Interferons (IFNs) — the body's first line of antiviral defence — are cytokines that are secreted by host cells in response to virus infection. By inducing the expression of hundreds of IFN-stimulated genes, several of which have antiviral functions, IFNs block virus replication at many levels.
The global antiviral state of the cell involves cross-talk between IFN signalling and pathways that regulate apoptosis, inflammation and cellular stress-response programmes.
Viruses counteract the antiviral response by encoding mechanisms to control IFN signalling, block the actions of IFN-stimulated gene products and disrupt the various levels of cross-talk between IFNs and other cellular pathways. Studies of influenza virus, hepatitis C virus, herpes simplex virus and vaccinia virus highlight the importance of IFNs for the control of virus replication and pathogenesis.
Studies of both host antiviral pathways and viral-counteracting strategies will greatly benefit from the recent development of functional-genomic technologies, such as microarrays, proteomics and DNA shuffling. Our 'virus compendium' — a multi-faceted, functional genomics effort focusing in the field of virus–host interactions — will be useful to assimilate these data.
The action of interferons (IFNs) on virus-infected cells and surrounding tissues elicits an antiviral state that is characterized by the expression and antiviral activity of IFN-stimulated genes. In turn, viruses encode mechanisms to counteract the host response and support efficient viral replication, thereby minimizing the therapeutic antiviral power of IFNs. In this review, we discuss the interplay between the IFN system and four medically important and challenging viruses — influenza, hepatitis C, herpes simplex and vaccinia — to highlight the diversity of viral strategies. Understanding the complex network of cellular antiviral processes and virus–host interactions should aid in identifying new and common targets for the therapeutic intervention of virus infection. This effort must take advantage of the recent developments in functional genomics, bioinformatics and other emerging technologies.
Interferons (IFNs), although best known for their antiviral properties1,2, are potent regulators of cell growth3 and have immunomodulatory activity4. Indeed, an emerging theme is that these cytokines are important regulators of innate and adaptive immune responses. Furthermore, studies now highlight the importance of cross-talk between cellular regulatory pathways that control IFN signalling, apoptosis, inflammation and the stress response (Box 1). There are two main types of IFN, type I and type II. Type I or 'viral' IFNs include IFN-α, IFN-β, IFN-ω and IFN-τ; type II IFN is IFN-γ. Most types of cell can produce IFN-α and IFN-β, which are the best-characterized type I IFNs, whereas IFN-γ is produced only by certain cells of the immune system, including natural killer (NK) cells, CD4+ T helper 1 (TH1) cells and CD8+ cytotoxic T cells. There are 14 different IFN-α genes, but only one IFN-β and one IFN-γ gene. IFNs mediate their effects through interactions with type-specific receptors, which are different and non-redundant for the type I and type II IFNs5. IFNα/β-receptor-knockout mice (as well as IFN-γ-receptor knockouts) cannot mount effective antiviral responses6,7. The IFN receptors do not have enzymatic activity, but they set in motion a complex signalling pathway that ultimately results in the transcription of hundreds of IFN-stimulated genes (ISGs) (Fig. 1 and Box 2). It is now clear that although IFN levels markedly increase in response to virus infection, the sequence of events, types of IFN that are produced and ISGs that are targeted have an important effect on the outcome.
The regulation of IFN-β synthesis is well characterized and requires the participation of several transcription-factor complexes, such as nuclear factor-κB (NF-κB), ATF/JUN and, in particular, the IFN-regulatory factors (IRFs)5,8. These factors are often activated by phosphorylation on serine residues. However, the crucial feature is that activation of the IRFs is triggered by virus infection, probably through the production of viral double-stranded (ds)RNA and other virus-specific signals. Reflecting the intimate relationship between viruses and their host, host cells have evolved signalling mechanisms to sense and respond to virus infection. As described below, such mechanisms involve cross-talk between different cellular pathways to modulate the expression and antiviral function of IFNs and specific IFN-induced gene products. Similarly to IFN-β, the IFN-α genes are also activated in response to virus infection and the induced serine phosphorylation of specific transcription factors. However, the IFN-α and IFN-β genes are not expressed at the same level or with the same kinetics after virus infection. There seems to be a crucial positive-feedback loop that depends on many IRFs to control IFN expression, although recent in vivo experiments indicate that there is an even more complex system of regulation9. The expression of IFN-β and IFN-α4 seems to be induced early through the action of IRF3 (Ref. 10). However, the other IFN-α genes require that IRF7 is synthesized and activated for their expression11,12. Unlike IRF3, IRF7 is not constitutively expressed and it needs to be transcriptionally activated through the IFN receptor/Janus kinase–signal transducer and activator of transcription (JAK–STAT) pathway, as do most IFN-induced genes (Box 2). IFN-β and IFN-α4 provide the initial signal that allows IRF7 to be produced, thereby leading to the expression of the full spectrum of IFNs and ISGs.
The workhorses of the type I IFN system are the many ISGs1,13 (Fig. 1). Until the advent of DNA microarrays, it was thought that there were perhaps 30–40 ISGs, a small number of which were thought to have antiviral properties. Now, we know that there are hundreds of IFN-regulated genes, many of which are repressed or downregulated by IFN14,15 (G. Geiss et al., unpublished observations). We must, therefore, completely re-evaluate our thoughts on how IFNs interfere with virus infection and how, in turn, viruses fight back. Clearly, the host repertoire that is involved in host defence is much more extensive than was previously thought.
Perhaps the most intensely studied type I IFN-induced gene is the dsRNA-activated serine/threonine protein kinase, which is now known as PKR16. Activated PKR can negatively affect cell-regulatory pathways, primarily messenger-RNA translation and transcriptional events. As for IFN itself, viral-specific RNAs can activate PKR, which inhibits virus replication and the production of virion progeny. Nearly all viruses have developed strategies to downregulate the activity of PKR so that virus replication is not compromised17. Moreover, there are several cellular regulators, both inhibitors and activators, of PKR. Another crucial pathway involves the IFN-mediated response that is responsible for mRNA degradation, which comprises two enzymes — 2′,5′-oligoadenylate synthetase (OAS) and RNase L18,19. This pathway also seems to be activated by dsRNA. Originally, it was thought that this pathway was focused only on the degradation of viral RNAs, as part of the IFN-mediated antiviral artillery. Clearly, however, cellular RNAs are also targets of this pathway, which indicates that it has an important cell-regulatory role. Both knockout mice20 and convincing in vitro experiments show that this pathway has an important antiviral role. The myxovirus-resistance (Mx) proteins were among the first IFN-induced proteins to be studied in the context of a virus infection21. Mx proteins are IFN-inducible GTPases; their antiviral activity requires enzymatic function. The function of the Mx proteins was determined primarily in the influenza- and Thogoto-virus systems. A recent study has shown that MxA binds to the nucleocapsid proteins of bunyaviruses and causes the redistribution of viral capsid proteins as a mechanism to inhibit bunyavirus replication22. This turns out to be a highly complicated story because of the differences between human and mouse Mx proteins, the differences between nuclear and cytoplasmic forms of Mx and the spectrum of viruses that are negatively affected by Mx proteins. A recently discovered IFN-induced gene is the RNA-specific adenosine deaminase ADAR23, although its potential antiviral function requires characterization. ADAR is involved in RNA editing by virtue of its ability to deaminate adenosine to yield inosine, which provides a mechanism to alter the functional activity of viral and cellular RNAs. Such RNA editing occurs on viral RNAs, particularly NEGATIVE-STRAND RNA GENOMES. These modifications might relate to the persistence of infection and/or be a mechanism by which mRNAs are inactivated late in virus infection.
As mentioned earlier, IFNs have potent immunomodulatory properties. It is probable that the complete IFN response involves both innate and adaptive immune responses4,24. MHC class I and II molecules present antigenic peptides, derived from the degradation of viral proteins, to CD8+ T cells and CD4+ T cells, respectively. Both class-I-restricted CD8+ T cells and class-II-restricted CD4+ T cells are activated during viral infection25. So, it is no accident that IFNs also upregulate the expression of MHC class I and II, thereby enhancing the cellular immune response to virus infection in vivo. This might be a later event in the host-response repertoire, primarily contributing to recovery from infection, rather than being an initial host defence.
