Multiple Functional Domains and Complexes of the Two Nonstructural Proteins of Human Respiratory Syncytial Virus Contribute to Interferon Suppression and Cellular Location

Abstract

Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKK, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo-as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKK reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression. Human respiratory syncytial virus (RSV) is a member of thePneumovirus genus in the Paramyxoviridae family (9, 25). RSV replicates primarily in the respiratory epithelium and is a leading cause of respiratory disease and asthma in the very young and the elderly (41, 42). The nonsegmented negative-strand RNA genome of RSV encodes 10 genes, of which the two most promoterproximal and abundantly transcribed genes code for nonstructural proteins 1 and 2 (NS1 and NS2), which are small proteins, 139 and 124 amino acid residues long, respectively (9, 25). The NS proteins circumvent the host innate immune system by preventing the induction of type I interferons (IFNs) as well as IFN-induced antiviral responses (6,12,20,22,23,29,(33)(34)(35)(36)39), thus allowing a more robust replication of the virus, leading to the severe respiratory disease that characterizes RSV infection.The IFN pathways of the cell can be divided into two functional ones, the IFN induction pathway, in which the cells produce IFN, and the IFN response pathway, in which the cells respond to exogenous IFN. One of the most proximal steps in the IFN induction pathway is the activation of the cytoplasmic RNA sensors of the RIG-I family (27,37,43,44). This triggers a cascade of signaling events whereby the CARD sequence of RIG-I interacts with and activates the CARD-like domaincontaining mitochondrial protein, MAVS (7, 18). Activated MAVS then activates TRAF3 (7, 31), which in turn activates two downstream kinases, IKKε and TBK1. The latter are Ser/ Thr kinases that phosphorylate the C-terminal domain of interferon regulatory factor 3 (IRF3) and IRF7, leading to...

Viral degradasome hijacks mitochondria to suppress innate immunity

“…When we tested the kinetics of the degradation, the pooled NSD fractions (#16-22, Figure 2A) were found to be active for only about 15 min ( Figure 2B). As we and others have recently found that a large portion of NS proteins are localized to the mitochondria [17,27], we speculated that mitochondrial association may be required for optimal NS functionality. However, as the purified NSD complex is ~300-750 kD in size, it cannot accommodate mitochondria; and thus, any mitochondria associated in vivo may have been dissociated from the complex during chromatography.…”

“…We co-transfected A549 cells with NS1-and NS2-expressing plasmids [27,28], harvested cell-free extracts 20 h later using a non-denaturing buffer and subjected them to size fractionation using a Superdex 200 10/300 GL resin pre-calibrated with size markers, as described in the Materials and Methods. Substantial amounts of NS1/2 proteins (roughly 60% of the total) were found in the void volume, but we did not characterize the fraction of the void volume further due to its crude nature and the likely presence of multiple large cellular structures such as cell membranes and intracellular organelles.…”

“…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).…”

“…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.…”

Role of non-motile microtubule-associated proteins in virus trafficking

“…The two NS proteins suppress both type I IFN induction and the IFN response pathways by interfering with various innate immunity actors such as RIG-I and STAT2. Recently, MAP1B was shown to interact with NS1 and NS2 and to contribute to the STAT2-degrading activity of NS2 (104). In the case of HIV infection, the viral transactivator Tat protein has been shown to contribute to neuronal damage observed in the brain of HIV-1 patients with neurological dysfunctions, by causing rapid proteasomemediated degradation of MAP2 leading to the collapse of cytoskeletal filaments, as well as aberrant splicing of tau (105,106).…”