1.4.3/1 NS1 and NS2 functions against the IFN-induction pathway
The vast majority of RSV anti-IFN properties are attributed to NS1 and NS2, which have joint, as well as, independent functions against the cellular IFN response. The molecular mechanisms by which NS1 and NS2 suppress IFN-induction pathway are currently under intense investigation and the existing experimental data is still inconclusive. NS1 and NS2 have been reported to interact with multiple steps of the RLR-mediated pathway but all of these interactions have yet to be elucidated (Figure 1.7). The first studies to report NS-mediated IFN antagonism were performed using BRSV (Schlender et al., 2000; Bossert et al., 2003). BRSV NS1 and NS2 have been found to interact with the IFN-induction pathway by blocking phosphorylation of IRF3 (Bossert et al., 2003). Given that human and bovine RSV share 71% similarity, regarding the amino acid sequences of their individual proteins, it is not surprising that NS1 and NS2 of human RSV have been reported to have a similar function against the IFN-induction pathway. Specifically, it has been shown that NS2 inhibits the nuclear accumulation of both IRF3 and NF-κB (Ling et al., 2009; Spann et al., 2004). However, the level of inhibition was significantly greater when both NS1 and NS2 were present,
suggesting that NS1 and NS2 act cooperatively to suppress activation and nuclear localization of both IRF3 and NF-κB (Spann et al., 2005). A more recent study suggested a different mechanism for IRF3 inhibition, according to which NS1 interferes with the interaction of IRF3 with its cofactor CBP, and subsequently inhibits IRF3 binding to the IFN-β promoter to suppress IFN-β induction (Ren et al., 2011).
In addition to the interactions with IRF3 and NF-κB, evidence suggests that NS1 and NS2 mediate a decrease in the expression levels of TRAF3, whereas NS1 also mediates a decrease in IKKε and IRF7 (Figure 1.7) (Swedan et al., 2009; Goswami et al., 2013). It is suggested that NS1 and NS2 reduce TRAF3 levels through a novel non- proteasomal mechanism, for which their common C-terminal tetrapeptides are not required, however the C terminus of NS1 is involved in lowering IKKε levels by a nonproteasomal mechanism (Figure 1.7) (Swedan et al., 2011; Swedan et al., 2009).
Interestingly, NS2 appears to antagonize the early activation of the RIG-I signalling cascade by binding to the N-terminal CARD of RIG-I, and thus inhibiting its interaction with the downstream component MAVS (Figure 1.7) (Ling et al., 2009). It is also speculated that the inhibition of the RIG-I pathway is caused by an interaction between NS1 and NS2 with MAVS in mitochondria. Specifically, intracellular localization studies have shown that NS2 and NS1-NS2 complexes localize in mitochondria, whereas singular expression of NS1 results in nuclear localization, suggesting that NS1-NS2 complex might directly interact with mitochondrial MAVS to block RIG-I mediated signalling (Swedan et al., 2011). In addition to the interaction of NS2 with RIG-I, NS1 was shown to degrade RIG-1 when constitutively expressed in A549 cells (Goswami et al., 2013), however the prevalence of these interactions during RSV infection remains to be elucidated. In conclusion, RSV NS1 and NS2 have
evolved a plethora of mechanisms with which they circumvent key steps of the IFN- induction signalling cascade, including early events like RIG-I activation, and latter events like IRF3 and NF-κB nuclear translocation.
Figure 1.7 RSV NS1 and NS2 interactions with the IFN-induction pathway. RSV NS1 and NS2 have been reported to interact with several effector molecules of the IFN-induction pathway to suppress the activation of the IFN-β promoter, and block the IFN response to RSV infection. There are a noteworthy number of interactions in the literature, most of which seem to target the RIG-I dependent IFN induction pathway.
1.4.3/2 NS1 and NS2 functions against the IFN-signalling pathway
RSV NS1 and NS2 proteins also counteract the IFN-signalling pathway and subsequently suppress the activation of the ISRE elements, which are present within the promoters of ISGs (Ramaswamy et al., 2004). The majority of studies that focus on the RSV-mediated IFN antagonism unanimously suggest that RSV infections and expression of recombinant NS1 and NS2 in epithelial cells causes an evident decrease in STAT2 levels, which outruns the downstream events of the IFN-α/β response. However, there is controversy regarding the molecular mechanism that RSV uses to mediate a decrease in STAT2 levels. In particular, the majority of evidence supports that STAT2 decrease is mainly driven by NS2 with NS1 having some effect (Spann et al., 2004; Lo et al., 2005; Swedan et al., 2011; Goswami et al., 2013), whereas other evidence suggests that STAT2 degradation requires only NS2 (Ramaswamy et al.,
2006). In contrast, Elliott et al., (2007) proposed a NS2-independent mechanism and suggested that NS1 has an E3 ligase activity that is crucial for STAT2 degradation. It is possible that the exact stoichiometry of the NS1, NS2 and NS1-NS2 heterodimer varies between these studies and that may account for some of the differences reporter in the literature, which are discussed in detail below.
