We previously found that the NS1 protein of influenza A virus inhibits the induction of IFN-β during influenza virus infection (15
). Expression of only the amino-terminal dsRNA-binding domain of NS1 appears sufficient to prevent the induction of IFN-β (51
). In agreement, mutations of the amino acids R38 and K41 responsible for binding to dsRNA result in mutant NS1 proteins with impaired abilities to inhibit the induction of IFN-β (11
). A possible mechanism then for inhibition of IFN induction by the NS1 protein is the sequestration of dsRNA induced during influenza virus infection. However, several lines of evidence suggest that additional mechanisms are involved in the NS1 inhibitory properties on IFN production. First, dsRNA-binding-deficient NS1 proteins are not completely deficient in inhibition of IFN induction (11
). Second, the IFN antagonist functions of the NS1 protein from some viral strains are more efficient than others in inhibiting the IFN system, depending on the host species (16
). This host specificity cannot be explained by merely sequestration of viral dsRNA and most likely reflects NS1 interactions with host factors. Third, dsRNA is likely not to be the only molecule produced during viral infection to trigger the IFN system; in fact, little dsRNA is detected in cells infected with influenza viruses (53
). Therefore, we postulated that, in addition to inhibiting dsRNA binding, the NS1 protein of influenza A virus may inhibit the induction of IFN-β by interacting with host factors involved in IFN-β expression.
The inhibition of IFN-α/β production by the NS1 protein is mediated by an inhibition of the activation of transcription factors involved in induction of the IFN-β promoter, including IRF-3, a key mediator of IFN-β production (49
). IRF-3 can be activated in response to different stimuli, including the activation of TLR pathways, but more recently, it was found that induction of IFN-β by RNA virus infection is mediated in most cell systems by a TLR-independent pathway based on activation of cellular RNA helicases, such as RIG-I and Mda-5 (23
). In fact, RIG-I was recently demonstrated to be essential for the induction of IFN-β by influenza virus in a mouse knockout system (23
). Our results with a dominant negative RIG-I and with RIG-I-specific siRNA are also consistent with this notion (Fig. ). It remains to be seen whether the NS1 protein can inhibit the activation of IFN in specialized cells that use TLR-7/8 pathways to induce IFN in response to RNA viruses, such as plasmacytoid dendritic cells (22
Based on the proposed central role for RIG-I as a cellular sensor of viral products resulting in activation of IRF-3 and induction of IFN-β and also based on the fact that the NS1 of influenza virus inhibits the activation of IRF-3, we investigated the possibility that NS1 and RIG-I interact. This was readily seen in coimmunoprecipitation experiments (Fig. ). Importantly, NS1 expression inhibited the RIG-I-mediated activation of IRF-3 and of the IFN-β promoter even when a constitutively activated mutant form of RIG-I, RIG-IN, was used (Fig. and ). Wild-type RIG-I is believed to be activated by binding of its C-terminal helicase domain to dsRNA. Binding of dsRNA results in a conformational change within RIG-I that exposes an N-terminal domain containing a CARD that acts as a protein-protein-interacting module, thus allowing the recruitment of downstream cellular factors and the induction of IRF-3 activation (55
). Expression of this RIG-I CARD or RIG-IN bypasses the requirement for dsRNA for activation and results in constitutive induction of IRF-3 and IFN-β. Since NS1 expression can inhibit signaling mediated by RIG-IN, it suggests a mechanism of action of NS1 independent of dsRNA binding. Future characterization of the domains of NS1 responsible for binding to and inhibiting RIG-I may shed light on the structural requirements for the IFN-antagonistic functions of the influenza A virus NS1 protein. In this respect, it is worth mentioning that the crystal structure of the N-terminal region of NS1 is known (29
) and that the structure of the C-terminal region of NS1 has recently been solved (8
). Once these domains are determined, the use of reverse genetics will allow the generation of mutant viruses expressing NS1 proteins impaired in RIG-I binding in order to study the phenotypic impact of this NS1 property.
Although we could demonstrate an interaction between RIG-I and NS1 in coimmunoprecipitation experiments, we cannot exclude the possibility that this interaction is not direct and could be mediated by a second cellular bridging protein. In fact, when we used NS1 and RIG-I purified from bacteria, we were unable to detect an interaction (data not shown). The downstream cellular effector of RIG-I is known to be a mitochondrial resident protein with a cytoplasmic CARD, known as IPS-1, MAVS, VISA, or CARDIF (25
). Interestingly, when we overexpressed IPS-1, we observed that both RIG-I and NS1 levels are enriched in a RIPA buffer-insoluble cellular fraction that is highly abundant in IPS-1. Overexpression of RIG-I did not affect the levels of NS1 recovered in the insoluble fraction when coexpressed with IPS-1, indicating that RIG-I does not compete with NS1 for binding to IPS-1. In fact, the results suggest that NS1 localizes in the same complex with RIG-I and IPS-1 during viral infection, resulting in inhibition of further downstream signaling to the IRF-3 kinases. We are currently exploring this possibility.
The RIG-I/IPS-1 pathway appears to be targeted by different viruses to achieve inhibition of the IFN-β system. One of the best characterized examples of such a virus is hepatitis C virus, whose viral protease, NS3-4A, cleaves IPS-1 and renders this cellular protein inactive (28
). Several paramyxoviruses encode a viral protein, the V protein, that interacts with mda-5, a cellular protein with high similarity to RIG-I and which has also been implicated in antiviral functions by triggering IFN-β (3
). In the case of influenza A virus, the NS1 protein is required for IFN-β antagonism, and this appears to be mediated by two independent mechanisms, one involving sequestration of dsRNA (11
) and the second one most likely mediated through an interaction with RIG-I (this study). Moreover, NS1 has also acquired mechanisms to inhibit the IFN response at steps after IFN transcription, including inhibitory effects of cellular RNA processing (26
) and inhibition of two important IFN antiviral effectors, PKR (5
) and OAS (38
). The presence of multiple mechanisms in the NS1 protein of influenza virus to inhibit the IFN-α/β system illustrates the importance of this system in the fight against virus infections in general and influenza virus infections in particular.
Despite the presence of inhibitors of IFN-β production in many viruses, it is also evident that hosts are able to induce IFN-β after viral infection. Influenza viruses are not an exception to this observation. Natural infection with influenza A virus results in IFN-α/β production (20
), and in fact, global patterns of host gene expression during influenza virus infection are characterized by an IFN signature with an upregulation of many IFN-α/β-responsive genes (21
). However, the absence of NS1 renders influenza virus an even higher IFN-α/β inducer, and this greatly limits its replication in vivo, resulting in attenuation (13
). Thus, hosts and viruses have evolved a very intricate and delicate balance of responses and counter responses, and the IFN-α/β system exemplifies many of these processes as a central mediator of innate and adaptive immunity (14
). A better understanding of these processes may help us in the design of novel therapeutic and prophylactic strategies.
In summary, we found that the NS1 protein of influenza virus interacts with RIG-I and inhibits the RIG-I pathway, preventing the activation of IFN-β by the cell. Targeting of the RIG-I/IPS-1 pathway leading to the activation of IFN appears to be a strategy shared by several viruses that we are just starting to understand. Our results are likely to stimulate more research on the interactions of different viruses with the RIG-I/IPS-1 pathway and research on how these interactions may modulate virulence and pathogenesis.