As a protein family, the IRFs have received much attention for their roles in regulating the host response to virus infection26. The first IRFs — IRF1 and IRF2 (we are up to IRF10 at the last count27) — were identified originally as a transcriptional activator and repressor, respectively. The IRFs are extremely important during virus infection and the host response8, and they are targeted by viruses for regulation during infection. Some viruses, such as human herpes virus 8 (HHV8) or Kaposi's sarcoma-associated herpesvirus (KSHV), encode IRF homologues to act as decoys and thereby evade host IFN-mediated defence28. At least four members of the IRF family — IRF1, IRF3, IRF5 and IRF7 — act as transducers of virus signalling. In response to infection, these transcription factors are phosphorylated on serine residues and transported to the nucleus, where they can activate or repress the transcription of either IFNs themselves or IFN-regulated genes.
Viruses fight back
Viruses have been reported to block nearly all aspects of the IFN regulatory pathway2,29,30,31. This includes the disruption of dsRNA and IFN receptor/JAK–STAT signalling events, the inhibition of IRF and NF-κB functions, and other mechanisms that target the antiviral actions of specific ISG products. As there have been several reviews published on this subject recently2,29,30,31, we do not attempt to cover the entire topic, but focus on four medically important virus systems.
Influenza virus, an orthomyxovirus that has a segmented negative-strand RNA genome, has a prominent role in the history of IFNs. After all, type I IFN was first discovered using heat-inactivated influenza-virus-infected chick cells32. Moreover, the antiviral Mx proteins inhibit influenza-virus replication at many levels, and this was one of the early prototype systems to study the antiviral effects of IFN33. Curiously, there are no reports of strategies of influenza virus to negate the various effects of Mx proteins, probably because no one has looked. There are, however, many reports of viral strategies to evade other aspects of the IFN response — in particular, the role of the viral NS1 gene product in disarming the host innate-defence system and blocking PKR activity34,35. Recently, Garcia-Sastre36 has reviewed these influenza-virus strategies; therefore, we discuss only the poorly understood, but emerging, theme that viruses can usurp a cellular stress response to fight the innate IFN response (in other words, viruses can turn the host on itself); and the anti-IFN effector non-structural protein NS1, the pandemic of 1918–1919 (Box 3) and mechanisms of pathogenesis.
Stressed out. P58IPK is a cellular molecular co-chaperone and member of the heat-shock 40 DnaJ family of proteins37. P52RIPK is a cellular protein that has homology to the heat-shock protein 90 kD (HSP90) family of HSPs38. P58IPK is known to interact with HSP40 and HSP70, and it can stimulate the ATPase activity of the latter39. So, what relevance does this have for influenza virus and the IFN response? P58IPK is a cellular inhibitor of PKR that is activated by influenza-virus infection40. P58IPK-mediated inhibition of PKR ensures that viral mRNA translation is not compromised due to excessive phosphorylation of eukaryotic initiation factor 2, α-subunit (eIF-2α). P52RIPK is a cellular inhibitor of P58IPK that indirectly potentiates PKR activity through its ability to regulate the function of P58IPK (Ref. 38). HSP40 is also a negative regulator of P58IPK, keeping it in an inactive complex until it is required. Furthermore, we found recently that the gene that encodes P58IPK has an endoplasmic-reticulum stress element (ERSE) in its promoter region (W. Yan et al., unpublished observations). This promoter is activated during the stress of the unfolded protein response (UPR). Moreover, it now seems that P58IPK can interact with and inhibit the eIF-2α kinase PERK, which controls protein synthesis during the stress of the UPR (W. Yan et al., unpublished observations). This can result in the translation of specific mRNAs, such as the transcription factor C-EBP-homologous protein (CHOP)41. So, it seems that P58IPK is at the centre of a cellular stress pathway that is related to the IFN response. What is truly remarkable from the point of view of the virus is that it outsmarts the host at its own game. The host cell has set up several sensors to deal with stresses of all kinds (including virus infection and the UPR). The P58IPK stress-regulatory pathway has been taken over by the virus to downregulate the host-defence IFN system (Fig. 2). The end result is that the virus does not need to devote important coding capacity to the regulation of the host response.
NS1. The influenza-virus-encoded non-structural NS1 protein is a viral gene product that seems to do everything (reminiscent of the simian virus 40 (SV40) T antigen) — such as controlling mRNA transport, splicing, polyadenylation and translation, to mention only a few of its ascribed functions36. So, one would never predict (from the literature at least) that a virus that lacks the complete NS1 gene could ever be viable. However, the only 'real' phenotype seems to be that the NS1-deleted virus is exquisitely sensitive to the antiviral effects of IFN. The deleted virus can replicate effectively only in knockout cells that lack key components of the IFN regulatory pathway35. So, NS1 has been identified as an important viral component of the influenza-virus armament against IFN. Indeed, our recent microarray study42 showed that during influenza-A/PR/8/34-virus infection of human lung epithelial cells, deletion of the NS1 gene increased the number and level of expression of cellular genes that are implicated in the IFN pathway and other antiviral pathways. Specifically, viruses that were deleted of the NS1 gene induced a general increase in the transcription of ISGs and NF-κB-mediated gene expression compared with wild-type virus, and were observed to differentially regulate genes of the suppressor of cytokine signalling (SOCS) family, which modulate cytokine and growth-factor signalling in a classic negative-feedback loop43. The complete mechanisms by which NS1 disrupts the IFN response are not known yet, although clearly, the inhibition of PKR (in addition to cellular P58IPK) and regulation of the IRFs are involved36.
Hepatitis C virus
No other virus has received more attention than hepatitis C virus (HCV) with regard to IFNs44,45,46. The reason for this is simple — type I IFN is used as the main antiviral therapeutic against HCV in humans. At present, IFN is prescribed in combination with ribavirin (a guanine nucleotide analogue. This virus is interesting because: HCV infects 2–4% of the world population; IFN therapy is a billion-dollar industry; IFN is ineffective in most treated individuals, the explanations for which are controversial; nearly every large pharmaceutical and biotechnology company has a programme to develop more-effective HCV antiviral therapeutics; and there is no robust animal or tissue-culture model to study the natural history of HCV infection, replication and pathogenesis47,48.
HCV and IFN therapy. HCV resistance to IFN therapy is defined loosely as the continued presence of HCV RNA in patient serum after treatment49. As with many RNA viruses, HCV circulates in the host as a population of QUASISPECIES, most probably selected from mutations that have accumulated in the HCV genome due to the infidelity of the NS5B RNA polymerase during viral replication50. Different HCV isolates or genotypes have different levels of sensitivity to IFN treatment. In particular, HCV genotypes 1 and 4 are less sensitive to type I IFN therapy than are HCV genotypes 2 and 3; the latter genotypes have a response to pegylated IFN and ribavirin combination therapy in up to 85% of individuals, whereas the former genotypes show IFN resistance in most patients. This is a problem in the United States, where HCV genotype 1 is the predominant form. As differences in the HCV genome might affect the structure and function of viral genome and proteins, these modifications might, in turn, affect interactions with many host-cell functions, including those that are involved in the antiviral actions of IFNs in infected cells. To elucidate the mechanisms by which selected HCV variants escape the antiviral effects of type I IFN, Enomoto and colleagues compared full-length sequences of IFN-α-responsive and -unresponsive viruses from HCV-infected patients51,52. Genotype-1b-HCV-infected patients who had a sustained response to type I IFN therapy were found to carry isolates that contain many recurring mutations in a region of 40 amino acids in the carboxyl half of NS5A, which corresponds to residues 2209 to 2248 of the HCV polyprotein. These observations indicate that this NS5A region, termed the IFN-sensitivity-determining region (ISDR), might have a role in HCV resistance to IFN treatment (Fig. 3). However, the predictive value of the ISDR in determining the outcome of IFN therapy, particularly for European and North American HCV isolates, has been questioned by other studies53. Nevertheless, a recent study based on a new statistical META-ANALYSIS of a database of 675 published individual ISDR sequences strongly indicates that there is a significant correlation between the NS5A ISDR and the IFN response54. Despite this controversy, NS5A and the ISDR provide the first hint of a potential molecular mechanism by which specific HCV genotypes might escape the IFN response. Although the correlation is not universally accepted, it is clear that NS5A is important in conferring IFN resistance to HCV.
