Journal ClubVirulence

TRIM Proteins: Another Class of Viral Victims

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Science Signaling  20 Apr 2010:
Vol. 3, Issue 118, pp. jc2
DOI: 10.1126/scisignal.3118jc2


TRIM (tripartite motif) proteins are a family of RING (really interesting new gene) domain–containing proteins comprising more than 70 human members, with new members still being described. In addition to their involvement in cell proliferation, differentiation, development, morphogenesis, and apoptosis, roles in immune signaling and antiviral functions are emerging. In response to viral infection, TRIM25 ubiquitinates the N terminus of the viral RNA receptor retinoic acid–inducible gene-I (RIG-I), and this modification is essential for RIG-I to interact with its downstream partner mitochondrial antiviral signaling (MAVS). TRIM25 activity thus leads to activation of the RIG-I signaling pathway, which results in type I interferon production to limit viral replication. Recently, it has been demonstrated that influenza A viruses target TRIM25 and disable its antiviral function, thereby suppressing the host interferon response. This Journal Club article highlights the emerging roles of TRIM proteins in antiviral defense mechanisms and an immune evasion strategy in which influenza viruses target a member of the TRIM family.

Eukaryotes have evolved an intrinsic capacity to restrict viral replication mainly through the induction of type I interferons (IFNs), which are secreted by virus-infected cells or certain immune cells, such as plasmacytoid dendritic cells, and trigger cell defense mechanisms in either an autocrine or a paracrine manner. By binding to a common receptor, the IFN-α/β receptor (IFNAR), these cytokines induce a JAK (Janus kinase)–STAT (signal transducer and activator of transcription) signaling cascade, resulting in the transcription of hundreds of IFN-stimulated genes that encode transcription factors or other molecules involved in IFN signaling, as well as antiviral effector molecules that interfere with distinct stages of viral replication. Among the latter, the myxoma resistance (Mx) proteins, the zinc finger antiviral protein (ZAP), Friend virus susceptibility factor 1 (Fv1), the S-adenosyl-l-methionine enzyme viperin, proteins involved in RNA editing and silencing, and tripartite motif (TRIM) proteins have been recognized as important molecules involved in antiviral innate immune responses (1).

The number of members in the TRIM family has grown since the description of Xenopus laevis nuclear factor 7 (XNF7) as the first identified TRIM protein (2). To date, more than 70 genes encoding TRIM proteins have been documented in the human genome; however, only a few of these have been well characterized (3, 4). Members of the TRIM family contain at least three domains—an N-terminal RING domain, one or two B-boxes, and a central coiled-coil domain (CCD)—and are also known as the RBCC (RING, B-box, and coiled coil) family. The RING domain suggests potential ubiquitin E3 ligase activity, the B-box is a zinc-binding motif of unknown function, and the CCD is involved in homomeric and heteromeric protein-protein interactions (3, 4). At their C-termini, TRIM proteins usually possess one or two domains of variable length and composition. In about 60% of human TRIM proteins, the RBCC motif is followed by a B30.2/SPRY domain, which is related to the B30.2 protein-protein interaction domain of the immunoglobulin superfamily member butyrophilin (5). The B30.2/SPRY domain of various TRIM proteins has been shown to mediate specific protein-protein interactions.

TRIM proteins are involved in diverse cellular functions, including cell proliferation, differentiation, apoptosis, and morphogenesis (3, 4). Given that IFNs are critical mediators of the innate immune response to viral infection, the observation that genes encoding TRIM proteins are transcriptionally up-regulated by type I IFNs suggests a role for TRIM proteins in innate immunity (4). Indeed, several TRIM proteins have been shown to have antiviral activity (3, 4). TRIM1 and TRIM22 interfere with the replication of N-tropic murine leukemia virus (N-MLV) and human immunodeficiency virus 1 (HIV-1), respectively (4). TRIM19, better known as PML (promyelocytic leukemia protein), inhibits the replication of a variety of RNA and DNA viruses, including herpes simplex virus type 1 (HSV-1) and HIV-1 (3). TRIM5α was recently shown to inhibit the replication of lentiviruses including HIV-1 (3, 4). Small interfering RNA (siRNA)–mediated knockdown analysis indicates that TRIM27 negatively regulates the activities of several transcription factors involved in the antiviral response. TRIM27 represses nuclear factor κB (NF-κB) activity by modulating the activity of inhibitor of NF-κB kinase (IKK) family members IKKα, IKKβ, and IKKε and represses interferon regulatory factors 3 and 7 (IRF3 and IRF7) by interacting with TANK binding kinase 1 (TBK1). TRIM30α negatively regulates Toll-like receptor (TLR)–mediated NF-kB activation (4). TRIM21 positively regulates the strength and duration of the primary antiviral response by interfering with the interaction between Pin1 (peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1) and IRF3, thus preventing ubiquitination and degradation of IRF3 (4). Although TRIM proteins are involved in many cellular processes, the precise mechanisms of their antiviral functions have not been well characterized. Here, I focus on the involvement of TRIM proteins in the signaling pathways that lead to type I IFN induction, as well as on the mechanisms by which viruses counteract the antiviral function of TRIM proteins.

