Respiratory Syncytial Virus NS1 Protein Degrades STAT2 by Using the Elongin-Cullin E3 Ligase

Abstract

Respiratory syncytial virus (RSV) infection causes bronchiolitis and pneumonia in infants.RSV has a linear single-stranded RNA genome encoding 11 proteins, 2 of which are nonstructural (NS1 and NS2). RSV specifically downregulates STAT2 protein expression, thus enabling the virus to evade the host type I interferon response. Degradation of STAT2 requires proteasomal activity and is dependent on the expression of RSV NS1 and NS2 (NS1/2). Here we investigate whether RSV NS proteins can assemble ubiquitin ligase (E3) enzymes to target STAT2 to the proteasome. We demonstrate that NS1 contains elongin C and cullin 2 binding consensus sequences and can interact with elongin C and cullin 2 in vitro; therefore, NS1 has the potential to act as an E3 ligase. By knocking down expression of specific endogenous E3 ligase components using small interfering RNA, NS1/2, or RSV-induced STAT2, degradation is prevented. These results indicate that E3 ligase activity is crucial for the ability of RSV to degrade STAT2. These data may provide the basis for therapeutic intervention against RSV and/or logically designed live attenuated RSV vaccines.Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infections in infants and young children (28,31). RSV belongs to the genus Pneumovirus in the subfamily Pneumovirinae of the family Paramyxoviridae. It is an enveloped, nonsegmented negative-strand RNA virus encoding 11 proteins, including nucleocapsid proteins (N, P, and L), surface proteins (F and G), and a matrix protein (M). In addition, the genome encodes two nonstructural proteins (NS1 and NS2), the functions of which are less clearly defined. RSV primarily infects epithelial cells of the respiratory tract and replicates exclusively in the cytoplasm. Progeny RSV particles exit the host cell by budding through the apical surfaces of polarized cells (35).In order to combat such infections, the immune system has evolved a potent antiviral response. Mediators, known as the type I interferons (alpha interferon [IFN-␣] and IFN-␤), stimulate the production of a range of antiviral gene products that limit virus replication and spread (4, 22). The type I IFN receptor consists of two subunits, IFNAR1 and IFNAR2, which are associated with the Janus kinases JAK1 and TYK2, respectively (23). Activation of these receptor tyrosine kinases results in tyrosine phosphorylation of signal transducer and activator of transcription 2 (STAT2) and STAT1. Activated STAT2 and STAT1 associate with interferon regulatory factor 9 (IRF-9) to form the transcriptional activator complex interferon-stimulated gene factor 3 (ISGF-3). These complexes translocate to the nucleus and bind IFN-stimulated response elements (ISRE) to initiate gene transcription and therefore antiviral immunity (8).Wild-type RSV induces a weak type I IFN response following infection (27), suggesting that it has the capacity to evade this host defense mechanism in order to establish a successful infection. RSV is thought to block IFN-␣ and -␤ signaling...

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.…”

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).…”

“…RSV F and G proteins also bind to several other cell constituents, such as CX3CR1 molecules (Tripp et al, 2001), Toll-like receptor 4 (TLR4) (Kurt-Jones et al, 2000), RhoA (Budge & Graham, 2004;Pastey et al, 1999), intercellular adhesion molecule 1 (Mastrangelo & Hegele, 2013;Mata et al, 2012) and nucleolin (Tayyari et al, 2011). These interactions may facilitate infection and contribute to tropism.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) …”