inhibits the expression of Stat-1 and Jak-2 proteins but has little effect on Jak-1; the expression of two key transcription factors regulated by IFN- signaling, IRF-1 and the MHC class II transactivator (CTIIA), is inhibited in VZV-infected cells, as is the induction of the MHC class II molecules (1). A different strategy of antagonism occurs in CMV-infected cells, where MHC class II expression also is inhibited. There is a specific decrease in the level of Jak-1 due to enhanced protein degradation in CMV-infected fibroblasts (283).

Several nonsegmented negative-strand RNA viruses encode gene products that antagonize IFN receptor-mediated signaling from both type I / and type II  IFN receptors. For example, infection with simian virus 5 or mumps virus leads to an increased proteosome-mediated degradation of Stat-1 (94) whereas in cells infected with parainfluenza virus type 2 there is a degradation of Stat-2 (442). In the case of Sendai virus, the C proteins interfere with IFN action in at least two ways. C proteins prevent the synthesis of Stat-1 and they also induce an increased turnover of Stat-1 (130, 201). The VP35 protein of Ebola virus, a negative-strand RNA virus, functions as a type I IFN antagonist although the precise biochemical mechanism of the antagonism has not yet been defined (21). VP35 inhibits virus induction of the IFN- promoter and dsRNA- and virus-mediated activation of ISRE-driven gene expression (21).

The replication of a wide range of different DNA and RNA animal viruses is inhibited by IFN, both in cell culture and in animals (318, 351, 394). For many of the viruses that are sensitive to the antiviral action of IFN in cell culture systems, the primary step of the virus multiplication cycle inhibited typically is the synthesis of viral polypeptides (36, 351). Exceptions do exist, however. Papovaviruses and certain retroviruses often are inhibited in IFN-sensitive cell systems at early and late steps, respectively, of their multiplication cycles (36, 349, 351), myxoviruses and rhabdoviruses may be inhibited in certain cell types at or before primary transcription (158, 393), and adenoviruses and poxviruses can be comparatively resistant to the antiviral actions of IFN because of virus-encoded antagonists (264, 351). Among the IFN-induced proteins implicated in the antiviral actions of IFNs in virus-infected cells are PKR, the 2',5'-oligoadenylate synthetase (OAS) and RNase L, the RNA-specific adenosine deaminase (ADAR), and the Mx protein GTPases. Double-stranded RNA plays a central role in modulating protein phosphorylation, RNA degradation, and RNA editing catalyzed by the IFN-inducible enzymes: the PKR kinase, the OAS synthetases, and the ADAR1 deaminase (181, 353). dsRNA interestingly also serves as a template for gene silencing (25). IFN also induces a form of nitric oxide synthase (iNOS2) and the MHC class I and II molecules, all of which play important roles in immune responses to infections.

PKR is an IFN-inducible, RNA-dependent protein kinase (68, 352) known in the earlier literature as DAI; dsI; P1 kinase; P1/eIF-2 kinase; p65, p67, or TIK (for the mouse enzyme); and p68 or p69 (for the human enzyme) (69). In IFN-treated cells, PKR is found predominantly in the cytoplasm and associated with ribosomes (318, 352, 412); however, small amounts of PKR have also been localized to the nucleus by cell fractionation and immunofluorescence analyses (183, 412). As summarized in the Fig. 3 schematic, PKR is activated by autophosphorylation, a process mediated by RNA with double-stranded character (68, 328, 352). Following activation, PKR catalyzes the intermolecular phosphorylation of at least six protein substrates: the PKR protein itself (410, 411); the  subunit of protein synthesis initiation factor 2, eIF-2 (348); the transcription factor inhibitor IB (211, 303); the Tat protein encoded by human immunodeficiency virus (HIV) (272); the 90-kDa NFAT protein (220); and the M-phase specific dsRNA-binding phosphoprotein MPP4 (310).

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Functions of selected IFN-inducible proteins. Among the IFN-induced proteins believed to affect virus multiplication within single cells are PKR kinase, which inhibits translation initiation through the phosphorylation of protein synthesis initiation factor eIF-2; the OAS synthetase family and RNase L nuclease, which mediate RNA degradation; the family of Mx protein GTPases, which appear to target viral nucleocapsids and inhibit RNA synthesis; and ADAR, which edits double-stranded RNA by deamination of adenosine to yield inosine. IFN-induced expression of MHC class I and class II antigens and NOS may contribute to the antiviral responses observed within whole animals. Ag, antigen. Adapted from reference 351 with permission of the publisher.

