Molecular mechanisms of influenza A virus nonstructural protein (NS1) in supressing RIG-I signal transduction


Ligaza ubiquitara TRIM 25 mediaza ubiquitarea Lizinei 63-a domeniilor CARD N terminal a senzorului ARN viral RIGI pentru a facilita productia de interferon tip I si imunitatea antivirala.

Acesta este un review al datelor din literatura cu privire la mecanismele moleculare ale inhibarii de catre NS1 ale virusului gripal A a TRIM 25 mediata de ubiquitarea RIG- I CARD ,supresand in felul acesta transducerea semnalului RIG-I.Un nou domeniu in NS1 continand reziduuri E96/E97 mediaza interactiunea sa cu domeniul TRIM25 blocand astfel multimerizarea si ubiquitarea domeniului RIG-I CARD.

Mecanismul prin care virusul gripal A inhiba raspunsul IFN al gazdei este discutat si de asemenea este evidentiat rolul vital al TRIM25 in modularea apararii antivirale.

Cuvinte-cheie: proteina 1 nonstructurala a virusului gripal A, domeniul TRIM25,multimerizare, tipul I de interferon


The ubiquitin ligase TRIM25 mediates Lysine 63-linked ubiquitination of the N-terminalCARDdomains of the viral RNA sensor RIG-I to facilitate type I interferon (IFN) production and antiviral immunity.

Here, we report that the influenza A virus nonstructural protein 1 (NS1) specifically inhibits TRIM25-mediated RIG-I CARD ubiquitination, thereby suppressing RIG-I signal transduction. A novel domain in NS1 comprising E96/E97 residues mediates its interaction with the coiled-coil domain of TRIM25, thus blocking TRIM25 multimerization and RIG-I CARD domain ubiquitination.

The mechanism by which influenza virus inhibits host IFN response and also emphasize the vital role of TRIM25 in modulating antiviral defenses.

Keywords: influenza virus A nonstructural protein 1,TRIM25 domain,multimerisation, interferon typeI


The production of type I interferons(IFNS) is accounted for as an integral component of innate immunity , which is a family of antiviral cytokines that functions to prevent completion of the virus lifecycle as well as virus dissemination in vivo [1] To elicit IFN responses, mammalian hosts have evolved a variety of cellular pattern recognition receptors (PRRs) including Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), which sense the presence of viral nucleic acids or other conserved molecular components of invading microbes [2].

With respect to the respiratory pathogens influenza and respiratory syncytial viruses, analysis of infected RIG-I_/_ or MDA5_/_ cells illustrated the essential role of RIG-I in initiating antiviral responses against these RNA viruses [3]. Upon viral infection, the cytosolic receptor RIG-I recognizes viral RNA in a 50-triphosphate- dependent manner and initiates an antiviral signaling cascade by interacting with the downstream partner MAVS/ VISA/IPS-1/Cardif [4]. For MAVS binding and downstream signaling, the N-terminal Caspase recruitment domains (CARDs) of RIG-I are responsible. In addition, tripartite motif (TRIM) proteins, containing an N-terminal RING domain with potential ubiquitin E3 ligase activity, represent a new class of antiviral molecules involved in innate immunity[5].

TRIM5a has been extensively studied as a host restriction factor for HIV-1 infection [6]. TRIM25 has recently been shown to induce Lys63-linked ubiquitination of the N-terminal CARDs of RIG-I, which is crucial for the cytosolic RIG-I signaling pathway to elicit host antiviral innate immunity [7]. Specifically, TRIM25 interacts with the first CARD of RIG-I, and this interaction effectively delivers the Lys63-linked ubiquitin moiety to the second CARD of RIG-I, leading to efficient interaction with MAVS/VISA/IPS-1/Cardif. Additionally, a splice variant of RIG-I that carries a deletion (amino acids 36–80) within the first CARD loses TRIM25 binding, CARD ubiquitination, and downstream signaling ability, demonstrating the critical role of TRIM25-mediated ubiquitination for RIG-I antiviral activity [7]. On the other hand, viruses have evolved sophisticated mechanisms to evade or counteract the host IFN system.

Specifically, virus-encoded IFN antagonists inhibit host innate antiviral responses by targeting IFN gene expression or IFN-induced host effector molecules [8]. Indeed, the NS1 is the main IFN antagonist encoded by influenza A viruses[1], and was shown to antagonize the assembly of the IFN-b enhanceosome by inhibiting activation of IRF-3 [9], NF-kB , and ATF-2/c-Jun [10] transcription factors. The ability of NS1 to prevent the nucleation of the IFN-b enhanceosome appears to be due at least in part to its binding to double-stranded RNA (dsRNA), likely resulting in the sequestration of this viral mediator from cellular sensors including RIG-I. Indeed, an influenza virus expressing an NS1 mutant defective in RNA-binding activity induces high levels of IFN in vitro and is attenuated in vivo[11]. In addition, it has recently been shown that NS1 interacts with RIG-I and efficiently suppresses its signal transducing activity [12].

However, the precise details of the molecular mechanism by which the NS1 protein antagonizes RIG-I function remains unknown. Specifically, it is not known whether the interaction of NS1 with RIG-I directly antagonizes RIG-I function (i.e., sensing viral RNA), or whether the NS1-RIG-I interaction precludes RIG-I interactions with and/or modifications by additional cellular proteins, such as TRIM25. In this study,the authors describe the long-sought mechanism by how the influenza A virus NS1 achieves this inhibition of the host IFN system. Surprisingly,this involves direct inhibition of the ubiquitin ligase activity of TRIM25, a mechanism not yet described for any other viral protein.


