Species-Specific Inhibition of RIG-I Ubiquitination and IFN Induction by the Influenza A Virus NS1 Protein

Abstract

Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1) proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN) production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling. In order to understand possible mechanisms of viral adaptation and host tropism, we examined the ability of NS1 encoded by human (Cal04), avian (HK156), swine (SwTx98) and mouse-adapted (PR8) influenza viruses to interact with TRIM25 orthologues from mammalian and avian species. Using co-immunoprecipitation assays we show that human TRIM25 binds to all tested NS1 proteins, whereas the chicken TRIM25 ortholog binds preferentially to the NS1 from the avian virus. Strikingly, none of the NS1 proteins were able to bind mouse TRIM25. Since NS1 can inhibit IFN production in mouse, we tested the impact of TRIM25 and NS1 on RIG-I ubiquitination in mouse cells. While NS1 efficiently suppressed human TRIM25-dependent ubiquitination of RIG-I 2CARD, NS1 inhibited the ubiquitination of full-length mouse RIG-I in a mouse TRIM25-independent manner. Therefore, we tested if the ubiquitin E3 ligase Riplet, which has also been shown to ubiquitinate RIG-I, interacts with NS1. We found that NS1 binds mouse Riplet and inhibits its activity to induce IFN-β in murine cells. Furthermore, NS1 proteins of human but not swine or avian viruses were able to interact with human Riplet, thereby suppressing RIG-I ubiquitination. In conclusion, our results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production.

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

“…In such an arrangement, RING dimerization could potentially be enhanced by substrate binding, thereby ensuring that TRIM25 activity is only available in the correct setting. This is particularly interesting in the light of the observation that viral proteins such as NS1 bind to the coiled‐coil region of TRIM25, which could prevent intramolecular RING dimerization in this model and hence activity (Gack et al , 2009; Rajsbaum et al , 2012). In contrast, in TRIM32 the RING acts as an independent dimerization module and promotes formation of a TRIM tetramer, in which the RING dimers are located on either side of the central coiled‐coil (Figs 7 and EV4).…”

Influenza virus-induced lung injury: pathogenesis and implications for treatment

“…The sialic acid specificity of different viruses has been suggested to explain why some viruses appear to be more lethal than others [5, 73,75]. [41,42] It also induces the downregulation of the IFN-α receptor [43] and upregulates inhibitors of JAK/STAT signalling (SOCS1 and SOCS3) [43,44] During infection, non-structural protein-1 also blocks pro-apoptotic signalling by protein kinase R [45,46] and prevents the activation of NF-κB [47] Nonstructural protein-2 Expressed during replication, not part of the mature virion…”

RIG-I from waterfowl and mammals differ in their abilities to induce antiviral responses against influenza A viruses

“…This suggests that only waterfowl-derived RIG-Is are functional in chicken DF-1 cells and some factors necessary for mammal-derived RIG-I activation are missing or unrecognizable in chicken cells. For example, RIPLET, which ubiquitinates Lys849 and Lys851 in the C-terminal domain of RIG-I (Gao et al, 2009;Oshiumi et al, 2009Oshiumi et al, , 2010, is absent from chicken and duck genomes (Magor et al, 2013;Rajsbaum et al, 2012).…”

“…2a-f). Another study reported that only avian influenza virus-derived NS1 can bind to chicken TRIM25, thereby suppressing the induction of IFN (Rajsbaum et al, 2012). Mouse-adapted IAV NS1 specifically binds to mouse RIPLET in mouse cells (Rajsbaum et al, 2012).…”