Protein synthesis factor eIF-2 so far is the best characterized of the PKR substrates, in both structural and functional terms. Serine phosphorylation of eIF-2 catalyzed by PKR occurs at Ser-51 (311, 348). This phosphorylation of eIF-2 leads to an inhibition of translation by impairing the eIF-2B-catalyzed guanine nucleotide exchange reaction (68, 125, 352, 366). A variety of physiologic conditions, including IFN treatment and virus infection (351, 352, 355), cause the phosphorylation state of eIF-2 to increase and mRNA translation to subsequently decrease. Activation of PKR and subsequent phosphorylation of eIF-2 change the translational pattern of the host cell.

Human PKR is a 551-amino-acid protein with a molecular mass of about 62 kDa as deduced from the cDNA open reading frame (ORF) (209, 279, 352, 412). By contrast, the mouse and rat PKR proteins are somewhat smaller, 515 amino acids (178, 401) and 514 amino acids (276), respectively. The 11 conserved catalytic subdomains characteristic of protein serine/theonine kinases are, without exception, located in the C-terminal half of PKR (352). Replacement of the subdomain II-invariant Lys-296 with arginine (K296R) eliminates both autocatalytic and eIF-2 protein kinase activities of PKR (17, 409). The N-terminal region of the PKR protein possesses a repeated motif (dsRBM, or R), whose core is about 20 amino acid residues and is responsible for the dsRNA-binding activity (108, 144, 189, 269, 309). Mutational analyses established that the N-terminal proximal copy of dsRBM is both necessary and sufficient for the RNA-binding activity of PKR, although both copies of dsRBM are required for optimal kinase activity (145, 268, 273, 352).

The RNA-dependent autoactivation of PKR involves autophosphorylation of PKR (68, 188, 350, 352, 370, 387). Biochemical and genetic studies suggest that the autophosphorylation can occur by either intramolecular (31, 120) or intermolecular (204, 342, 410, 411) mechanisms. There are multiple sites of autophosphorylation on PKR, predominantly serine residues but also including threonine residues (120, 221, 348). Phosphopeptide analysis suggests a multiple of about four major phosphorylation sites per PKR molecule (411). No PKR phosphorylation on tyrosine is detectable (178, 221).

A number of RNA effectors of PKR function, that is, RNA activators and RNA inhibitors, have been identified. RNA activators include both synthetic and natural dsRNA, for example (rI)_n-(rC)_n and reovirus genome dsRNA, respectively (352). Undistorted A-form dsRNA has its sequence-rich information buried in the major groove, and indeed, no sequence specificity has been observed in interactions between PKR and dsRNA per se (68, 352). Certain highly structured single-stranded viral RNA species are also activators, as exemplified by HIV TAR RNA, reovirus s1 mRNA, and hepatitis delta virus RNA (67, 351). RNA inhibitors of PKR autophosphorylation include dsRNA at high concentration and also three highly structured viral single-stranded RNA (ssRNA) species: adenovirus VAI RNA, EBV EBER RNA, and HIV TAR RNA (68, 264, 351, 352). Synthetic aptamer RNAs selected from a library of ~10¹⁴ RNA sequences containing a randomized region of 50 nucleotides (nt) include both activators and inhibitors of PKR autophosphorylation and eIF-2 phosphorylation (33).

The basis of the RNA selectivity of PKR activation remains an important question. While kinase activation is associated with the formation of a stable PKR dsRNA complex that requires ~30 to 50 bp of duplex RNA and is optimal with about 80 bp, the PKR protein appears to interact with as little as 11 bp of dsRNA (32, 254). Both the full-length PKR protein and the truncated N-terminal region of the PKR protein containing the two copies of the dsRBM motif bind VAI RNA (267) and an 85-bp dsRNA (365) with comparable affinity, with an apparent K_D between 2 and 4 nM. Mutations in PKR that impair RNA binding have similar effects on the binding of both activator and inhibitor RNAs (144, 267, 268). This suggests that the discrimination between activator and inhibitor RNAs presumably takes place after RNA binding. Curiously, the cellular protein activator PACT (308a) and the carbohydrate heparin (136, 170) can substitute for RNA in mediating the autophosphorylation and activation of PKR. Heparin oligosaccharide with 8 sugar residues is nearly as efficient as heparin with 16 residues in activating PKR, whereas heparin with 6 residues is a very poor activator (136). The RNA-binding activity of wild-type PKR is not competed by heparin, and PKR mutants that fail to bind to and be activated by dsRNA can be activated by heparin and by PACT (136, 309).

