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NEWS AND VIEWS RIGing a virus trap Chris A Benedict & Carl F Ware A new player in the innate defense system has recently emerged, RIG-I (retinoic acidinducible gene I). RIG-I recognizes the RNA of RNA viruses and has a more famous counterpart in innate immune defense, the Toll-like receptors (TLRs), which also recognize conserved molecular components of pathogens. RIG-I operates differently than the TLRs—for instance, RIG-I functions in the cytoplasm, whereas TLRs function at the cell surface or in endosomal compartments. But both TLR and RIG-I signaling culminate in the induction of the type I interferon (IFN-α/β) response—one of the earliest alarm bells of the immune system, which counteracts viral replication without killing the infected cell. In a recent issue of Immunity, Kato et al. report that RIG-I induces IFN-α/β in several cell types infected with RNA viruses, but TLR signaling is dominant in plasmacytoid dendritic cells, highly specialized cells that make extraordinarily high levels of IFN-α/β1. Earlier this year, two reports2,3 showed that hepatitis C virus (HCV) targets the RIG-I pathway, thereby promoting virus replication. Together, these results place RIG-I at a crucial position in the innate response to viral infection, and highlight the diverse pathways used to recognize pathogen-specific molecules. RIG-I consists of two caspase recruitment (CARD) domains and an RNA-binding helicase domain, and is one several proteins consisting of these fused domains. CARD domains are present in various proteins involved in proinflammatory or cell-death pathways. Previous work has shown that RIG-I recognizes viral doublestranded RNA through its helicase domain, and induces downstream signals leading to IFN-α/β production through its CARD domains4. To assess the importance of RIG-I in regulating the IFN-α/β response, Kato et al. generated RIG-I–deficient mice1. Most of these mice died a mysterious death during Chris A. Benedict and Carl F. Ware are in the Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA. e-mail: [email protected] or [email protected] RNA virus Helicase CARD CARD Viral dsRNA TLR7/8 IRF7 88 MyD IRAK4 RIG-I Viral ssRNA HCV NS3/4a ? Endosome TRAF6 IRF5 TBK1, IKKε NF-κB JNK p38 IRF7 IRF3 IFN-β IFN-β IFN-α Katie Ris © 2005 Nature Publishing Group http://www.nature.com/naturemedicine Molecules that recognize pathogens and activate the immune response are being discovered at a rapid rate. RIG-I, a new protein in this category, recognizes viral RNA. Recent studies show that RIG-I operates independently of Toll-like receptors and that it is targeted for inactivation by the hepatitis C virus. IL-6 IL-12 Hepatocyte (fibroblast, conventional dendritic cell) Plasmacytoid dendritic cell Figure 1 TLR- and RIG-I–dependent induction of type I interferon during RNA virus infection. In plasmacytoid dendritic cells, the interaction of viral single-stranded RNA (ssRNA) with TLR7 (TLR7 or TLR8 in humans) probably occurs in an early endosomal compartment after virus uptake into the cell. The cytoplasmic adaptor protein MyD88 and interferon response factor-7 (IRF7) are required for downstream induction of IFN-α/β gene expression. Expression of additional inflammatory cytokines, such as interleukin (IL)-6 and IL-12, is also dependent upon MyD88, but this pathway uses the adaptor proteins TNF receptor-associated factor 6 (TRAF6), IL-1 receptor-associated kinase 4 (IRAK4) and IRF5. In other cell types (right panel) cytosol-localized RIG-I recognizes viral RNA, such as found in hepatitis C virus. RIG-I uses a C-terminal RNA-helicase domain to bind RNA with double-stranded structure, and transduces downstream signals leading to IFN-β transcription through two N-terminal caspase recruitment domains (CARD). The HCV nonstructural 3/4a (NS3/4a) protease inhibits RIG-I induction of IFN-β, but does not cleave RIG-I or the downstream IKKε and TBK1 kinases directly, indicating the existence of an as yet unidentified protein targeted by NS3/4a. embryogenesis due to massive liver apoptosis, but a few offspring survived to several weeks of age, allowing for analysis of their cells in culture. Fibroblasts derived from embryos of RIG-I–deficient mice mounted a poor IFN-β response when infected with different RNA viruses, and did not activate transcription factors that mediate this response, such as NF-κB and interferon response factor (IRF)-3. Vesicular stomatitis virus, a negative-strand RNA virus, replicated to higher levels in RIG-I–deficient fibroblasts. Treatment of RIG-I–deficient cells with IFN-β restored resistance, confirming a role for RIG-I in the NATURE MEDICINE VOLUME 11 | NUMBER 9 | SEPTEMBER 2005 induction of IFN-β not in its downstream antiviral effects. The authors then examined the induction of IFN-α/β by a distinct RNA-based virus, Newcastle disease virus (NDV), in dendritic cells. There are several subsets of dendritic cells: some capture and present antigen to T lymphocytes (conventional dendritic cells) and others are known for their robust production of IFN-α/β (plasmacytoid dendritic cells). Conventional dendritic cells from the spleen or derived from bone marrow of RIG-I– deficient mice produced almost no IFN-α/β when infected with NDV. In contrast, 929 IFN-α/β production by RIG-I–deficient plasmacytoid dendritic cells was essentially normal after NDV infection. In these cells, TLR instead mediated the induction of IFN-α/β. Thus, RIG-I seems to induce IFN-α/β production in several cell types, but TLRs remain the key pathway