Respiratory Syncytial Virus Nonstructural Proteins Decrease Levels of Multiple Members of the Cellular Interferon Pathways

Abstract

Viruses of the Paramyxoviridae family, such as the respiratory syncytial virus (RSV), suppress cellular innate immunity represented by type I interferon (IFN) for optimal growth in their hosts. The two unique nonstructural (NS) proteins, NS1 and NS2, of RSV suppress IFN synthesis, as well as IFN function, but their exact targets are still uncharacterized. Here, we investigate if either or both of the NS proteins affect the steady-state levels of key members of the IFN pathway. We found that both NS1 and NS2 decreased the levels of TRAF3, a strategic integrator of multiple IFN-inducing signals, although NS1 was more efficient. Only NS1 reduced IKK, a key protein kinase that specifically phosphorylates and activates IFN regulatory factor 3. Loss of the TRAF3 and IKK proteins appeared to involve a nonproteasomal mechanism. Interestingly, NS2 modestly increased IKK levels. In the IFN response pathway, NS2 decreased the levels of STAT2, the essential transcription factor for IFN-inducible antiviral genes. Preliminary mapping revealed that the C-terminal 10 residues of NS1 were essential for reducing IKK levels and the C-terminal 10 residues of NS2 were essential for increasing and reducing IKK and STAT2, respectively. In contrast, deletion of up to 20 residues of the C termini of NS1 and NS2 did not diminish their TRAF3-reducing activity. Coimmunoprecipitation studies revealed that NS1 and NS2 form a heterodimer. Clearly, the NS proteins of RSV, working individually and together, regulate key signaling molecules of both the IFN activation and response pathways.Respiratory syncytial virus (RSV) is by far the most significant agent of pediatric respiratory disease for which no reliable antiviral or vaccine yet exists (45, 59). The lukewarm success of traditional active-immunization-based strategies has drawn focus to cellular innate immunity, which acts as a broad antiviral defense. A major arm of innate antiviral immunity is the type I IFN family, represented by alpha interferon (IFN-␣) and 46,58). In counterdefense, however, members of the Paramyxoviridae family have evolved accessory gene products that neutralize or inhibit various steps of the IFN pathway, thus permitting robust virus growth (18,27,57,71). It is now appreciated that a better understanding of the viral IFN suppression mechanism(s) is essential for the prudent design of attenuated vaccine strains and better overall therapy.RSV encodes unique anti-IFN genes not found in any other member virus of this family. While other viruses generate IFNsuppressive proteins, mainly the V protein (1,12,18,19,21,22,27,33,50,51,57), from alternative translational reading frames in the P gene through "RNA editing," the P gene of RSV codes for the P protein only. Instead, the RSV genome contains two promoter-proximal genes that code for nonstructural (NS) proteins, NS1 and NS2, which are so named because they are synthesized in RSV-infected cells but are not packaged in the mature virion structure. The predicted primary structures of the NS proteins do not share any sig...

IFNβ/TNFα synergism induces a non-canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH Oxidase-mediated airway antiviral response

“…In this line, we unveil for the first time that DUOX2 is critical for the outcome of respiratory virus infections through its key function in the establishment of the antiviral state specifically induced by the synergistic action of IFNβ and TNFα secreted during SeV infection. The capacity of RSV to counteract this novel antiviral pathway adds to its previously recognized capacity to interfere with other key antiviral events, including RIG-I-dependent signaling or IKKε, TRAF3 or STAT2 stability [33,34]. Altogether, these evasion mechanisms contribute to the development of RSV-associated diseases.…”

Viral degradasome hijacks mitochondria to suppress innate immunity

“…These results also reveal that although NS1 and NS2 display some preferences, there is a significant degree of overlap in their targets. The inhibitory activity is nonetheless specific, as the expression levels of MAVS, LGP2, STAT1, TRIF, MyD88 and numerous IFN signaling-unrelated cellular proteins that we have tested, such as L13a ( Figure 1A), actin [28], GAPDH and SP6 (data not shown), were not affected by the ectopic expression of NS1 and/or NS2. We proposed that this is indicative of the ability of NS proteins to form specific degradative complex(s), which we set out to study.…”

“…We and others [16][17][18][19][20][21][22][23][24][25][26][27][28][29] have recently shown that NS1 and NS2 inhibit and/or degrade a number of proteins of the IFN induction and response pathways. We have since screened other members of these pathways, confirmed and extended the repertoire of NS targets to include: RIG-I, TRAF3, IKKε, IRF3, IRF7 and STAT2 ( Figure 1A).…”

“…However, RSV∆NS2, which contains NS1, but not NS2, degraded RIG-I and IRF7 but not STAT2. Reciprocally, RSV∆NS1, which contains NS2, but not NS1, degraded STAT2, but left The experiment was performed as described previously [28]. A549 cells in 24-well plates were transfected with 0.6 µg of the indicated recombinant plasmids of various targets (shown on left) and 0.6 µg of either the vector (pCAGGS), or pCAGGS-NS1 or pCAGGS-NS2, or both.…”

“…With the sole exception of pneumoviruses, members of this family produce IFN-suppressor proteins such as V, W and C through co-transcriptional RNA editing of the viral P gene [10,[13][14][15]. Pneumoviruses, represented by RSV, do not use RNA editing; instead, they uniquely encode two nonstructural (NS) proteins, NS1 and NS2, which strongly suppress both IFN induction and IFNresponse pathways by inhibiting or degrading a number of signaling proteins involved in these two pathways [16][17][18][19][20][21][22][23][24][25][26][27][28][29], and thus act as essential virulence factors [30][31][32]. In pursuing the mechanism by which the NS proteins target such a diverse array of immune proteins that share little or no sequence identity, we wondered whether there is a common location of NS proteins and their targets.…”