Southern blot and nucleotide sequence analyses are consistent with a single Pkr gene, and fluorescence in situ hybridization shows that genomic clones colocalize to human chromosome 2p21-22 and mouse chromosome 17E2 (16, 208, 390). The human Pkr gene consists of 17 exons and spans about 50 kb (208), whereas the mouse gene is 16 exons and spans about 28 kb (401). The organization of the regulatory and catalytic subdomains of the PKR protein are remarkably preserved between the human and mouse Pkr genes; the amino acid junction positions for 13 of the 15 protein coding exons are exactly conserved (208).

The TATA-less Pkr promoter possesses an ISRE as well as a novel 15-nt KCS element, which so far has been identified only in the human and mouse Pkr promoters (206, 401). KCS affects basal as well as IFN-inducible expression of PKR (207, 210). IFN- is an efficient inducer of two Pkr mRNAs in human cells, of ~2.5 and ~6.0 kb (279, 412), but IFN- is a relatively poor inducer of them (412). Tissue-specific differences in the ratios of the three PKR transcripts are observed in mice; for example, a 2.5-kb mRNA is the predominant species in testes, but in both lung and heart tissues a 4.0-kb species is predominant over the 2.5- and 6.0-kb mRNAs (178). The molecular basis and functional significance of the three forms of PKR mRNA transcripts remain unresolved. For human PKR, alternative exon 2 splice variants with different translational activity and abundance have been found in placental tissue, but their relationship to the 2.5- and 6.0-kb transcripts has not been defined (190). In addition to the regulation of PKR by IFN-inducible transcriptional activation, the synthesis of PKR in transfected mammalian cells is autoregulated primarily at the level of translation by a mechanism dependent on catalytically active PKR (20, 409).

Several studies establish that changes in protein phosphorylation mediated by the IFN-inducible PKR play an important role in the antiviral actions of IFNs as well as the control of cell growth mediated by IFNs (68, 228, 352, 356). Evidence for the involvement of the IFN-inducible PKR in the antiviral actions of IFN and the control of translation in virus-infected cells comes from three types of analyses: (i) the study of virus replication in mammalian cells expressing PKR cDNAs (29, 224, 281, 293); (ii) the analysis of mutant mice and MEF deficient in PKR by targeted disruption of the Pkr gene (2, 13, 436; L. Basu and C. E. Samuel, unpublished data); and (iii) the analysis of virus-encoded inhibitors of the PKR kinase (188, 264, 350, 387). For example, the replication of encephalomyocarditis (EMC) virus (281), HW (29, 293), vaccinia virus (224), and VSV (398; Basu and Samuel, unpublished) is reduced in cell culture by overexpression of the cDNA encoding wild-type PKR but not by expression of the catalytic subdomain II point mutant PKR (1-551)K296R, which lacks kinase activity. Furthermore, in replication-competent HIV-1, chimeric genomes that express the wild-type PKR but not the K296R mutant in place of nef inhibited their own expression in cis and pNL4-3 in trans (29). A systematic analysis designed to identify the precise point(s) of virus replication blocked in transfected cells overexpressing wild-type PKR cDNA, similar to earlier studies carried out with cells treated with purified cloned IFNs (262, 415), has not been reported yet. Mutant Pkr^0/0 mice with a targeted disruption in the N-terminal region of PKR that deletes the methionine start codon and RNA-binding motif I show a reduced antiviral response to EMC virus induced by IFN- (436). In mutant Pkr^0/0 mice with the disruption in the catalytic domain, the antiviral responses to influenza virus and vaccinia virus are normal (2). However, these mutant mice lacking PKR are predisposed to lethal intranasal infection by VSV (13), and MEF derived from these knockout mice treated with recombinant IFN- display a reduced antiviral state against VSV compared to those from wild-type parental mice (398; Basu and Samuel, unpublished). Tat-mediated activation of transcription factor NF-B and transcriptional induction of the HIV-1 long terminal repeat LTR also are impaired in mouse cells in which the Pkr gene is knocked out. Both functions are restored by cotransfection of Tat with the cDNA for PKR (81). The HSV ICP34.5 gene product mediates neurovirulence in the mouse model by antagonizing the function of PKR. ICP34.5 is not required for HSV-1 multiplication in nonneuronal cells in culture but is required for replication in the central nervous system. In mice with the PKR gene disrupted in the N-terminal RNA-binding regions, mutant HSV lacking ICP34.5 show wild-type replication and neurovirulence (226).