One of the earliest studies to support NS-mediated STAT2 antagonism was published by Lo et al., (2005), who showed that constitutive expression of NS2 is related to a significant reduction in STAT2, whereas NS1 expression had a less significant impact on STAT2 levels. Interestingly, co-expression of NS1 and NS2 suppressed STAT2 below baseline levels, suggesting that NS1 and NS2 work together to achieve robust inhibition of STAT2 (Lo et al., 2005). Furthermore, it was shown that NS2 interacts with the host microtubule-associated protein 1B (MAP1B) through its C-
terminus DNLP tetrapeptide (Figure 1.5), and this interaction was found to be essential for the STAT2-decreasing activity of NS2 (Swedan et al., 2011). In fact, it is suggested that MAPIB could be part of the NS1-NS2 complex, hence it might be important for the synergistic functions of NS1 and NS2 (Swedan et al., 2011).
Shedding more light on the mechanism behind STAT2 decrease, other studies demonstrated that STAT2 antagonism is caused by a NS2-mediated proteasomal degradation (Ramaswamy et al., 2006; Ramaswamy et al., 2004). In general, proteasome-mediated degradation of proteins usually occurs after protein ubiquitylation. The process of protein ubiquitylation is catalyzed by coordinated enzymatic reactions that are mediated by enzymes known as E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) (Da Fonseca et al., 2012; Jiang & Chen 2011). E3 ligases are responsible for targeting ubiquitylation to specific substrate proteins, by covalently attaching ubiquitin to lysine side chains of the substrate protein (Jiang & Chen 2011). Some of the E3 ubiquitin ligases belong to the suppressor of cytokine signalling (SOCS) family of proteins, which are involved in inhibiting the JAK/STAT pathway (Yoshimura et al., 2007). Interestingly, RSV- induced STAT2 degradation was prevented by knocking down expression of endogenous E3 ligase components like Cul2 and Rbx1 (Elliott et al., 2007). The same study has also shown that NS1 associates with Cul2, suggesting that NS1 can assemble ubiquitin ligase enzymes to target STAT2 to the proteasome (Elliott et al., 2007). Notably, internal sequences of NS1 and NS2 shared distant homology to the consensus sequence for elongin C and cullin 2 binding motif (BC box), which occurs in E3 ligases such as SOCS, proposing that this motif might be responsible for the E3 ligase activity of NS1 and NS2 that permits STAT2 degradation (Elliott et al., 2007; Swedan et al.,
2009). However, the role of this motif still remains unclear, since mutations within the BC box motif did not inhibit STAT2 degradation or any other function of either NS1 or NS2 protein (Swedan et al., 2011). Interestingly, a more recent study have demonstrated that RSV NS1 protein upregulates SOCS1 mRNA independently of the RLR signalling pathway, suggesting that SOCS1 might be important for the degradation activity of NS1 against STAT2 or other innate immune proteins (Xu et al., 2014).
The first model to describe the NS-degradasome has recently been proposed by Goswami et al., (2013), who suggested that NS proteins assemble a heterogeneous degradation complex (~300 – 750 kDa in size), which translocates to mitochondria upon RSV infection. Their controversial findings suggest that optimal RSV suppression of cellular interferon response requires mitochondrial MAVS to be part of the NS- degradasome, hence MAVS facilitates the NS-mediated RIG-1 inhibition, and STAT2 degradation (Goswami et al., 2013). To date, no other studies have been reported that either support or refute this observation, however a few studies have reported association of RSV NS1 and NS2 with mitochondria. Specifically, proteomic analyses of the RSV NS1 interactome indicated that NS1 is associated with a number of mitochondrial proteins (Wu et al., 2012). Consistent with these findings, Boyapalle et al., (2012) have demonstrated that RSV NS1 directly binds to mitochondrial MAVS, however the domains of interaction have not been mapped. Interestingly, another recent study has shown that during RSV infection proteins involved in innate antiviral immune response (e.g.Tom70) accumulate on mitochondria, supporting the hypothesis that mitochondria are likely to be hijacked by NS1 and NS2 for the assemblage of the NS- degradasome (Munday et al., 2015). The role of mitochondria in RSV infection remains
to be further elucidated, however current evidence unanimously suggests that they have important implications for RSV biology, and perhaps IFN antagonism.
Although the function of RSV NS1 and NS2 against STAT2 is well documented, the precise mechanism behind STAT2 degradation is undetermined. Our up-to-date knowledge suggests that STAT2 degradation is a synergistic event that requires both NS1 and NS2, and remarkably, this function still remains the only documented interaction of RSV NS1 and NS2 with the type I IFN signalling cascade.
1.4.3/3 NS1 and NS2 interactions with antiviral ISGs
Current investigations suggest that RSV NS1 and NS2 can also directly suppress ISGs. For instance, NS1 and NS2 were shown to antagonize the RSV-mediated upregulation of the let-7i and miR-30b miRNAs, which have an antiviral effect and are induced by an IFN- or NF-κB-dependent mechanism, respectively (Thornburg et al.,
2012). More recently, another study have shown that NS1 can suppress the function of an IFN-induced antiviral protein, namely 2'-5'-oligoadenylate synthetase-like (OASL) by mediating proteasomal degradation of specific OASL isoforms (Dhar et al.,
2015). RSV infection also activates several ISGs of the IFIT family, namely ISG56, ISG54 and ISG60 (Hastie et al., 2012; Janssen et al., 2007), however it is still unclear whether RSV interferes directly with these IFITs, the antiviral role of which is also not clear yet.