HCV adapts to pressure imposed by the cell. HCV cannot be propagated in vitro. This limitation has been overcome partially by the development of the HCV replicon, which is an autonomously replicating subgenomic viral RNA that encodes a drug-selectable marker and the viral components that are required for authentic HCV RNA replication55,56. This system allows the selection of stable cell lines that support HCV RNA replication. Recent in vitro studies using the HCV replicon system are beginning to shed some light on how HCV interacts with the host cell and the IFN system. Sequence analysis of stable HCV-replicon populations has shown that the process of viral RNA replication in culture involves selection for adaptive mutations, many of which occur in the NS5A coding region47. Evidence indicates that such mutations do not arise stochastically, but are, in part, determined by specific antiviral pressures from the host cell that are induced as a result of viral RNA replication. Although initial reports concluded that the HCV replicon is simply sensitive to IFN56,57, we now know that the actual picture is not so simple. Type I IFN treatment results in a marked reduction in viral RNA levels, but extremely high and prolonged doses of IFN are required to completely ablate replicon replication58. These observations raise two main questions: is IFN resistance of HCV an acquired trait; and what are the molecular mechanisms by which IFN suppresses HCV replication? The second question is most relevant considering that type I IFN has been approved as a drug to treat HCV infection, yet the actual mechanisms of drug action are unknown. To address these issues, we have isolated several distinct replicon quasispecies that have been selected in IFN-treated cells59. These HCV replicons have incorporated further mutations, including many in NS5A, that confer increased resistance to the IFN-induced antiviral response when passaged in fresh cells that have not previously been exposed to IFN. Comparison of the phenotypic properties of such IFN-adapted replicons and those replicons isolated in the absence of IFN has shed light on specific stages of HCV replication that are likely to be targeted by IFN action, including, most importantly, the assembly of the viral replicase complex on the substrate RNA. What is now required is a genetic dissection of the viral sequences that confer IFN resistance and a comprehensive identification of host ISGs that function to limit HCV replication. Such an approach will help to define the molecular mechanisms of the action of IFN on HCV replication, and might lead to improved therapies for the millions of HCV-infected individuals worldwide.
Herpes simplex virus
Herpes simplex virus type 1 (HSV1) is a neurotropic DNA virus that infects a large proportion of the human population60. Direct contact of HSV1 with host mucosal tissues results in primary infection and eventual dissemination of progeny virions from the site of infection to neuronal tissue, where they persist in a latent state in the sensory ganglia. This latent state is punctuated by sporadic reactivation of virus replication in peripheral mucosal tissue that is innervated by the infected ganglia. Genetic studies have defined various virus–host interactions that induce or counteract the cellular antiviral state to affect HSV replication and latency. These studies show that viral modulation of the IFN system, and PKR in particular, is the main basis for neurovirulence and pathogenesis associated with HSV1 infection61.
HSV1 triggers the IFN response. The virion structure of HSV1 includes a lipid envelope that contains 11 viral-encoded glycoproteins, at least a subset of which are thought to facilitate interaction with and attachment to the host cell62. The infection of cultured human cells with HSV1 induces the production and secretion of IFN-α, which is attributed to specific interactions between the virus and cell-surface proteins. In particular, IFN-α production can be induced in human mononuclear cells by culturing cells in the presence of purified, recombinant HSV1 glycoprotein D (gD), perhaps through a mechanism that involves gD-mediated stimulation of intracellular signalling through engagement of CC-chemokine receptor 3 (CCR3) or CXC-chemokine receptor 4 (CXCR4)63. So, it seems that host–viral-glycoprotein interactions might induce cellular signalling events that culminate in IFN production. The mechanisms by which chemokine receptors might signal the host antiviral response and induce IFN production remain unclear, but in a similar manner to tumour-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF signalling64, this might well be attributable to receptor cross-talk with IFN signalling pathways. Relevant to this idea, the interactions of HSV1 with host cells have now been shown to activate IRF3 and to trigger the production of IFN in cultured human fibroblasts65. IRF3 activation was attributed to the (gD-dependent) viral-glycoprotein-mediated entry of HSV1 into the host cell, possibly through chemokine-receptor interactions with the viral glycoproteins. The potential involvement of CCR3 and CXCR4 in the host response to HSV1 is intriguing, as both types of receptor are present on the surface of the dendritic-cell-like major IFN-producing cells. So, one might propose that as the primary host for HSV infection, humans have evolved cellular mechanisms to 'sense' and rapidly respond to HSV before it gains a foothold in the cell. This would indicate that viral-glycoprotein interactions might be a potential cellular sensing mechanism that signals the presence of HSV to induce the rapid production of IFN and the expression of ISGs, thereby preparing the cell to counter a possible HSV infection. Collectively, these results raise the questions of how IFN affects HSV replication and how the virus counteracts the potentially deleterious effects of IFN to persist in the infected cell.
HSV–IFN interactions. Through the functions of various viral proteins and host interactions, HSV1 can regulate the host response at many levels (Fig. 4). The processes of cellular exposure to HSV1 and viral binding or entry stimulate the production of IFN and expression of ISGs, perhaps through the activation of IRF3 (Ref. 65). This poses an early threat to viral replication, which is, in part, countered by infected cell protein 0 (ICP0). ICP0 can interact with the host proteasome-mediated degradation pathway to alter the stability of certain ISG products66, and the temporal expression of ICP0 correlates with a block in host JAK–STAT signalling processes67,68. However, ICP0-mediated regulation of ISG expression is not sufficient to counter the antiviral actions of PKR, the activity of which is probably induced by viral dsRNA products of the HSV1 transcriptome67. Unless the virus counteracts PKR function, the host cell will undergo a block in protein synthesis due to the high levels of eIF-2α phosphorylation that are catalysed by active PKR. The primary mechanism by which HSV1 circumvents PKR action might be through the recruitment of protein phosphatase 1α (PP1α) by viral ICP34.5 into a high-molecular-weight complex that efficiently dephosphorylates eIF-2 (Ref. 69), or through a direct interaction with PKR itself70. It is this ability to functionally counteract the effects of PKR that gives HSV1 its neurovirulent phenotype and allows virus growth in neuronal cells61,71. Leib et al.71 have elegantly shown that a virus that is attenuated by deletion of ICP34.5 has wild-type replication capacity and virulence in a host from which the PKR gene has been deleted, which provides a formal genetic test for identifying the in vivo mechanisms and targets of microbial virulence genes. Perhaps an evolutionarily more ancient system for inhibiting PKR resides in the Us11 coding region. In suppressor mutants of ICP34.5-deleted viruses, Us11 protein expression occurs early after infection72. This new expression pattern allows Us11 to inhibit PKR function, thereby supporting the growth of mutant HSV1 and partially restoring its neurovirulence. Overall, these results form an intriguing model to indicate that ICP34.5 and PKR are the key players in regulating HSV growth and pathogenesis. However, viral proteins are often multifunctional, and it is probable that ICP34.5 has other functions that contribute to HSV1 neurovirulence. In addition, such a model does not consider the contributions of other HSV1 proteins, which might function alone or cooperatively to interact with host antiviral systems. What other viral proteins might interact with the IFN system; do HSV gene products target specific ISGs for regulation; and what other ISGs might specifically affect HSV1 replication? Until the complex and pleiotropic actions of IFN on the host cell are completely understood, our understanding of the actions of IFN on viral replication and pathogenesis will remain incomplete.