Adenovirus virus-associated (VA) RNA antagonizes the antiviral action of IFN by preventing the activation of PKR (264, 352). The growth of wild-type adenovirus that produces VA RNA is not inhibited by IFN, but IFN treatment inhibits the growth of adenovirus mutants that are unable to produce VA RNA (197). In the absence of functional VA RNA, adenovirus produces virus-specific RNAs that activate PKR (197, 264), which appears to be responsible for the observed inhibition of translation and the IFN sensitivity of VA mutant virus growth. Additional examples of escape strategies from the antiviral actions of IFN involving antagonism of PKR by virus-encoded gene products will be considered separately (see "Viral antagonists of IFN-induced proteins" below). While adenovirus VA RNA illustrates a well-defined example of how a single viral gene product can drastically affect IFN sensitivity of a virus by targeting a specific IFN-inducible protein (PKR), studies with other viral systems have also shown that the IFN response can differ markedly for a given type of virus. For example, the effect of IFN treatment on the multiplication of reovirus is dependent on the kind of host cell, the type of IFN, and the serotype of reovirus. Reovirus multiplication is sensitive to the antiviral action of IFN in some cell lines but is not significantly inhibited by IFNs in other lines (354). The principal step of reovirus macromolecular synthesis inhibited in reovirus-infected IFN-sensitive mouse fibroblast and monkey kidney cells is the translation of viral mRNA into viral protein. Analysis by two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting reveals that the phosphorylation of eIF-2 is increased in IFN-treated cells following infection with reovirus (355).

In addition to playing a role in the antiviral actions of IFNs (36, 351), PKR is implicated in the control of cell proliferation (228, 356). For example, stable transformants of NIH 3T3 cells overexpressing catalytically inactive human PKR proteins display a transformed phenotype and are highly tumorigenic when injected into nude mice (19, 203, 280). Further evidence in support of the notion that PKR-mediated perturbation of the homeostatic balance of translation can cause malignant transformation comes from overexpression of either mutated eIF-2 which cannot be phosphorylated (100) or the 58-kDa cellular inhibitor which impairs PKR kinase activity (18). Overexpression of either the PKR inhibitory protein p58 (18) or the nonphosphorylatable mutant Ser51Ala eIF-2 (100) causes malignant transformation of NIH 3T3 cells. The tumor suppressor activity of IRF-1 also appears to be mediated, in part, by PKR (196). And in yeast, the wild-type but not mutant PKR cDNA mediates a growth suppression phenotype (65, 342). Somewhat unexpectedly, however, no evidence of tumor suppressor activity of PKR was observed in two independent mouse mutants devoid of functional PKR by targeted gene disruption (2, 436).

The IFN-inducible 2-5A response leading to the degradation of RNA requires two enzymes, OAS and RNase L (Fig. 3). OAS catalyzes the synthesis of oligoadenylates of the general structure ppp(A2'p)_nA, commonly abbreviated 2-5A. As their name implies, they possess a 2',5'-phosphodiester bond linkage (192). RNase L, a latent endoribonuclease, becomes activated by binding 2-5A oligonucleotides. A third enzymatic activity, that of a phosphodiesterase, also is involved in the metabolism and action of 2-5A oligoadenylates (Fig. 3). The phosphodiesterase catalyzes the hydrolysis of oligonucleotides possessing 2',5'-phosphodiester bonds, thereby attenuating the 2-5A response. The expression, regulation, and function of the OAS and the 2-5A-dependent RNase L has been characterized extensively in IFN-treated and virus-infected cells (191, 322, 334, 357, 394).

