TRIM proteins, so named due to the presence of RING, B-box, and coiled-coil domains, constitute a family of proteins that are involved in a broad range of biological processes including cell cycle progression and anti-viral responses (Meroni and Diez-Roux, 2005
; Nisole et al., 2005
; Ozato et al., 2008
). Among them, TRIM19 (promyelocytic leukemia protein (PML)) and TRIM5α have been shown to exhibit potent anti-viral activity. Whereas TRIM19, a component of nuclear bodies, inhibits the replication of a wide variety of DNA and RNA viruses, TRIM5α has been demonstrated to interfere with the uncoating of the pre-integration complex of lentiviruses including HIV-1 (Kratovac et al., 2008
; Stremlau et al., 2004
; Takeuchi and Matano, 2008
). The importance of TRIM molecules in host defense against virus infection has also been illustrated by the finding that the RING ubiquitin E3 ligase TRIM25 is crucial for RIG-I-mediated induction of type I IFN (Gack et al., 2007
). Due to their anti-viral activities, TRIM molecules might be targeted by viral proteins enabling viruses to evade the host immune response. Although a few viral proteins have been shown to associate with TRIM family members (Ahn et al., 1998
; Hoppe et al., 2006
; Yondola and Hearing, 2007
), the biological consequences of these interactions have not been elucidated. Our study is the first demonstration of how a viral protein directly interferes with the activity of a TRIM molecule to disrupt IFN-mediated innate immunity, which ultimately leads to viral pathogenesis.
For influenza viruses, the main IFN-antagonistic action is encoded by the NS1 gene. Several functions have been documented that may account for NS1 inhibition of type I IFN production, including sequestration of dsRNA, interaction with RIG-I, and inhibition of downstream processes after RIG-I-MAVS interactions such as the processing and trafficking of cellular mRNAs and the activities of IFN-inducible enzymes PKR and OAS (for recent reviews see (Albrecht and Garcia-Sastre, 2009
; Hale et al., 2008b
)). However, the precise mechanism by which the NS1 avoids recognition of virus infection by the host and prevents IFN production was not elucidated. In this report, we describe that the NS1 protein directly binds TRIM25; specifically, the NS1 targets the TRIM25 CCD, thus interfering with TRIM25 multimerization. TRIM25 multimerization is crucial for ubiquitination of RIG-I CARDs, a modification that was found to be necessary for maximal IFN production in response to virus infection (Gack et al., 2007
). Thus, by directly binding to and inhibiting the enzymatic activity of TRIM25, the NS1 suppresses RIG-I signal transduction and ultimately IFN-β production.
Since NS1 and RIG-I bind to non-overlapping domains of TRIM25, the CCD and SPRY domains (Gack et al., 2007
), respectively, our protein interaction studies suggest a NS1/TRIM25/RIG-I triple complex in influenza virus-infected cells. In further support of this, we did not observe a competition between RIG-I and NS1 for TRIM25 binding (data not shown). The detailed molecular architecture of the RIG-I/TRIM25/NS1 triple complex is currently being investigated. We have shown that the interaction of NS1 with TRIM25 is dependent on amino acids located both in the NS1 RNA-binding domain (basic residues 38 and 41) and outside of this domain (acidic residues 96 and 97). The recently published crystal structure of full-length NS1 reveals that these four residues are not in close proximity (Bornholdt and Prasad, 2008
). Interestingly, the crystal structure could only be determined for an RNA-binding NS1 mutant; R38 and K41 were determined to govern aggregation and stability of the full-length NS1. It is plausible that the R38A/K41A mutations have created conformational changes that have resulted in the unanticipated loss of binding to TRIM25. Further studies are then required to understand the structural contribution of these amino acids to TRIM25 binding. Nevertheless, the use of a recombinant influenza virus bearing the NS1 E96A/E97A mutations and of TRIM25 knockout cells allowed us to conclude that NS1 binding to TRIM25 is needed for optimal inhibition of IFN production in virus-infected cells. This mutant virus showed attenuated replication in human cells and pathogenesis in vivo
, and since amino acids 96 and 97 of NS1 have not been implicated in additional NS1 functions, this strongly suggests that TRIM25 binding by NS1 is required for virulence. Interestingly, the NS1 R38A/K41A mutant virus, impaired in both NS1 binding to dsRNA and TRIM25, also shows reduced IFN production in TRIM25−/− cells. The lack of a complete loss of IFN induction indicates that TRIM25 is not completely required for IFN induction by influenza virus infection, and that NS1 functions other than binding to TRIM25, such as binding to dsRNA, also contribute to maximal inhibition of type I IFN synthesis during infection.
It is interesting that despite sequence variations, NS1 proteins encoded by human, avian, and swine influenza viruses interacted with TRIM25 in infected or transfected cells, indicating that targeting TRIM25 is a conserved function of NS1 of various influenza viruses. However, it is conceivable that sequence variations in the NS1 proteins of different virus strains influence the affinity for TRIM25 binding, which may correlate with viral pathogenesis. This concept is important when considering the adaptation of an influenza virus to a new host species since cellular factors targeted by NS1 may have divergent sequences from one host to the other. Indeed, human and avian TRIM25 show a detectable degree of sequence variation, such that the human TRIM25 coiled-coil domain, which is targeted by NS1, exhibits only 33% identity with the corresponding domain of its avian orthologue. Thus, further studies are directed to address the influence of sequence variations in TRIM25 and NS1 on their interactions and on the NS1-mediated IFN antagonizing activity.
Our study demonstrates that the influenza A virus NS1 targets multiple checkpoints of the IFN-mediated signaling pathway by sequestering RNA from cellular sensors like RIG-I and by inhibiting TRIM25 E3 ligase. These NS1 activities, combined with the ability of the NS1 of several viral strains to suppress host gene expression collectively establish a comprehensive suppression of IFN production during viral infection. Our findings also provide the first example of a virus suppressing IFN production by directly antagonizing the enzymatic function of a TRIM family member, in this case the RING-mediated E3 ligase activity of TRIM25. In addition, by identifying NS1 mutants lacking TRIM25 inhibition and by testing their replication in mice, we demonstrate the important role of the NS1-mediated TRIM25 inhibition in influenza A virus virulence. Finally, the crucial role of TRIM25 for maximal type I IFN production in response to influenza A virus was demonstrated by viral infection studies in TRIM25 knock-out cells. Thus, our findings not only describe a new class of viral immune evasion mechanism being crucial for in vivo virulence, but also provide detailed insights into the biological role of TRIM25 in anti-viral host defense. These observations should stimulate the search for additional viral antagonists of innate immune responses that target TRIM proteins and for additional cellular proteins modified by TRIM25 which may play specific roles in anti-viral immunity.