Vaccinia virus is a member of the poxvirus family — a family of complex, large dsDNA viruses that replicate in the cytoplasm and have had important roles in the history of medicine73,74. Vaccinia virus was used as the vaccine to eliminate smallpox — a devastating disease caused by variola virus that is currently an important bioterrorism concern75,76. Poxviruses have evolved various mechanisms to interfere with the activity of host cytokines, including IFN77,78,79,80 (Fig. 5). Uniquely, poxviruses encode soluble versions of cytokine receptors ('viroceptors') that intercept the normal activities of the target cytokines81,82,83. For example, the vaccinia-virus B8R protein binds soluble IFN-γ and prevents it from binding to cellular receptors81. This strategy to block the action of IFN-γ enables poxviruses to inhibit both the antiviral effects and, more importantly, the immune-regulatory functions of IFN-γ, simultaneously. The vaccinia-virus B18R open reading frame also encodes a soluble IFN-α/β receptor that blocks the binding of IFN-α/β to cell-surface receptors84,85. The IFN-γ receptor is highly conserved between members of the poxvirus family. Vaccinia virus that lacks the IFN-γ receptor is attenuated in vivo, although this deletion has no effect on virus replication in vitro86,87.
Vaccinia virus uses at least two known functions to target PKR, which highlights the important role of this kinase in virus–host interactions. The vaccinia-virus proteins E3L88,89,90,91 and K3L91,92,93, which are conserved in variola virus94,95, block IFN-mediated antiviral responses. E3L encodes a dsRNA-binding protein that is involved in the inhibition of PKR89,90 by interfering with the binding of PKR to dsRNA96. E3L might also prevent PKR activation by masking the substrate-binding domain97. K3L, a vaccinia-virus-encoded eIF-2α homologue92 that potentiates translation by inhibiting PKR and eIF-2α phosphorylation93,98, acts by means of its homology to eIF-2α to interfere with the interaction of eIF-2α with PKR96. Deletion of the vaccinia-virus K3L gene reduced the ability of the virus to grow in IFN-treated cells92, and vaccinia virus devoid of the E3L gene was also sensitive to the antiviral effects of IFN91.
As a dsRNA-binding protein, vaccinia virus E3L can also block other dsRNA-mediated antiviral pathways, such as the IFN-induced OAS enzyme99 and IRF3 and IRF7 phosphorylation100, which indicates that there are other mechanisms for the anti-IFN effects of E3L that are distinct from its inhibition of PKR. Furthermore, E3L inhibits the adenosine-to-inosine editing activity of IFN-induced ADAR101. Although the amino-terminal domain of E3L is dispensable for infection of cells in culture, both the carboxy-terminal domain (which is required for IFN resistance, binds to dsRNA and inhibits PKR) and the amino-terminal domain of E3L were required for full pathogenesis in intranasal infections in a mouse model102, which indicates the existence of dsRNA- and PKR-independent functions of E3L. Interestingly, vaccinia-virus virion-contained phosphatase (VH1) can bind to and dephosphorylate STAT1, which indicates a new mechanism by which vaccinia virus interferes with the onset of host immune responses by blocking the IFN signalling cascade through the dephosphorylating activity of the viral phosphatase VH1 (Ref. 103).
IFN and functional genomics
The new technologies of functional genomics have had a marked impact on human biomedical research. In particular, global gene-expression analysis is now in widespread use in cancer and infectious-disease research, and it has become an integral part of the drug-discovery process104,105,106. Recent advances in proteomics have augmented this approach, making it possible to identify and quantify virtually all proteins that are present in a particular cell or tissue107, and to characterize global protein–protein interaction networks in an organism108. Together, these technologies provide a global perspective on the complex interactions that occur between all levels of biological information ('systems biology'), from gene expression to protein production109,110. Making sense of the huge amounts of data that are generated by these approaches requires highly sophisticated information technologies, which is the domain of the relatively new discipline of bioinformatics. This confluence of genomic and information technologies brings a powerful new approach to the study of biological systems, as exemplified by recent studies111. The study of virus–host interactions and viral evasion of host defences will be revolutionized by these approaches. Reports from the Cleveland Clinic on DNA microchip analysis of IFN-treated cells have already changed the field14,15. Before this high-throughput analysis was possible, it was thought that there might be, at most, 30, 40 or perhaps 50 IFN-regulated genes. Now, their studies and our own (G. Geiss et al., unpublished observations) show that there are probably hundreds of IFN-regulated genes, many of which are 'repressed' during IFN treatment. This must change the way that a virologist thinks about viral strategies to evade host defences.
The ever-increasing amount of microarray data that has been generated by examining virus–host systems has consistently shown that the expression of IFN and IFN-induced genes is differentially regulated in many systems (and this phenomenon is not only limited to studies of virus–host interactions). Studies with different members of the herpesvirus family best exemplify this point. IFN-induced genes are transcriptionally activated during human cytomegalovirus (CMV) infection. Perhaps more interestingly, the addition of gB — the virion attachment protein — to cell cultures induces basically the same subset of genes that are induced by replication of the virus during a normal infection112. After infection with another herpesvirus, HSV, IFN-regulated genes were induced by a non-replicating mutant, but were inhibited by the wild-type virus, which indicates that virus replication is necessary to suppress the host-defence IFN response in this HSV system113. In related studies, we have found by microarray analysis that heat- or UV-inactivated influenza virus, which attaches to host cells, but does not replicate, also induces the expression of a substantial subset of the same genes that are dysregulated during a productive infection114. Interestingly, the cellular IFN response to influenza-virus infection is viral-strain specific. The PR8 strain induces the synthesis of several IFN-induced genes, whereas the WSN strain dysregulates very few IFN-induced genes. We speculate that this might account for the neurovirulence of WSN (a reduced IFN response allows increased viral replication), which might be due, in part, to the NS1 gene product.
Little is known about how certain viruses trigger the IFN response or how this might occur as a result of viral attachment. It is widely thought that viral dsRNA intermediates that accumulate during the course of replication are the primary mediators that trigger IFN production. However, dsRNA is not present when inactivated virus or a viral attachment protein is used (unless contamination is a factor). We have carried out microarray analysis of dsRNA-treated cells that lack all type I IFN genes115. So, the possibility of gene induction by autocrine actions of IFN was eliminated. More than 175 genes were stimulated and nearly 100 genes were repressed — all in the absence of an IFN response. Different inflammatory cytokines and viruses also induced a subset of these dysregulated genes, which shows that there are interconnections between disparate pathways. Induction (and repression) of such a diverse family of genes has profound implications for virus–host interactions: this is a lot for the virus to cope with.
Another advantage of these high-throughput approaches is best illustrated by our work on HCV and microarrays. Many groups, including our own, have shown that the HCV NS5A protein confers type I IFN resistance, at least in part, through the inactivation of PKR116,117,118,119,120. To get a wider view, we carried out microarray analysis on cells that had been treated with type I IFN in the presence or absence of NS5A (or a mutant NS5A that is unable to bind PKR) (G. Geiss et al., unpublished observations). Our goal was to define the molecular mechanisms that make HCV resistant to IFN treatment. At the same time, we were able to carry out a high-throughput analysis of the global effects of treating cells with IFN. Remarkably, we observed that a distinct subset of IFN-regulated genes were downregulated after treatment with NS5A, some of which were not downregulated by the NS5A mutant that is unable to bind PKR. Another microarray approach was used to examine transcriptional profiles in chimpanzees infected with HCV121. A progressive increase in the number of genes with altered expression profiles occurred until the peak in alanine transaminase (a liver enzyme, increased levels of which indicate liver damage/inflammation), at which time the expression of more than 180 genes was significantly altered. Prominent among these were many IFN-regulated genes, including STATs, IRFs, Mx proteins and OAS. Remarkably, the level of expression of some of these genes was altered nearly 100-fold. Therefore, it is not surprising that HCV must encode anti-IFN strategies.