Three size forms of OAS, designated OAS1, OAS2, and OAS3 (334), have been identified in human cells by immunoblotting and by characterization of cDNA and genomic clones. OAS proteins of 40, 46, 69, 71, and 100 kDa are detectable by Western analysis (60, 255, 258). The 40- and 46-kDa forms of OAS, designated OAS1, are identical to each other over their N-terminal 346 amino acids but differ at their C-terminal regions. They are encoded by alternatively spliced 1.6- and 1.8-kb transcripts derived from a single gene (26, 174, 432). The differential splicing of OAS1 occurs between exons 5 and 6 (27, 359). The 69- and 71-kDa medium forms of human OAS, designated OAS2, likewise appear to be generated by differential splicing. The OAS2 isoforms, identical over their N-terminal 683 amino acids, consist of two adjacent domains each homologous to the 40-kDa isoform of OAS1 (255). Several RNA transcripts hybridize to OAS2 cDNAs, with sizes of 2.8, 3.3, 3.9, and 4.5 kb (258). The 100-kDa large isoform of OAS, designated OAS3, is a 1,087-amino acid protein as deduced from cDNA sequence; it is composed of three adjacent repeat domains, each homologous to OAS1 (215, 336). An IFN-inducible transcript of ~7 kb encodes OAS3. Based on analysis of cDNA and genomic clones, the small (40- and 46-kDa), middle-sized (69-kDa), and large (100-kDa) OAS proteins are products of three distinct genes, clustered over ~130 kb on human chromosome 12 in the region 12q24.2 (60, 174, 175, 255, 258). Thus, it appears that mammalian OAS genes underwent successive gene duplication events, resulting in the three sizes of enzymes containing one (OAS1), two (OAS2), or three (OAS3) homologous domains (215).

Oligomerization of OAS1 and OAS2 appears necessary for enzymatic activity (137, 358). In their native forms, the small OAS1 isoform exists as a ~180-kDa tetramer, the middle-sized OAS2 isoform exists as a ~160-kDa dimer, and the large OAS3 protein exists as a monomer (258). Mutations affecting a tripeptide sequence CFK impair subunit association and are characterized by a reduction in OAS enzymatic activity (137). The three different-sized forms of OAS are associated with different subcellular fractions, including membranes, cytoplasm, and nucleus; they differ in the concentration of dsRNA required for their activation; they differ in the reaction conditions required for their optimal enzymatic activity; and they differ in the size pattern of the 2-5A products produced (172, 173, 179, 334, 433). The full physiological significance of these differences is not yet known. While three forms of OAS are seen in human cells, the cDNAs so far isolated and characterized in other species, including mouse, rat, pig and chicken, appear to correspond most closely to the OAS1 form (334).

Although OAS proteins are activated by dsRNA, there is no obvious structural homology between the dsRNA-binding domains of the OAS proteins and those of PKR or ADAR (139, 269). However, like PKR and ADAR1 (68, 312, 352), the OAS enzymes possess separate subdomain regions responsible for their RNA-binding activity and for their catalytic activity (334, 357). OAS enzymes are activated during viral infection (334). Although in most instances the activator RNA has not been defined precisely, it is presumed to be of viral origin. Conceivably, viral "dsRNA" activators might include single-stranded transcripts which possess significant double-stranded character in addition to the obvious potential sources of dsRNA structures such as RNA duplexes as part of their replicative intermediates, RNA duplexes derived by symmetric transcription from opposing promoters, and RNA duplexes that are genomic dsRNAs released from unstable virions or subviral particles of Reoviridae family members. Two well-characterized viral RNAs that do affect OAS enzymatic activity are the HIV TAR RNA and adenovirus VA RNA. The TAR RNA sequence, present at the 5'-termini of HIV transcripts, forms a stable secondary structure and possesses an intrinsic ability to activate both OAS and PKR (250). By contrast, a mutant form of TAR RNA with disrupted secondary structure does not activate either IFN-induced enzyme. The activation of OAS and PKR by TAR RNA suggests a mechanism for the control of HIV replication by the IFN system (29, 250, 293). Curiously, adenovirus VA RNA that antagonizes PKR has just the opposite effect on the OAS; VA RNA can both bind and activate OAS (86). The ATP-binding domain of recombinant human 40-kDa OAS identified by photoaffinity labeling and sequence analysis includes Lys-K199 that is photolabeled with 8-azido-[-³²P]ATP; Lys-199 is present in a dodecapeptide sequence that is highly conserved among all OAS proteins as deduced from human, mouse, and rat cDNAs (202).

