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...

Although production of cytokines by TLR is essential for viral and bacterial clearance, overproduction can be detrimental, thus controlling these responses is essential. CD33-related sialic acid binding Ig-like lectin receptors (Siglecs) have been implicated in the control of leukocyte responses. In this study, we report that murine Siglec-E is induced by TLRs in a MyD88-specific manner, is tyrosine phosphorylated following LPS stimulation, and negatively regulates TLR responses. Specifically, we demonstrate the Siglec-E expression inhibits TLR-induced NF-κB and more importantly, the induction of the antiviral cytokines IFN-β and RANTES. Siglec-E mediates its inhibitory effects on TIR domain containing adaptor inducing IFN-β (TRIF)-dependent cytokine production via recruitment of the serine/threonine phosphatase SHP2 and subsequent inhibition of TBK1 activity as evidenced by enhanced TBK1 phosphorylation in cells following knockdown of Siglec-E expression. Taken together, our results demonstrate a novel role for Siglec-E in controlling the antiviral response to TLRs and thus helping to maintain a healthy cytokine balance following infection.

Suppressors of cytokine signaling (SOCS) are important regulators of lipopolysaccharide (LPS) and cytokine responses but their role in macrophage polarization is unknown. We have shown here that myeloid-restricted Socs3 deletion (Socs3(Lyz2cre)) resulted in resistance to LPS-induced endotoxic shock, whereas Socs2(-/-) mice were highly susceptible. We observed striking bias toward M2-like macrophages in Socs3(Lyz2cre) mice, whereas the M1-like population was enriched in Socs2(-/-) mice. Adoptive transfer experiments showed that responses to endotoxic shock and polymicrobial sepsis were transferable and macrophage dependent. Critically, this dichotomous response was associated with enhanced regulatory T (Treg) cell recruitment by Socs3(Lyz2cre) cells, whereas Treg cell recruitment was absent in the presence of Socs2(-/-) macrophages. In addition, altered polarization coincided with enhanced interferon-gamma (IFN-γ)-induced signal transducer and activator of transcription-1 (STAT1) activation in Socs2(-/-) macrophages and enhanced interleukin-4 (IL-4) plus IL-13-induced STAT6 phosphorylation in Socs3(Lyz2cre) macrophages. SOCS, therefore, are essential controllers of macrophage polarization, regulating inflammatory responses.

CD33-related Siglecs (sialic acid-binding immunoglobulin-like lectins) 5-11 are inhibitory receptors that contain a membrane proximal ITIM (immunoreceptor tyrosine-based inhibitory motif) (I/V/L/)XYXX(L/V), which can recruit SHP-1/2. However, little is known about the regulation of these receptors. SOCS3 (suppressor of cytokine signaling 3) is up-regulated during inflammation and competes with SHP-1/2 for binding to ITIM-like motifs on various cytokine receptors resulting in inhibition of signaling. We show that SOCS3 binds the phosphorylated ITIM of Siglec 7 and targets it for proteasomal-mediated degradation, suggesting that Siglec 7 is a novel SOCS target. Following ligation, the ECS E3 ligase is recruited by SOCS3 to target Siglec 7 for proteasomal degradation, and SOCS3 expression is decreased concomitantly. In addition, we found that SOCS3 expression blocks Siglec 7-mediated inhibition of cytokine-induced proliferation. This is the first time that a SOCS target has been reported to degrade simultaneously with the SOCS protein and that inhibitory receptors have been shown to be degraded in this way. This may be a mechanism by which the inflammatory response is potentiated during infection.The Siglec 2 family of receptors consists of sialoadhesin (Siglec 1/Sn), CD22 (Siglec 2), CD33 (Siglec 3), myelin-associated glycoprotein (MAG or Siglec 4), and Siglecs 5-11. CD33-related Siglecs (Siglecs 5-11) are characterized by an N-terminal V-set Ig domain that mediates sialic acid binding and varying numbers of C2-set Ig domains. Sialic acids are a family of 9-carbon sugars that are derivatives of neuraminic acid or ketodeoxynonulosonic acid. The CD33-related Siglecs contain an ITIM and an ITSM in their cytoplasmic tails (1). The ITIM consists of a tyrosine with a leucine or valine at the ϩ3 position and a hydrophobic base at the Ϫ2 position ((I/L/V)XYXX(L/ V)), where X denotes any amino acid. The ITIM tyrosine is phosphorylated by Src family protein tyrosine kinases followed by recruitment of SH2-containing phosphatases such as the inositol polyphosphate 5-phosphatase, SHIP, and protein tyrosine phosphatases SHP-1 and SHP-2 to inhibit signaling (2). Engagement of Siglec 7 exerts an inhibitory signal mediated by tyrosine phosphorylation of the ITIM and recruitment of SHP-1 resulting in regulation of NK-cell-mediated target cell lysis, reduced phosphorylation of ZAP-70, and transcriptional activity of NFAT (nuclear factor of activated T cells) (4 -6). Cross-linking Siglec 7 positive chronic myeloid leukemia cells leads to an inhibitory effect on cell proliferation, suggesting that Siglec 7 may regulate normal myelopoiesis and could be a potential therapeutic target for chronic myeloid leukemia cells (7).Suppressor of cytokine signaling (SOCS) molecules inhibit the JAK/STAT pathway, ensuring that cytokine responses are regulated. SOCS proteins (CIS and SOCS1-7) contain an SH2 domain and a SOCS box, which binds Elongin B/C and Cul5/ Rbx1/2 (8). This complex can act as an E3 ligase and degrade associated proteins via the...

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