Final thoughts toward a strategic plan
The greatest challenges for the future will involve designing and developing better antiviral therapeutics, perhaps in conjunction with an IFN that has been made more potent by molecular-breeding technologies (Box 4). But, we desperately need a strategic plan. One possibility is to determine whether all viruses (or all RNA viruses, or all DNA viruses, or all respiratory viruses and so on) use common strategies to survive in the host and successfully replicate. We propose the establishment of a 'virus compendium' or a database that summarizes the events that occur during infection by all mammalian viruses (Fig. 6). This database should comprise both microarray transcriptional-profiling data and high-throughput proteomics information. Data should be assembled from various experimental systems, including in vitro infection systems, animal and human models, and surrogate systems such as the HCV replicon system. Using sophisticated software, such as ResolverTM and the proteomics software that has been developed at the Institute for Systems Biology, it should be possible to define common cellular pathways that are affected by virus infection or by overexpression of a viral protein. If specific genes/proteins are consistently up- or downregulated after infection with highly pathogenic viruses, we might be in a position to target these host-cell genes as a way to influence the outcome of a viral infection. Such an approach might lead us to the development of broadband antivirals — 'virus silver bullets' — which would be effective against a wide variety of viruses, just as antibiotics are effective against many different species of bacteria.
Samuel, C. E. Antiviral actions of interferons. Clin. Microbiol. Rev. 14, 778–809 (2001).
Levy, D. E. & Garcia-Sastre, A. The virus battles: IFN induction of the antiviral state and mechanisms of viral evasion. Cytokine Growth Factor Rev. 12, 143–156 (2001).
Grander, D., Sangfelt, O. & Erickson, S. How does interferon exert its cell growth inhibitory effect? Eur. J. Haematol. 59, 129–135 (1997).
Biron, C. A. Interferons-α and -β as immune regulators — a new look. Immunity 14, 661–664 (2001).
Doly, J., Civas, A., Navarro, S. & Uze, G. Type I interferons: expression and signalization. Cell. Mol. Life Sci. 54, 1109–1121 (1998).
Hwang, S. Y. et al. A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons-α and -β and alters macrophage responses. Proc. Natl Acad. Sci. USA 92, 11284–11288 (1995).Mice with a null mutation in the Ifnar1 gene were generated and it was shown that the type I IFN system is an important acute antiviral defence.
Kamijo, R. et al. Biological functions of IFN-γ and IFN-α/β: lessons from studies in gene knockout mice. Hokkaido Igaku Zasshi. 69, 1332–1338 (1994).
Barnes, B., Lubyova, B. & Pitha, P. M. Review: on the role of IRF in host defense. J. Interferon Cytokine Res. 22, 59–71 (2002).
Barnes, B. J., Moore, P. A. & Pitha, P. M. Virus-specific activation of a novel interferon regulatory factor, IRF-5, results in the induction of distinct interferon-α genes. J. Biol. Chem. 276, 23382–23390 (2001).
Juang, Y. et al. Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3. Proc. Natl Acad. Sci. USA 95, 9837–9842 (1998).This study identified IRF3 and CBP/p300 as integral components of the virus-induced complex that stimulates type I IFN gene transcription, and indicated a new mechanism by which adenovirus might overcome the antiviral effects of the IFN pathway.
Levy, D. E., Marie, I., Smith, E. & Prakash, A. Enhancement and diversification of IFN induction by IRF-7-mediated positive feedback. J. Interferon Cytokine Res. 22, 87–93 (2002).
Yeow, W. S. et al. Reconstitution of virus-mediated expression of interferon-α genes in human fibroblast cells by ectopic interferon regulatory factor-7. J. Biol. Chem. 275, 6313–6320 (2000).
Biron, C. A. & Sen, G. C. in Fields Virology (eds Knipe, D. M. & Howley, P. M.) 321–352 (Lippincott, Williams & Wilkins, Philadelphia, 2001).
de Veer, M. J. et al. Functional classification of interferon-stimulated genes identified using microarrays. J. Leukocyte Biol. 69, 912–920 (2001).
Der, S. D., Zhou, A., Williams, B. R. & Silverman, R. H. Identification of genes differentially regulated by interferon-α, -β or -γ using oligonucleotide arrays. Proc. Natl Acad. Sci. USA 95, 15623–15628 (1998).Using microarray-based mRNA profiling of IFN-treated human cells, this study showed the usefulness of oligonucleotide arrays for monitoring mammalian gene expression and provided new insights into the basic mechanisms of IFN actions.
Meurs, E. et al. Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell 62, 379–390 (1990).
Gale, M. Jr & Katze, M. G. Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase. Pharmacol. Ther. 78, 29–46 (1998).
Ghosh, S. K. et al. Cloning, sequencing and expression of two murine 2′-5′-oligoadenylate synthetases. Structure–function relationships. J. Biol. Chem. 266, 15293–15299 (1991).
Zhou, A., Hassel, B. A. & Silverman, R. H. Expression cloning of 2-5A-dependent RNAase: a uniquely regulated mediator of interferon action. Cell 72, 753–765 (1993).
Zhou, A. et al. Interferon action and apoptosis are defective in mice devoid of 2′,5′-oligoadenylate-dependent RNase L. EMBO J. 16, 6355–6363 (1997).
Staeheli, P. & Haller, O. Interferon-induced Mx protein: a mediator of cellular resistance to influenza virus. Interferon 8, 1–23 (1987).
Kochs, G., Janzen, C., Hohenberg, H. & Haller, O. Antivirally active MxA protein sequesters La Crosse virus nucleocapsid protein into perinuclear complexes. Proc. Natl Acad. Sci. USA 99, 3153–3158 (2002).
Patterson, J. B., Thomis, D. C., Hans, S. L. & Samuel, C. E. Mechanism of interferon action: double-stranded RNA-specific adenosine deaminase from human cells is inducible by α- and γ-interferons. Virology 210, 508–511 (1995).
Biron, C. A. Role of early cytokines, including α- and β-interferons (IFN-α/β), in innate and adaptive immune responses to viral infections. Semin. Immunol. 10, 383–390 (1998).
Guidotti, L. G. & Chisari, F. V. Noncytolytic control of viral infections by the innate and adaptive immune response. Annu. Rev. Immunol. 19, 65–91 (2001).
Taniguchi, T. & Takaoka, A. The interferon-α/β system in antiviral responses: a multimodal machinery of gene regulation by the IRF family of transcription factors. Curr. Opin. Immunol. 14, 111–116 (2002).
Nguyen, H., Hiscott, J. & Pitha, P. M. The growing family of interferon regulatory factors. Cytokine Growth Factor Rev. 8, 293–312 (1997).
Zimring, J. C., Goodbourn, S. & Offermann, M. K. Human herpesvirus 8 encodes an interferon regulatory factor (IRF) homolog that represses IRF-1-mediated transcription. J. Virol. 72, 701–707 (1998).
Cebulla, C. M., Miller, D. M. & Sedmak, D. D. Viral inhibition of interferon signal transduction. Intervirology 42, 325–330 (1999).
Goodbourn, S., Didcock, L. & Randall, R. E. Interferons: cell signalling, immune modulation, antiviral response and virus countermeasures. J. Gen. Virol. 81, 2341–2364 (2000).
Garcia-Sastre, A. Mechanisms of inhibition of the host interferon-α/β-mediated antiviral responses by viruses. Microbes Infect. 4, 647–655 (2002).
Isaacs, A. & Lindenmann, J. Virus interference. I. The interferon. Proc. R. Soc. Lond. B 147, 258–267 (1957).
Haller, O., Frese, M. & Kochs, G. Mx proteins: mediators of innate resistance to RNA viruses. Rev. Sci. Tech. 17, 220–230 (1998).
Bergmann, M. et al. Influenza virus NS1 protein counteracts PKR-mediated inhibition of replication. J. Virol. 74, 6203–6206 (2000).
Garcia-Sastre, A. et al. Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 252, 324–330 (1998).
Garcia-Sastre, A. Inhibition of interferon-mediated antiviral responses by influenza A viruses and other negative-strand RNA viruses. Virology 279, 375–384 (2001).