Influenza A Virus NS1 Suppresses RIG-I CARD Ubiquitination Given that TRIM25-induced ubiquitination of the N-terminal CARDs is essential for RIG-I to elicit antiviral signal transduction [13], we postulated that virus-encoded IFN antagonists might specifically inhibit this step to prevent RIG-I activation.

To test this, the authors have examined the effect of expressing various viral proteins known to prevent IFN-b promoter activation on the ubiquitination of GST-RIG-I 2CARD in HEK293T cells.

Among the three viral IFN antagonist proteins tested, influenza A virus NS1 was unique in that it potently inhibited the RIG-I 2CARD ubiquitination, whereas the other IFN antagonists Ebola virus VP35[14] and vaccinia virus E3L[15] showed no effect under the same conditions. Consistently, influenza A NS1 effectively suppressed the ubiquitination of full-length RIG-I in a dose-dependent manner . Furthermore, the ubiquitination levels of endogenous RIG-I markedly decreased in cells infected with wild-type (WT) influenza A/PR/8/34 virus compared to mock-infected cells. In contrast, cells infected with influenza A/PR/8/34 virus containing a deletion of the NS1 gene (DNS1virus) exhibited a slight increase in RIG-I ubiquitination compared to mock-infected cells.

Since the Lys63-linked ubiquitination of RIG-I CARDs is crucial for efficient MAVS interaction, they further tested the effect of increasing amounts of influenza A NS1 on the CARD-dependent binding of RIG-I to MAVS . Consistent with its ability to suppress the RIG-I CARD ubiquitination, NS1 inhibited the RIG-I-MAVS interaction in a dose-dependent manner. These results indicate that influenza A virus NS1 specifically inhibits the RIG-I CARD ubiquitination, resulting in the suppression of RIG-I-MAVS complex formation.

NS1 Interacts with TRIM25 ;in order to elucidate the mechanism by which influenza A virus NS1 inhibits RIG-I ubiquitination, they tested the potential interaction between NS1 and TRIM25 in HEK293T cells. Coimmunoprecipitation (coIP) studies in transfected cells showed an interaction between NS1 and TRIM25 . TRIM25 is composed of an N-terminal RING domain, two B-boxes, a central coiled-coil domain (CCD), and a C-terminal SPRY domain[16]. The RING and the SPRY domains of TRIM25 have been shown to interact with E2 ubiquitin-conjugating enzymes and with the N-terminal CARDs of RIG-I, respectively [13].

TRIM25 polypeptides corresponding to RING, B-boxes/ CCD, B-boxes, CCD, and SPRY domains were examined for NS1 binding. This showed that NS1 specifically interacted with the central CCD (aa 180–450) of TRIM25 . Accordingly a TRIM25 mutant in which the CCD was deleted (TRIM25 DCCD) was incapable of binding NS1.

HEK293T cells infected with various human influenza A virus strains, including A/PR/8/34 and human virus isolates
A/Texas/36/91, A/New Caledonia/20/99, A/Wyoming/3/2003, and A/Panama/2007/99, also showed an interaction between NS1 and endogenous TRIM25 . Furthermore, the NS1 protein of several avian and swine influenza A virus strains and of the 1918 pandemic strain of influenza A virus readily bound TRIM25. Confocal microscopy confirmed an interaction between TRIM25 and NS1.

While expression of NS1 in HeLa cells resulted in nuclear localization with a minor cytoplasmic component, TRIM25 overexpression led to a marked increase of NS1 cytoplasmic localization . However, expression of a TRIM25 DCCD mutant that no longer interacted with NS1 showed little or no effect on the subcellular localization of NS1. In addition, they have observed an in vitro interaction between bacterially purified GST-PR8 NS1 and MBP-TRIM25-Flag , suggesting the direct binding of influenza A NS1 to TRIM25.
WT NS1 but Not R38A/K41A and E96A/E97A NS1Mutants Inhibit TRIM25-Mediated RIG-I Ubiquitination and Signal TransductionThe structure of the N-terminal RNA-binding domain (RBD; aa1–73) and the C-terminal effector domain (ED; aa 74–230) of the influenza A virus NS1 has been obtained [17]. The NS1 RBD likely sequesters viral dsRNA from cytoplasmic RNA sensors, such as PKR, OAS, and RIG-I, while the ED mediates interactions with several cellular factors, including CPSF (binding cleavage and polyadenylation specificity factor),PAB II (inhibiting poly-[A]-binding protein), and eIF4G-I to regulate viral and host gene expression [18]. An initial mapping study showed that both domains were required to bind TRIM25.

We then mutated conserved amino acids within both NS1 domains to analyze their impact on TRIM25 binding. The R38A/K41A NS1 mutant is known to be deficient in dsRNA-binding activity and in IFN antagonism [11]. The E96A/E97A mutation is located in a highly conserved putative protein-protein interacting motif (S/T-x-E-E), identified by a computational analysis 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 ortholog.
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.


Authors 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.

Their findings also provide an elegant 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 knockout cells. Thus, their findings not only describe a viral immune evasion mechanism that is crucial for in vivo virulence,but also provide detailed insights into the biological role of TRIM25 in antiviral 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 antiviral immunity.


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