In the current study we identified NOD2 as a viral PRR that can sense viral ssRNA genomes to activate IFN production and antiviral defense. Like RLH receptors NOD2 associated with MAVS to activate IRF3 and promote IFN production. The importance of NOD2 in host defense was evident from the ability of both immune (e.g. macrophages) and non-immune (e.g. epithelial cells, MEFs) cells to utilize NOD2 for IFN production. The in vivo importance of NOD2 in antiviral responses was evident from the enhanced RSV-induced pathogenesis in infected NOD2-KO mice.
It is important to mention that other PRRs including RIGI may also be involved in activating an antiviral response against paramyxoviruses like RSV 20
. For quite some time, the in vivo
relevance of RIGI in antiviral function was not documented, due to the embryonic lethal phenotype of majority of RIGI-KO mice 38
. However, one strain of RIGI-KO mice generated by crossing RIGI heterozygous mice with ICR outbred mice, followed by intercrossing of the resulting RIGI heterozygous mice 38
, survived to adulthood. These RIGI-KO mice exhibited impaired IFN production and enhanced susceptibility to two positive sense ssRNA viruses: Encephalomyocarditis virus (EMCV) and Japanese encephalitis virus (JEV) 38
. However no studies were conducted to demonstrate the importance of RIGI in activating the antiviral host defense apparatus against negative sense ssRNA viruses (e.g. paramyxoviruses).
NOD2 is expressed in low amounts in uninfected mice 39
, and we showed that its expression increased after viral infection. Our observation is similar to previous studies demonstrating that majority of PRRs (for e.g. RIGI) are expressed at low abundance, but their expression is elevated following pathogen invasion 40, 41
. Similarly, we observed viruses mediated induction of NOD2 in various cells. Although in our studies we noted induction of NOD2 in virus infected MEFs; one study reported an inability of MDP to induce NOD2 expression in wild-type MEFs, this observation may have been due to a defect in MDP transport to the cytoplasm 42
. Several other studies showed that NOD2 expression is stimulated by various bacteria and bacterial components 40, 41
. Similarly, expression of RIGI 43
and Mda-5 38
in uninfected, unstimulated wild-type MEFs is negligible, but treatment of cells with PAMPs results in up-regulation of RIGI expression 43
. This mechanism of restricting expression of PRRs in un-stimulated cells may be critical to prevent uncontrolled inflammation.
Although NOD2 can be activated by MDP 7, 8, 9, 10
, so far no studies have demonstrated direct binding of MDP to NOD2; it is only known that lack of NOD2 expression results in loss of MDP responsiveness. Thus, MDP could directly interact with NOD2, or associate with protein(s) that form a complex with NOD2. In that context, the interaction of viral-ssRNA with NOD2 demonstrated here could also be mediated indirectly via “bridging” protein(s). Interaction of ssRNA genomes of viruses with RIGI has also been noted previously 44, 45
. In addition to NOD2, cryopyrin (also called Nalp3), another NLR protein, activates the inflammasome and leads to IL-1 production upon stimulation with bacterial and viral (influenza A) RNA 46, 47, 48
. Although NLRs like cryopyrin 46,47, 48
and NLRX1 11
play an important role in innate immunity by activating the inflammasome and inhibiting IFN production, respectively, no studies have determined whether other NLRs, like NOD2, can directly contribute to antiviral responses by inducing IFN production.
Previous studies 49
have shown that transfection of NOD2 alone (in the absence of any stimulant) in wild-type MEFs resulted in substantial NF-κB activation. However, we found that overexpression of NOD2 in 293 cells did not induce marked activation of IRF3 in the absence of external stimuli (e.g. RSV, synthetic or viral ssRNA). Thus, it appears that NOD2-mediated activation of IRF3 is stimulus dependent, whereas RICK activation is stimulus independent.
In summary, our findings demonstrated that in addition to RIGI, Mda5 and TLRs, NOD2 can also function as a viral PRR and participate in inducing antiviral signaling. Distinct temporal activation of various PRRs may be required to generate optimal antiviral responses, and various viruses may trigger induction of different classes of PRRs.