Lee, T. G., Tang, N., Thompson, S., Miller, J. & Katze, M. G. The 58,000-dalton cellular inhibitor of the interferon-induced double-stranded RNA-activated protein kinase (PKR) is a member of the tetratricopeptide-repeat family of proteins. Mol. Cell. Biol. 14, 2331–2342 (1994).
Gale, M. Jr et al. Regulation of interferon-induced protein kinase PKR: modulation of P58IPK inhibitory function by a novel protein, P52rIPK. Mol. Cell. Biol. 18, 859–871 (1998).
Melville, M. W. et al. The cellular inhibitor of the PKR protein kinase, P58(IPK), is an influenza virus-activated co-chaperone that modulates heat-shock protein 70 activity. J. Biol. Chem. 274, 3797–3803 (1999).
Polyak, S. J., Tang, N., Wambach, M., Barber, G. N. & Katze, M. G. The P58 cellular inhibitor complexes with the interferon-induced, double-stranded RNA-dependent protein kinase, PKR, to regulate its autophosphorylation and activity. J. Biol. Chem. 271, 1702–1707 (1996).
Wang, X. Z. et al. Signals from the stressed endoplasmic reticulum induce C/EBP-homologous protein (CHOP/GADD153). Mol. Cell. Biol. 16, 4273–4280 (1996).
Geiss, G. K. et al. Cellular transcriptional profiling in influenza virus-infected lung epithelial cells: the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza. Proc. Natl Acad. Sci. USA 99, 10736–10741 (2002).This study examined the effects of NS1 protein expression during influenza A virus infection on global cellular mRNA levels using high-density microarrays. It indicated that the cellular IFN response to influenza A virus infection in lung epithelial cells is influenced markedly by the sequence of the NS1 gene, and it characterized a virus that contains the 1918 pandemic influenza NS1 gene.
Krebs, D. L. & Hilton, D. J. SOCS proteins: negative regulators of cytokine signaling. Stem Cells 19, 378–387 (2001).
He, Y. & Katze, M. G. To interfere and to anti-interfere: the interplay between hepatitis C virus and interferon. Viral Immunol. 15, 95–119 (2002).
Tan, S. L. & Katze, M. G. How hepatitis C virus counteracts the interferon response: the jury is still out on NS5A. Virology 284, 1–12 (2001).
Taylor, D. R. Hepatitis C virus and interferon resistance: it's more than just PKR. Hepatology 33, 1547–1549 (2001).
Bartenschlager, R. & Lohmann, V. Novel cell-culture systems for the hepatitis C virus. Antiviral Res. 52, 1–17 (2001).
Gale, M. Jr & Beard, M. R. Molecular clones of hepatitis C virus: applications to animal models. ILAR J. 42, 139–151 (2001).
Pawlotsky, J. M. Hepatitis C virus resistance to antiviral therapy. Hepatology 32, 889–896 (2000).
Bukh, J., Miller, R. H. & Purcell, R. H. Genetic heterogeneity of hepatitis C virus: quasispecies and genotypes. Semin. Liver Dis. 15, 41–63 (1995).
Enomoto, N. et al. Comparison of full-length sequences of interferon-sensitive and -resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino-acid substitutions in the NS5A region. J. Clin. Invest. 96, 224–230 (1995).
Enomoto, N. et al. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection. N. Engl. J. Med. 334, 77–81 (1996).This study analysed the HCV NS5A ISDR sequences in patients with chronic HCV1b infection before and after IFN therapy, and concluded that there was a substantial correlation between responses to IFN and mutations in the NS5A gene.
Nakano, I. et al. Why is the interferon sensitivity-determining region (ISDR) system useful in Japan? J. Hepatol. 30, 1014–1022 (1999).
Witherell, G. W. & Beineke, P. Statistical analysis of combined substitutions in nonstructural 5A region of hepatitis C virus and interferon response. J. Med. Virol. 63, 8–16 (2001).
Lohmann, V. et al. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113 (1999).
Blight, K. J., Kolykhalov, A. A. & Rice, C. M. Efficient initiation of HCV RNA replication in cell culture. Science 290, 1972–1974 (2000).
Frese, M., Pietschmann, T., Moradpour, D., Haller, O. & Bartenschlager, R. Interferon-α inhibits hepatitis C virus subgenomic RNA replication by an MxA-independent pathway. J. Gen. Virol. 82, 723–733 (2001).
Guo, J. T., Bichko, V. V. & Seeger, C. Effect of α-interferon on the hepatitis C virus replicon. J. Virol. 75, 8516–8523 (2001).
Sumpter, R. Jr & Gale, M. Jr. in American Society for Virology 21st Annual Meeting W33-3 (Lexington, Kentucky, 2002).
Whitley, R. J. in Fields Virology (eds Knipe, D. M. & Howley, P. M.) 2461–2509 (Lippincott, Williams & Wilkins, Philadelphia, 2001).
Tan, S. L. & Katze, M. G. HSV.com: maneuvering the internetworks of viral neuropathogenesis and evasion of the host defense. Proc. Natl Acad. Sci. USA 97, 5684–5686 (2000).
Roizman, B. & Knipe, D. M. in Fields Virology (eds Knipe, D. M. & Howley, P. M.) 2399–2460 (Lippincott, Williams & Wilkins, Philadelphia, 2001).
Ankel, H., Westra, D. F., Welling-Wester, S. & Lebon, P. Induction of interferon-α by glycoprotein D of herpes simplex virus: a possible role of chemokine receptors. Virology 251, 317–326 (1998).
Kumar-Sinha, C., Varambally, S., Sreekumar, A. & Chinnaiyan, A. M. Molecular cross-talk between the TRAIL and interferon signaling pathways. J. Biol. Chem. 277, 575–585 (2002).
Preston, C. M., Harman, A. N. & Nicholl, M. J. Activation of interferon response factor-3 in human cells infected with herpes simplex virus type 1 or human cytomegalovirus. J. Virol. 75, 8909–8916 (2001).
Eidson, K. M., Hobbs, W. E., Manning, B. J., Carlson, P. & DeLuca, N. A. Expression of herpes simplex virus ICP0 inhibits the induction of interferon-stimulated genes by viral infection. J. Virol. 76, 2180–2191 (2002).
Mossman, K. L. & Smiley, J. R. Herpes simplex virus ICP0 and ICP34.5 counteract distinct interferon-induced barriers to virus replication. J. Virol. 76, 1995–1998 (2002).
Harle, P., Sainz, B. Jr, Carr, D. J. & Halford, W. P. The immediate-early protein, ICP0, is essential for the resistance of herpes simplex virus to interferon-α/β. Virology 293, 295–304 (2002).
He, B., Gross, M. & Roizman, B. The γ(1)34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1α to dephosphorylate the α-subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase. Proc. Natl Acad. Sci. USA 94, 843–848 (1997).This study indicated a unique mechanism by which HSV γ(1)34.5 interacts with and redirects protein phosphatase 1α to dephosphorylate eIF-2α to allow continued protein synthesis despite the presence of activated PKR.
Cassady, K. A. & Gross, M. The herpes simplex virus type 1 U(S)11 protein interacts with protein kinase R in infected cells and requires a 30-amino-acid sequence adjacent to a kinase substrate domain. J. Virol. 76, 2029–2035 (2002).
Leib, D. A., Machalek, M. A., Williams, B. R., Silverman, R. H. & Virgin, H. W. Specific phenotypic restoration of an attenuated virus by knockout of a host resistance gene. Proc. Natl Acad. Sci. USA 97, 6097–6101 (2000).Using recombinant viruses to infect animals that have null mutations in host-defence genes, this study showed that a virus that was attenuated by deletion of ICP34.5 showed wild-type replication and virulence in a host from which the PKR gene had been deleted, exemplifying a formal genetic test for identifying in vivo mechanisms and targets of microbial virulence genes.
Poppers, J., Mulvey, M., Khoo, D. & Mohr, I. Inhibition of PKR activation by the proline-rich RNA-binding domain of the herpes simplex virus type 1 US11 protein. J. Virol. 74, 11215–11221 (2000).
