Respiratory Syncytial Virus Nonstructural Proteins NS1 and NS2 Mediate Inhibition of Stat2 Expression and Alpha/Beta Interferon Responsiveness

Abstract

Respiratory syncytial virus (RSV) subverts the antiviral interferon (IFN) response, but the mechanism for this evasion was unclear. Here we show that RSV preferentially inhibits IFN-␣/␤ signaling by expression of viral NS1 and NS2. Thus, RSV infection or expression of recombinant NS1 and NS2 in epithelial host cells causes a marked decrease in Stat2 levels and the consequent downstream IFN-␣/␤ response. Similarly, NS1/NS2-deficient RSV no longer decreases Stat2 levels or IFN responsiveness. RSV infection decreased human but not mouse Stat2 levels, so this mechanism of IFN antagonism may contribute to viral host range, as well as immune subversion.Paramyxoviruses appear to effectively antagonize the interferon (IFN) system through either inhibition of IFN generation or blockade of IFN signal transduction components such as Stat1 and Stat2 (8). For Sendai and measles viruses, the viral V and C proteins inhibit Stat1 expression and activation (7,11,14,22), whereas the V protein of human parainfluenza virus 2 affects Stat2 responses (1, 15). However, these proteins are not present in respiratory syncytial virus (RSV), the most common cause of serious respiratory infection in childhood (12). Nonetheless, RSV is also relatively resistant to the IFN-induced antiviral state (2). Recombinant deletion mutants of bovine RSV demonstrated that the nonstructural proteins NS1 and NS2 are cooperatively necessary for this resistance (21). These proteins also appear to inhibit induction of IFN, since deletion mutants of NS1 and NS2 for both human and bovine RSV induce more IFN-␣/␤ production than does wild-type RSV (23,24). However, these previous approaches have not addressed a possible action of RSV proteins on IFN signaling that might also inhibit the initial induction of an IFN-dependent antiviral state.In the present study, we found that infection of primaryculture human tracheobronchial epithelial cells (hTECs) (13) or a human alveolar epithelial cell line (A549 cells) with a low inoculum (multiplicity of infection [MOI] of 2) of RSV slightly increased baseline Stat1 and Stat2 expression and tyrosine phosphorylation but markedly suppressed any subsequent response to IFN-␤ (Fig. 1A and data not shown). A higher inoculum of RSV (MOI of 10) decreased the level of Stat2 expression and fully inhibited IFN-␤ responsiveness, as described by others (19). Neither level of RSV inoculation caused any significant decrease in Stat1 expression or IFN-␥ responsiveness ( Fig. 1A and data not shown). In concert with these effects, RSV infection also increased baseline ISRE promoter activity without affecting control Renilla luciferase activity and suppressed IFN-␤ responsiveness of the ISRE promoter (Fig. 1B). Thus, at a low inoculum, RSV-driven increases in baseline Stat1 and Stat2 expression and phosphorylation are likely due to endogenous IFN-␤ production acting in a paracrine manner on uninfected cells. A high inoculum of RSV causes selective down-regulation of Stat2 with consequent inhibition of IFN-␣/␤ signaling that becomes apparen...

Viral degradasome hijacks mitochondria to suppress innate immunity

“…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.…”

Human respiratory syncytial virus non-structural protein NS1 modifies miR-24 expression via transforming growth factor-β

“…RSV non-structural proteins have been shown to interact with several host nuclear, cytosolic and mitochondrial factors, leading to suppression of the antiviral response, cell cycle regulation, DNA damage repair and culminating in G 0 /G 1 phase cell cycle arrest (Atreya et al, 1998;Bossert et al, 2003;Boyapalle et al, 2012;Elliott et al, 2007;Lo et al, 2005;Munday et al, 2010;Munir et al, 2011;Ren et al, 2011;Schlender et al, 2000;Spann et al, 2004Spann et al, , 2005Wu et al, 2012;Xu et al, 2014). TGF-b expression during RSV infection has been shown to be important for cell cycle inhibition in the G 0 /G 1 phase (Gibbs et al, 2009;McCann & Imani, 2007;Wu et al, 2011) and miR-24 has been shown to be suppressed by TGF-b (Sun et al, 2008), as well as regulate TGF-b precursor processing (Luna et al, 2011).…”

“…Codon deoptimized RSV is attenuated in vitro, but evokes a strong humoral response upon vaccination and challenge (Meng et al, 2014). These and other immune modulators inhibit the development of the antiviral state (Bossert et al, 2003;Elliott et al, 2007;Goswami et al, 2013;Lo et al, 2005;Moore et al, 2008;Oshansky et al, 2009a;Spann et al, 2005).…”

“…The virus-host interactions that follow RSV binding are modified and regulated by several RSV proteins, including non-structural and G proteins, which modulate proinflammatory and antiviral cytokine expression by host cells (Atreya et al, 1998;Bossert et al, 2003;Boyapalle et al, 2012;Elliott et al, 2007;Kotelkin et al, 2006;Liesman et al, 2014;Ling et al, 2009;Lo et al, 2005;Moore et al, 2008;Munir et al, 2011;Ren et al, 2011;Schlender et al, 2000;Spann et al, 2004Spann et al, , 2005Wright et al, 2006;Wu et al, 2012;Xu et al, 2014). RSV non-structural proteins inhibit minigenome transcription and viral RNA replication (Atreya et al, 1998), and abrogate the innate host response to infection in part by controlling expression of host cell proteins involved in the cell cycle and replication (Atreya et al, 1998;Liesman et al, 2014;Wu et al, 2012).…”

“…Codon deoptimized RSV is attenuated in vitro, but evokes a strong humoral response upon vaccination and challenge (Meng et al, 2014). These and other immune modulators inhibit the development of the antiviral state (Bossert et al, 2003;Elliott et al, 2007;Goswami et al, 2013;Lo et al, 2005;Moore et al, 2008;Oshansky et al, 2009a;Spann et al, 2005).The role of microRNAs (miRNAs) in post-transcriptional regulation of host genes responding to RSV infection is not fully understood. miRNA expression is known to be regulated by multiple processes including TLR4 signalling (Nahid et al, 2009(Nahid et al, , 2011a O'Connell et al, 2007;Pauley et al, 2010;Schulte et al, 2013;Taganov et al, 2006) …”