Among the plethora of cellular functions controlled by TRIM proteins, recent studies have demonstrated that TRIM proteins regulate signaling pathways that lead to type I IFN induction in response to viral infection (4). In vitro and in vivo studies have demonstrated the physiological importance of two cytoplasmic receptors, retinoic acid–inducible gene-I (RIG-I) and melanoma differentiation–associated gene 5 (MDA5), in the detection of RNA virus infections in most cells (6, 7). Both RIG-I and MDA5 consist of two N-terminal caspase recruitment domains (CARDs), a central helicase domain, and a C-terminal regulatory-repressor domain (RD) (8, 9). Structural analysis has demonstrated that the C-terminal RD of RIG-I is essential for binding to viral RNA (10), which leads to multimerization of RIG-I and stimulation of its helicase activity, resulting in a conformational change that exposes the N-terminal CARDs (9). The CARDs of RIG-I and MDA5 interact with the CARD of the adaptor protein mitochondrial antiviral signaling [MAVS; also known as IPS-1 (IFN-β promoter stimulator 1), VISA (virus-induced signaling adaptor), or CARDIF (CARD adaptor inducing IFN-β)], which is localized to the mitochondrial outer membrane. MAVS then activates the IKKα-IKKβ-IKKγ complex and the kinases TBK1 and IKKε, thus provoking activation of the NF-κB, IRF3, and IRF7 transcription factors, respectively. NF-κB, IRF3, and IRF7, in turn, induce transcription of type I IFNs (IFN-α and IFN-β) to limit viral replication (Fig. 1) (11).

Fig. 1

IFN induction pathways and their regulation by TRIM proteins. Viral RNA induces the interaction of RIG-I and MDA5 with the mitochondrial adaptor protein MAVS, thereby activating two kinase complexes that phosphorylate transcription factors induce the transcription of IFN-α and IFN-β. These IFNs initiate a positive feedback loop by activating a JAK-STAT pathway that induces transcription of hundreds of genes containing IFN stimulatory response elements (ISREs). IFN-stimulated genes encode TRIM proteins plus other antiviral proteins such as ZAP, Mx protein, and viperin. The dashed line indicates that the activity occurs through intermediate steps not shown here, whereas continuous lines indicate direct interactions. P indicates phosphorylation, and Ac indicates acetylation.

The interaction of RIG-I with its downstream partner, MAVS, requires polyubiquitination of RIG-I, and this polyubiquitination is induced by the ubiquitin E3 ligase TRIM25 (12). TRIM25 was identified as a RIG-I binding partner by coimmunoprecipitation with the N-terminal CARDs of RIG-I (12). TRIM25 positively regulates RIG-I by adding a Lys63-linked ubiquitin chain to Lys172 in the second CARD of RIG-I (12). This unique type of ubiquitination does not lead to proteasomal degradation of RIG-I but instead substantially increases RIG-I’s ability to initiate signaling. Specifically, the ubiquitination of RIG-I at Lys172 was shown to be critical for efficient interaction of RIG-I with MAVS and therefore also for RIG-I’s ability to induce antiviral IFN production (12). However, the exact molecular mechanism by which TRIM25-mediated ubiquitination of RIG-I facilitates the RIG-I-MAVS interaction, and whether other cellular proteins are recruited to the RIG-I-MAVS complex through this polyubiquitin chain, remain to be determined.

Given that the type I IFN system is a powerful defense mechanism, it is no surprise that most viruses have evolved IFN-antagonizing strategies to ensure their successful replication (13). For example, it had long been recognized that influenza viruses have IFN-antagonizing activity (14) before it was determined that influenza A viruses produce a specialized anti-IFN factor called nonstructural protein 1 (NS1). A crucial role for NS1-mediated IFN suppression in efficient viral replication has been demonstrated by generation of a recombinant influenza A virus lacking the gene encoding NS1. Remarkably, this recombinant virus was unable to replicate in cells with an intact IFN system and established only a highly attenuated infection in mice (15). NS1 is a multifunctional protein that contains both an N-terminal RNA binding domain and a C-terminal effector domain that mediates interactions with various cellular proteins. Although several other functions have been described for NS1, its IFN-antagonistic action is the most well-characterized activity of this multifunctional protein (16). NS1 suppresses the host IFN response at multiple levels, including the inhibition of RIG-I, which is essential for the detection of influenza virus in most cells. The molecular mechanism by which NS1 exerts its effect on RIG-I signaling remained unknown until recent work demonstrated that influenza A NS1 inhibits host IFN production by targeting a TRIM protein (17).