for innate recognition of RNA viruses in plasmacytoid dendritic cells. The use of these two pathways in different cell types may reflect adaptation of host defense to different pathogens. Earlier this year, Foy et al. and Breiman et al. found that HCV has developed a mechanism to counteract RIG-I2,3. They traced this mechanism to the HCV nonstructural protease 3/4a (NS3/4a). When expressed in cell cultures with RIG-I, NS3/4a could prevent RIG-I from inducing IFN-β. Breiman et al. showed that overexpression of the cytosolic kinases IKKε or TBK1 involved in the activation of IFN-β could circumvent the NS3/4a-mediated block in IFN-β induction. Both groups concluded that RIG-I is not a proteolytic substrate for NS3/4a, placing the HCV block somewhere between RIG-I and IKKε, perhaps acting upon an unidentified adaptor protein (Fig. 1). Foy et al. went on to harness this information to generate a small peptide that inhibits the active site of the NS3/4a protease. The peptide inhibitor enhanced the IFN-β response and decreased HCV replication in cell culture. Similar inhibition of HCV replication was observed in cells that overexpressed IKKε3. These results suggest that antagonizing NS3/4a proteolytic activity in HCV infected individuals may increase the antiviral immune response, hopefully limiting the ability of this virus to establish a persistent infection. Over the last few years, TLR-dependent signaling pathways have stood in the spotlight of innate immunity, and deservedly so, given the importance and diversity of this system in pathogen recognition. The characterization of multiple receptors and cytoplasmic adaptor proteins required for TLR signaling has facilitated the discovery of additional pathways, such as RIG-I, which function independently of TLR but use overlapping cytoplasmic signaling cascades. RIG-I, for instance, operates in the cell cytoplasm, whereas TLRs are localized at the cell surface or in endosomal compartments. The plasmacytoid dendritic cell, or natural IFN-producing cell as it is known in humans, appears to be the major IFN-α/β–producing cell type. This may be because of the high level of expression of TLR7 and TLR9 and constitutive expression of IRF7, a downstream effector necessary for the type I IFN response5. Additionally, plasmacytoid dendritic cells retain TLR ligands for extended periods of time in early endosomal compartments, perhaps allowing for sustained signaling6. So why don’t all cell types have the capability to respond with the vigor of a plasmacytoid dendritic cell when confronted with an invading pathogen? Perhaps, more is not always better. A growing body of evidence indicates that dysregulated IFN-α/β production leads to autoimmune disorders, a price paid for too vigorous of a response7. This ‘new’ class of RNA helicase-CARD domain proteins shows functional diversity, as indicated by studies of a homolog of mouse RIG-I known as Helicard. Helicard operates during apoptosis, is a substrate for caspases, and increases DNA degradation through its helicase domain8. Another link between cell-death pathways and the innate response to virus is that the Fas-associated death domain–containing protein is required in some cells for the induction of IFN-α/β by doublestranded RNA9. It is unclear what regulates the balance between cell survival and death pathways by this family of helicase-CARD proteins. The emergence of RIG-I as a conduit between recognition of viral nucleic acid and the innate immune response adds insight into how these various signaling pathways interconnect. More practically, these results provide a new opportunity to modulate the interferon pathway in individuals infected with HCV or other pathogens10,11. 1. Kato, H. et al. Immunity 23, 19–28 (2005). 2. Foy, E. et al. Proc. Natl. Acad. Sci. USA 102, 2986– 29891 (2005). 3. Breiman, A. et al. J. Virol. 79, 3969–3978 (2005). 4. Yoneyama, M. et al. Nat. Immunol. 5, 730–737 (2004). 5. Liu, Y.J. Annu. Rev. Immunol. 23, 275–306 (2005). 6. Honda, K. et al. Nature 434, 1035–1040 (2005). 7. Theofilopoulos, A.N., Baccala, R., Beutler, B. & Kono, D.H. Annu. Rev. Immunol. 23, 307–336 (2005). 8. Kovacsovics, M. et al. Curr. Biol. 12, 838–843 (2002). 9. Balachandran, S., Thomas, E. & Barber, G.N. Nature 432, 401–405 (2004). 10. Zhong, J. et al. Proc. Natl. Acad. Sci. USA 102, 9294– 9299 (2005). 11. Sumpter, R. Jr. et al. J. Virol. 79, 2689–2699 (2005). Hatching a drug Human monoclonal antibodies have hatched from chicken eggs (Nat. Biotechnol. 23, 1159–1169). Generating antibodies in eggs could prove more efficient than more traditional methods of cell culture. To generate antibody-producing eggs, Lei Zhu et al. created an antibody transgene hooked up to regulatory elements for albumin, which is expressed at high levels in eggs. The investigators then transfected that gene into chicken embryonic stem cells; these cells, in turn, were used to create chickens that pumped out antibodies in the tubular gland cells of the oviduct (cells derived from stem cells are labeled in green (GFP), antibody in red and DNA in blue). The egg-derived antibodies largely live up to the quality standards of antibodies made in cultured cells—having, for instance, similar affinity to antigen. Some 17 monoclonal antibody–based drugs have been approved by the US Food and Drug Administration in the last 20 years and many more are on deck. Charlotte Schubert 930 Courtesy of Origen Therapeutics © 2005 Nature Publishing Group http://www.nature.com/naturemedicine NEWS AND VIEWS VOLUME 11 | NUMBER 9 | SEPTEMBER 2005 NATURE MEDICINE