The induction of OAS has been extensively characterized in a variety of different human and mouse cell lines and tissues (36, 351, 394). Early studies revealed that the magnitude of induction is dependent on the type of IFN, the type of cell, and the growth state of the cell. For example, induction levels range from about 10-fold for human HeLa cells that often show a high basal enzyme level (11) to about 10,000-fold for chicken embryo cells that have a low basal enzyme level (14). The three different-sized forms of OAS are all induced by IFN-, IFN-, and IFN- (60, 433). The 5'-flanking region of the OAS1, OAS2, and OAS3 genes all contain an ISRE (28, 335, 422). However, the induction by IFN-, IFN-, and IFN- of different isoforms of OAS, as well as total OAS enzymatic activity, differs among human cell lines in vitro and in primary sources, for example among donors of normal peripheral blood mononuclear cells (433). This no doubt reflects differences in the organization of additional regulatory elements between the promoters and the abundance of the trans-acting factors in different types of cells and tissues. For example, the OAS3 promoter possesses elements conferring direct inducibility not only by IFNs but also by TNF and retinoic acid (335). Consensus binding sites for IRF family members are present within OAS promoters (63, 335). Ectopic expression of IRF-1 leads to activation of the OAS gene promoter, whereas expression of IRF-2 leads to repression of the OAS promoter (63).

RNase L, the endoribonuclease activated by 2-5A oligonucleotides, is also variously known in the earlier literature as the 2-5A-dependent RNase and RNase F (322). The presence of a functional 2-5A oligomer is required for the conversion of RNase L from an inactive monomeric form to the active dimeric form. The RNase L protein acquires endoribonuclease catalytic activity after binding 2-5A, a process associated with the formation of stable homodimers (97, 98, 160). The ability of 2-5A derivatives to activate RNase L correlates with their ability to mediate dimer formation (97). However, RNase L has also been reported as a dimer of regulatory and catalytic subunits (347), a state which possibly corresponds to a heteromeric complex of the RNase L protein with the RNase L inhibitor protein RLI (38). Activated RNase L catalyzes the degradation of both viral and cellular RNAs, including cellular rRNA, by cleaving on the 3' side of -UpXp- sequences (113, 434).

The gene for RNase L, designated RNS4, maps to human chromosome 1q25 by fluorescence in situ hybridization (389). The mRNA transcript of the human RNS4 gene is about 5.0 kb as measured by Northern blot analysis; the protein-coding sequence constitutes only about 40% of the nucleotide sequence of the mRNA (450). The human RNase L protein is 741 amino acids, about 83 kDa as deduced from the cDNA sequence (450). Analysis of aligned human and mouse RNase L sequences suggests several intriguing features of the proteins. RNase L displays a similarity to RNase E, an RNase implicated in the control of mRNA stability in Eschericia coli (403, 450). The N-terminal half of RNase L possesses nine ankyrin-like domains that may be involved in protein-protein interactions (160). A duplicated phosphate-binding loop motif within the N-terminal half of RNase L functions in the binding of the 2-5A oligonucleotides as established from cDNA mutagenesis studies (160, 322). Antagonists that impair 2-5A-dependent RNase L activity have been described. These include a cellular protein of 68 kDa, designated RLI (38), and oligonucleotide derivatives of 2-5A that accumulate in certain types of virus-infected cells (55).

RNase L is constitutively present in most types of cells (112), although treatment with IFN-/ enhances RNase L activity in some types of cells (111, 182). Subsequent Northern blot analysis established that IFN-/ increases the steady-state amount of the RNase L transcript in mouse cells by about threefold (450). The RNase L inhibitor, RLI, is not regulated by IFN (38).