According to Gack et al. (17), NS1 inhibits oligomerization of TRIM25 by directly interacting with the CCD of TRIM25. Inhibition of TRIM25 oligomerization abolishes its E3 ligase activity and thus its ability to activate RIG-I, thereby suppressing the RIG-I–mediated induction of IFN (Fig. 1) (17). TRIM25 is the first and thus far only protein of the TRIM family that has been shown to be inhibited by NS1, and this mechanism of inhibition by targeting a TRIM protein has not been observed for any other viral protein. To test whether this mechanism is conserved among different strains of influenza A virus, NS1 proteins from human, avian, and porcine influenza A viruses, including the 1918 strain that killed about 50 million people around the world and the avian H5N1strain, were tested for the ability to interact with human TRIM25 (17). Despite the fact that the CCD of human TRIM25 exhibits only 33% identity with chicken TRIM25, the NS1 proteins from all influenza strains tested were able to interact with all three TRIM25 homologs (17). It is thus conceivable that this conserved function of NS1 is important for the adaptation of influenza A viruses to new host species.

Strain-specific structural differences in NS1 proteins have been described, implying that there are likely functional differences between the NS1 proteins from different influenza A strains as well [reviewed in (16)]. Interestingly, residues Glu96 and Glu97, which are highly conserved among different influenza A virus strains, are located in the region of NS1 responsible for binding to TRIM25. However, because some viral pathogenicity characters such as replication efficiency are strain-specific, it would be interesting to examine whether strain-specific mutations in NS1 affect its affinity for TRIM25, such as the Asp92 → Glu92 (D92E) mutation that has been observed in the highly pathogenic influenza H5N1 strain (18). In addition, it needs to be determined whether different affinities of NS1 for TRIM25 correlate with viral pathogenesis among different strains. Moreover, the exact architecture of the TRIM25-NS1 complex and the contribution of other viral or cellular proteins to this interaction remain to be determined. These studies will be important with regard to the possibility of developing live attenuated vaccines from influenza viruses carrying engineered mutations in NS1. In addition, structural analysis of the NS1-TRIM25 complex may help to identify small molecules that inhibit this interaction and thus may be used as anti-influenza drugs.

The multifunctionality of the NS1 protein is illustrated by the fact that NS1 not only inhibits the cellular antiviral state by limiting IFN production through inhibition of RIG-I, but it also sequesters viral RNA to prevent it from binding to viral RNA sensors of the host cell and disables IFN-induced antiviral effector proteins, such as the double-stranded RNA (dsRNA)–dependent protein kinase R (PKR) and 2'5′-oligoadenylate synthetase (OAS)-RNase L (14, 16). Consistent with this, Gack et al. did not observe complete inhibition of IFN induction in influenza virus–infected TRIM25−∕− mouse embryonic fibroblasts (17), suggesting that other functions of NS1 in addition to TRIM25 binding are required for maximal inhibition of type I IFN production. In vivo experiments in mice clarified the important role of NS1-mediated TRIM25 inhibition for the replication and pathogenesis of influenza A virus. Mice infected with wild-type influenza A virus succumbed by day 6 after infection, whereas all mice infected with Glu96 → Ala96 Glu97 → Ala97 (E96A;E97A) or Arg38 → Ala38 Lys41 → Ala41 (R38A;K41A) NS1 mutant viruses survived without showing any signs of morbidity (17). These mutations abolished the ability of NS1 to inhibit TRIM25, thereby eliminating the IFN-suppressive activity of NS1, which led to efficient IFN production and the complete loss of virulence. Considering the multifunctionality of NS1, it will be interesting to examine how each of the IFN-antagonistic functions of NS1 contributes to the potent IFN suppression by influenza A virus that permits its efficient replication in target cells.

The extent of innate immune defense mechanisms that hosts have developed to combat invading pathogens has become more understood and appreciated in recent years. This has led to a resurgence in research on the detailed mechanisms that viruses use to disarm the host antiviral response, of which targeting TRIM25 is only one example. The observation that genes encoding several TRIM proteins are transcriptionally up-regulated upon viral infection or IFN treatment may stimulate research on studying the roles of these TRIM proteins in antiviral signaling pathways. Lastly, this may also lead to the identification of additional viral proteins that antagonize TRIM molecules. Further advancement in this field may open new horizons for the development of novel antivirals, specifically anti-influenza drugs, to prevent and combat this deadly disease, which is responsible for 20,000 deaths and 114,000 hospitalizations, on average, in the United States alone (19).


Acknowledgments: I thank M. U. Gack and M. Berg for valuable commentary and critical reading of the manuscript.

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