Esposito, J. J. & Fenner, F. in Fields Virology (eds Knipe, D. M. & Howley, P. M.) 2849–2884 (Lippincott, Williams & Wilkins, Philadelphia, 2001).
Moss, B. in Fields Virology (eds Knipe, D. M. & Howley, P. M.) 2885–2922 (Lippincott, Williams & Wilkins, Philadelphia, 2001).
Cohen, J. Bioterrorism. Smallpox vaccinations: how much protection remains? Science 294, 985 (2001).
Smith, G. L. & McFadden, G. Smallpox: anything to declare? Nature Rev. Immunol. 2, 521–527 (2002).
Alcami, A. & Smith, G. L. Receptors for γ-interferon encoded by poxviruses: implications for the unknown origin of vaccinia virus. Trends Microbiol. 4, 321–326 (1996).
Alcami, A. & Smith, G. L. Soluble interferon-α receptors encoded by poxviruses. Comp. Immunol. Microbiol. Infect. Dis. 19, 305–317 (1996).
McFadden, G. & Murphy, P. M. Host-related immunomodulators encoded by poxviruses and herpesviruses. Curr. Opin. Microbiol. 3, 371–378 (2000).
Lalani, A. S. et al. Use of chemokine receptors by poxviruses. Science 286, 1968–1971 (1999).
Alcami, A. & Smith, G. L. Vaccinia, cowpox and camelpox viruses encode soluble γ-interferon receptors with novel broad species specificity. J. Virol. 69, 4633–4639 (1995).
Alcami, A. & Smith, G. L. Cytokine receptors encoded by poxviruses: a lesson in cytokine biology. Immunol. Today 16, 474–478 (1995).
McFadden, G., Lalani, A., Everett, H., Nash, P. & Xu, X. Virus-encoded receptors for cytokines and chemokines. Semin. Cell Dev. Biol. 9, 359–368 (1998).
Colamonici, O. R., Domanski, P., Sweitzer, S. M., Larner, A. & Buller, R. M. Vaccinia virus B18R gene encodes a type I interferon-binding protein that blocks interferon-α transmembrane signaling. J. Biol. Chem. 270, 15974–15978 (1995).
Symons, J. A., Alcami, A. & Smith, G. L. Vaccinia virus encodes a soluble type I interferon receptor of novel structure and broad species specificity. Cell 81, 551–560 (1995).This study characterized the vaccinia-virus soluble type I IFN receptor encoded by the B18R gene, which has broad species specificity and might have aided vaccinia-virus replication in many host species during evolution.
Verardi, P. H., Jones, L. A., Aziz, F. H., Ahmad, S. & Yilma, T. D. Vaccinia virus vectors with an inactivated γ-interferon receptor homolog gene (B8R) are attenuated in vivo without a concomitant reduction in immunogenicity. J. Virol. 75, 11–18 (2001).
Sroller, V., Ludvikova, V., Maresova, L., Hainz, P. & Nemeckova, S. Effect of IFN-γ receptor gene deletion on vaccinia-virus virulence. Arch. Virol. 146, 239–249 (2001).
Akkaraju, G. R., Whitaker-Dowling, P., Youngner, J. S. & Jagus, R. Vaccinia-specific kinase inhibitory factor prevents translational inhibition by double-stranded RNA in rabbit reticulocyte lysate. J. Biol. Chem. 264, 10321–10325 (1989).
Watson, J. C., Chang, H. W. & Jacobs, B. L. Characterization of a vaccinia virus-encoded double-stranded RNA-binding protein that may be involved in inhibition of the double-stranded RNA-dependent protein kinase. Virology 185, 206–216 (1991).
Chang, H. W., Watson, J. C. & Jacobs, B. L. The E3L gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded RNA-dependent protein kinase. Proc. Natl Acad. Sci. USA 89, 4825–4829 (1992).
Beattie, E., Paoletti, E. & Tartaglia, J. Distinct patterns of IFN sensitivity observed in cells infected with vaccinia K3L- and E3L-mutant viruses. Virology 210, 254–263 (1995).
Beattie, E., Tartaglia, J. & Paoletti, E. Vaccinia virus-encoded eIF-2α homolog abrogates the antiviral effect of interferon. Virology 183, 419–422 (1991).
Davies, M. V., Elroy-Stein, O., Jagus, R., Moss, B. & Kaufman, R. J. The vaccinia virus K3L gene product potentiates translation by inhibiting double-stranded-RNA-activated protein kinase and phosphorylation of the α-subunit of eukaryotic initiation factor 2. J. Virol. 66, 1943–1950 (1992).
Massung, R. F. et al. Analysis of the complete genome of smallpox variola major virus strain Bangladesh-1975. Virology 201, 215–240 (1994).
Shchelkunov, S. N. et al. Comparison of the genetic maps of variola and vaccinia viruses. FEBS Lett. 327, 321–324 (1993).
Davies, M. V., Chang, H. W., Jacobs, B. L. & Kaufman, R. J. The E3L and K3L vaccinia virus gene products stimulate translation through inhibition of the double-stranded RNA-dependent protein kinase by different mechanisms. J. Virol. 67, 1688–1692 (1993).
Sharp, T. V. et al. The vaccinia virus E3L gene product interacts with both the regulatory and the substrate-binding regions of PKR: implications for PKR autoregulation. Virology 250, 302–315 (1998).
Carroll, K., Elroy-Stein, O., Moss, B. & Jagus, R. Recombinant vaccinia virus K3L gene product prevents activation of double-stranded RNA-dependent, initiation factor 2α-specific protein kinase. J. Biol. Chem. 268, 12837–12842 (1993).
Rivas, C., Gil, J., Melkova, Z., Esteban, M. & Diaz-Guerra, M. Vaccinia virus E3L protein is an inhibitor of the interferon (IFN)-induced 2–5A synthetase enzyme. Virology 243, 406–414 (1998).
Smith, E. J., Marie, I., Prakash, A., Garcia-Sastre, A. & Levy, D. E. IRF3 and IRF7 phosphorylation in virus-infected cells does not require double-stranded RNA-dependent protein kinase R or IκB kinase but is blocked by vaccinia virus E3L protein. J. Biol. Chem. 276, 8951–8957 (2001).
Liu, Y., Wolff, K. C., Jacobs, B. L. & Samuel, C. E. Vaccinia virus E3L interferon resistance protein inhibits the interferon-induced adenosine deaminase A-to-I editing activity. Virology 289, 378–387 (2001).
Brandt, T. A. & Jacobs, B. L. Both carboxy- and amino-terminal domains of the vaccinia virus interferon resistance gene, E3L, are required for pathogenesis in a mouse model. J. Virol. 75, 850–856 (2001).
Najarro, P., Traktman, P. & Lewis, J. A. Vaccinia virus blocks γ-interferon signal transduction: viral VH1 phosphatase reverses Stat1 activation. J. Virol. 75, 3185–3196 (2001).
Cummings, C. A. & Relman, D. A. Using DNA microarrays to study host–microbe interactions. Emerg. Infect. Dis. 6, 513–525 (2000).
Fruh, K., Simmen, K., Luukkonen, B. G., Bell, Y. C. & Ghazal, P. Virogenomics: a novel approach to antiviral drug discovery. Drug Discov. Today 6, 621–627 (2001).
Manger, I. D. & Relman, D. A. How the host 'sees' pathogens: global gene expression responses to infection. Curr. Opin. Immunol. 12, 215–218 (2000).
Dongre, A. R., Opiteck, G., Cosand, W. L. & Hefta, S. A. Proteomics in the post-genome age. Biopolymers 60, 206–211 (2001).
Uetz, P. et al. A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627 (2000).
Kellam, P. Post-genomic virology: the impact of bioinformatics, microarrays and proteomics on investigating host and pathogen interactions. Rev. Med. Virol. 11, 313–329 (2001).
Ideker, T., Galitski, T. & Hood, L. A new approach to decoding life: systems biology. Annu. Rev. Genomics Hum. Genet. 2, 343–372 (2001).
