| Human respiratory syncytial virus (hRSV) is a primary cause of respiratory tract infection in infants and young children worldwide. To promote its own propagation, RSV has evolved mechanisms to evade cell antiviral responses. The interferon (IFN) system plays an important role in inducing cell antiviral responses and limiting virus spread at early infection. The nonstructural proteins of RSV NS1 and NS2 have been shown to inhibit both IFN production and IFN signaling. However, the mechanism is unknown. In our study, we defined the IFN induction pathway that NS1 and NS2 inhibit. Moreover, we provided evidence to show the mechanism of NS2 IFN antagonism. We found that NS1 and NS2 inhibit both retinoic acid inducible gene - I (RIG-I) and Toll-like receptor 3 (TLR3) mediated IFN induction, but in a different manner. NS1 blocked RIG-I and TLR3 mediated IFN production at the level of IFN regulatory factor -3 (IRF-3) kinases, Tank-binding kinase 1 (TBK1) and IkappaB kinase epsilon (IKKepsilon) or downstream. NS2 inhibited TLR3 mediated IFN production at the level of TIR-domain-containing adapterinducing interferon-beta (TRIF) or downstream, while it inhibited the RIG-I pathway at the level of RIG-I. In addition, we found that NS2 interacted with RIG-I at the N-terminus of RIG-I (N-RIG). We also found that NS2 disrupted the interaction between N-RIG and downstream mitochondrial antiviral signaling protein (MAVS), suggesting that NS2 inhibits RIG-I mediated IFN production by binding to RIG-I and blocking the RIG-I-MAVS interaction.;While we were studying the IFN antagonism of NS2, we found that NS2 induced transcriptional factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) activation. This phenomenon is consistent with published studies in which Akt (also known as protein kinase B, PKB) and NF-kappaB were shown to be activated by RSV infection and the activation was reduced when NS2 was knocked down by siRNA. Therefore, we investigated the role of Akt in NS2-induced NF-kappaB activation. We found that blockade of Akt with inhibitor or dominant negative form of Akt reduced NS2-induced NF-kappaB activation, suggesting that NS2 activates NF-kappaB through Akt. Subsequent analysis showed that NS2 led to the phosphorylation of Akt at Thr-308. Akt is activated mostly in a phosphatidylinositol 3-kinase (PI3K) dependent manner. We observed that PI3K inhibitor treatment had no effect on NS2-induced NF-kappaB activation, indicating that NS2 activates NF-kappaB independent of PI3K. Furthermore, co-immunoprecipitation (co-IP) experiment indicated that NS2 interacted with Akt, suggesting that Akt plays an important role for NS2-mediated NF-kappaB acitivation. Finally, we identified two amino acid residues in NS2 that are required for NF-kappaB induction. |