The availability of cDNA clones for OAS and for RNase L facilitated studies of the roles of these enzymes in biological processes including virus replication. Among the various families of animal viruses examined, the Picornaviridae shows the best correlation between activation of the 2-5A pathway and inhibition of virus replication (61, 70, 346, 349). Constitutive expression in hamster or mouse cells of cDNA clones encoding the small form of the OAS is sufficient to establish an antiviral state, and this state appears selective for picornaviruses (61, 70, 346). For example, Chinese hamster ovary cells constitutively expressing the 40-kDa form of the human OAS are resistant to infection by Mengo virus but not VSV or herpesviruses (61). Similar results are observed with human and mouse cells transfected to constitutively express a cDNA for OAS1. Human T98G cells that express the cDNA encoding the 40-kDa form of human OAS and mouse NIH 3T3 cells that express the cDNA encoding the 43-kDa form of murine OAS display a resistance to EMC virus replication but not to VSV replication (70, 346). The resistance to EMC virus replication correlates with the expression of OAS enzyme activity (70, 346). Likewise, mouse cells stably expressing either the 69- or 71-kDa isoform of human OAS2 show a partial antiviral response to EMC virus (257). Constitutive expression of the 69-kDa OAS2 in human HT1080 cells likewise causes inhibition of EMC virus replication but not of VSV, SeV, or reovirus replication (138). In agreement with the apparently selective inhibition of picornavirus replication observed for cells overexpressing the OAS cDNAs (61, 70, 346), overexpression of the RNase L inhibitor in stably transfected HeLa cells partially inhibits the antiviral activity of IFN against EMC virus but not against VSV (38). Likewise, overexpression of a dominant negative mutant of RNase L also antagonizes the antiviral activity of IFN against EMC virus (160). Conversely, treatment of cells with 2-5A oligomer molecules provides some protection against picornavirus infection (171, 334).

Targeted disruption of the RNase L gene effectively provides a functional knockout of the 2-5A pathway, because the only known activity of 2-5A is the activation of the latent RNase L protein. Studies carried out with mice with a homozygous RNase L^/ gene disruptions illustrate the importance of the 2-5A pathway in the antiviral actions of IFN. RNase L^/ mice die more rapidly in response to infections with EMC virus than do RNase L^+/+ wild-type mice (448, 449). Furthermore, RNase L^/ MEF are defective in apoptotic responses (449). Mice triply deficient in RNase L, PKR, and Mx, generated by combination of the RNase L null with the PKR null in a Mx1^/ background, show an added deficiency in the antiviral response to EMC virus (448). However, a substantial residual antiviral response is observed in MEF lacking all three proteins, RNase L, PKR, and Mx1, further illustrating the redundant nature of pathways that collectively constitute the innate antiviral response of IFN-/ (448). Somewhat surprising, the level of IFN-induced gene products even may be elevated in the absence of functional RNase L. Comparative kinetic analyses with RNase L^/ mutant and RNase^+/+ wildtype MEF suggest an RNase L-dependent destablization of IFN-induced mRNAs, consistent with a possible role for the 2-5A system in the attenuation of the IFN response (233).

Posttranscriptional RNA modifications such as deamination of adenosine to yield inosine provide an important mechanism by which the functional activity of viral and cellular RNAs can be altered and, hence, by which biological processes can be affected (22, 51, 252, 344, 384). One important RNA-editing enzyme, ADAR1, is an IFN-inducible RNA-specific adenosine deaminase (134, 312, 313). Several studies implicate the IFN-inducible ADAR1 deaminase in the editing of viral RNA transcripts and cellular pre-mRNAs. The biological importance of RNA editing in animal cells is significant and far ranging (22, 24, 51, 249, 384).

ADAR was first identified as a dsRNA-unwinding activity in Xenopus oocytes (23, 333). ADAR catalyzes the covalent modification of highly structured RNA substrates by hydrolytic C-6 deamination of adenosine to yield inosine. The resultant A-to-I transitions destabilize the dsRNA helix by disrupting base pairing; RNA becomes more single stranded in character because stable AU base pairs are changed to the considerably less stable IU pair (23, 419). Hypoxanthine, the base of the nucleotide inosine generated from adenosine by the deamination, is typically recognized as guanine by the translational and transcriptional machinery (4). Posttranscriptional conversion of adenosine to inosine has been demonstrated in both viral RNAs and nucleus-encoded cellular mRNAs.

The cDNA encoding the human ADAR1 deaminase (K88 cDNA) was isolated in a screen for IFN-regulated cDNAs (312). It hybridizes to a single major transcript of ~7 kb in human cells (313). Accumulation of ADAR1 transcripts is increased about fivefold by IFN treatment (312, 313). Both IFN- and IFN- induce ADAR1 transcript accumulation (313). As deduced from the cDNA ORF, human ADAR1 is a 1,226-amino-acid protein with a molecular mass of about 136 kDa (194, 312), but the protein migrates at about 150 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (312). Southern blot and sequence analyses are consistent with a single Adar1 gene. Genomic Adar1 clones colocalize to human chromosome 1q21.1-21.2 (423, 427) and mouse chromosome 3F2 (428) as shown by fluorescence in situ hybridization. Sequence analysis de