Ideker, T. et al. Integrated genomic and proteomic analyses of a systematically perturbed metabolic network. Science 292, 929–934 (2001).
Simmen, K. A. et al. Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B. Proc. Natl Acad. Sci. USA 98, 7140–7145 (2001).Using high-density microarrays, this study identified the specific viral component that triggers the cellular IFN response as the envelope glycoprotein B (gB), highlighting a pioneering paradigm for the consequences of virus–receptor interactions.
Mossman, K. L. et al. Herpes simplex virus triggers and then disarms a host antiviral response. J. Virol. 75, 750–758 (2001).
Geiss, G. K. et al. Global impact of influenza virus on cellular pathways is mediated by both replication-dependent and -independent events. J. Virol. 75, 4321–4331 (2001).
Geiss, G. et al. A comprehensive view of regulation of gene expression by double-stranded RNA-mediated cell signaling. J. Biol. Chem. 276, 30178–30182 (2001).
Gale, M. Jr et al. Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation. Mol. Cell. Biol. 18, 5208–5218 (1998).This study investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process, and proposed a model of PKR regulation by NS5A, which might have implications for therapeutic strategies against HCV.
Gale, M. J. Jr et al. Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology 230, 217–227 (1997).
Noguchi, T. et al. Effects of mutation in hepatitis C virus nonstructural protein 5A on interferon resistance mediated by inhibition of PKR kinase activity in mammalian cells. Microbiol. Immunol. 45, 829–840 (2001).
Polyak, S. J. et al. Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response. J. Virol. 75, 6095–6106 (2001).
Girard, S. et al. An altered cellular response to interferon and up-regulation of interleukin-8 induced by the hepatitis C viral protein NS5A uncovered by microarray analysis. Virology 295, 272–283 (2002).
Bigger, C. B., Brasky, K. M. & Lanford, R. E. DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection. J. Virol. 75, 7059–7066 (2001).
Toshchakov, V. et al. TLR4, but not TLR2, mediates IFN-β-induced STAT1α/β-dependent gene expression in macrophages. Nature Immunol. 3, 392–398 (2002).This study highlighted the cross-talk between TLRs and IFN — two pivotal host anti-microbial pathways — and provided the first explanation for the mechanistic basis of the differential patterns of gene expression that are activated by different TLR agonists.
Mita, Y., Dobashi, K., Shimizu, Y., Nakazawa, T. & Mori, M. Toll-like receptor 2 and 4 surface expressions on human monocytes are modulated by interferon-γ and macrophage colony-stimulating factor. Immunol. Lett. 78, 97–101 (2001).
Miettinen, M., Sareneva, T., Julkunen, I. & Matikainen, S. IFNs activate toll-like receptor gene expression in viral infections. Genes Immun. 2, 349–355 (2001).
Aderem, A. & Ulevitch, R. J. Toll-like receptors in the induction of the innate immune response. Nature 406, 782–787 (2000).
Alexopoulou, L., Holt, A. C., Medzhitov, R. & Flavell, R. A. Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3. Nature 413, 732–738 (2001).This study showed that mammalian TLR3 recognizes dsRNA and TLR3 activation induces type I IFN production. It was also found that TLR3-deficient mice have reduced responses to poly(inosine:cytosine).
Zhou, A., Paranjape, J. M., Der, S. D., Williams, B. R. & Silverman, R. H. Interferon action in triply deficient mice reveals the existence of alternative antiviral pathways. Virology 258, 435–440 (1999).
Horng, T., Barton, G. M. & Medzhitov, R. TIRAP: an adapter molecule in the Toll signaling pathway. Nature Immunol. 2, 835–841 (2001).
O'Shea, J. J., Gadina, M. & Schreiber, R. D. Cytokine signaling in 2002: new surprises in the Jak/Stat pathway. Cell 109, S121–S131 (2002).
Aaronson, D. S. & Horvath, C. M. A road map for those who know JAK–STAT. Science 296, 1653–1655 (2002).
Heim, M. H. The Jak–STAT pathway: cytokine signalling from the receptor to the nucleus. J. Recept. Signal Transduct. Res. 19, 75–120 (1999).
Yeh, T. C. & Pellegrini, S. The Janus kinase family of protein tyrosine kinases and their role in signaling. Cell. Mol. Life Sci. 55, 1523–1534 (1999).
Meraz, M. A. et al. Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK–STAT signaling pathway. Cell 84, 431–442 (1996).This study generated and characterized Stat1-deficient mice, which have a complete lack of response to IFNs and are highly sensitive to microbial and viral infection, showing that STAT1 has an obligate and dedicated role in mediating IFN-dependent biological responses.
Cox, N. J. & Subbarao, K. Global epidemiology of influenza: past and present. Annu. Rev. Med. 51, 407–421 (2000).
Patterson, K. D. & Pyle, G. F. The geography and mortality of the 1918 influenza pandemic. Bull. Hist. Med. 65, 4–21 (1991).
Basler, C. F. et al. Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc. Natl Acad. Sci. USA 98, 2746–2751 (2001).By generating recombinant influenza viruses from cloned cDNAs, the group tested the 1918 pandemic flu NS1 gene in a mouse model. The results indicate that interaction of the NS1 protein with host-cell factors is important for viral pathogenesis.
Taubenberger, J. K., Reid, A. H., Janczewski, T. A. & Fanning, T. G. Integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 Spanish influenza virus. Phil. Trans. R. Soc. Lond. B 356, 1829–1839 (2001).
Patten, P. A., Howard, R. J. & Stemmer, W. P. Applications of DNA shuffling to pharmaceuticals and vaccines. Curr. Opin. Biotechnol. 8, 724–733 (1997).
Harayama, S. Artificial evolution by DNA shuffling. Trends Biotechnol. 16, 76–82 (1998).
Pekrun, K. et al. Evolution of a human immunodeficiency virus type 1 variant with enhanced replication in pig-tailed macaque cells by DNA shuffling. J. Virol. 76, 2924–2935 (2002).
Chang, C. C. et al. Evolution of a cytokine using DNA family shuffling. Nature Biotechnol. 17, 793–797 (1999).This study used DNA shuffling of a family of human IFN-α genes to derive variants that have increased antiviral activities in mouse cells, and showed that diverse cytokine gene families can be used as starting material to rapidly evolve cytokines that are more active than the native form.
Weber, H., Valenzuela, D., Lujber, G., Gubler, M. & Weissmann, C. Single amino-acid changes that render human IFN-α2 biologically active on mouse cells. EMBO J. 6, 591–598 (1987).
This work was supported by grants from the National Institutes of Health to M.G.K. and M.G. We thank M. J. Korth for editorial assistance and N.-W. Soong for helpful discussions.
- NEGATIVE STRAND RNA GENOME
Genomic viral RNA that is complementary to the messenger RNA that is produced during infection.
A family of closely related, but slightly different, viral genomes. Viral genetics variants, derived from the original infecting virus, that are present during an infection.
A large-scale comparison of NS5A sequences isolated from IFN-resistant or IFN-sensitive HIV-infected patients.
About this article
Cite this article
Katze, M., He, Y. & Gale, M. Viruses and interferon: a fight for supremacy. Nat Rev Immunol 2, 675–687 (2002). https://doi.org/10.1038/nri888
Inhibition of miR-93 promotes interferon effector signaling to suppress influenza A infection by upregulating JAK1
International Immunopharmacology (2020)
Cholesterol 25-hydroxylase inhibits encephalomyocarditis virus replication through enzyme activity-dependent and independent mechanisms
Veterinary Microbiology (2020)
αvβ8 integrin-expression by BATF3-dependent dendritic cells facilitates early IgA responses to Rotavirus
Mucosal Immunology (2020)
CD8 T Cells and STAT1 Signaling Are Essential Codeterminants in Protection from Polyomavirus Encephalopathy
Journal of Virology (2020)
S-palmitoylation of swine interferon-inducible transmembrane protein is essential for its anti-JEV activity