Sendai virus C protein inhibits lipopolysaccharide-induced nitric oxide production through impairing interferon-β signaling
Introduction
Lipopolysaccharide (LPS) is present on the outer membranes of Gram-negative bacteria and known to cause the systemic inflammatory response syndrome, septic shock and multi-organ failure [1]. LPS leads to the production of large amounts of proinflammatory mediators, tumor necrosis factor (TNF)-α, interleukin (IL)-1 and 6, and nitric oxide (NO). A large amount of NO is produced by the expression of an inducible NO synthase (iNOS) [2], [3] and the excessive NO release is involved in LPS-induced cell death and tissue injury [4], [5]. LPS triggers the NO production through activating both MyD88-dependent and independent pathways after the binding to toll like receptor (TLR)-4 [6]. In particular, LPS-induced interferon (IFN)-β production in the MyD88-independent pathway is a key event and correlated with the transcription of iNOS gene, the level of iNOS protein expression, and finally, the production of NO [7], [8], [9]. The signaling molecules regulating LPS-induced NO production are potential targets for prevention of LPS-mediated inflammatory response and endotoxic shock.
Paramyxovirus employs accessory proteins affecting viral life cycle [10], [11]. Sendai virus (SeV), a prototype paramyxovirus, possesses V and C accessory proteins that are involved in the virus replication and pathogenicity [10], [11], [12], [13], [14]. The accessory proteins, such as C and V protein, are reported to interact with various host proteins in infected cells [15], [16], [17], [18]. Thus, SeV accessory proteins may modulate the cell function of infected cells, such as intracellular signaling, transcription, proliferation and cell death [15], [16], [17], [18]. Interestingly, we found that SeV inhibited LPS-induced NO production in macrophages. In the present study, we investigated the inhibitory action of SeV on LPS-induced NO production. Here, we report that SeV C accessory protein inhibits LPS-induced NO production through impairing IFN-β signaling.
Section snippets
Reagents
LPS from Escherichia coli O55:B5 was purchased from Sigma Chemicals (St Louis, MO, USA). Puromycin was obtained from Gibco-Invitrogen (Carlsbad, CA, USA). Antibodies to p65 nuclear factor (NF)-κB (#8242), phosphorylated p65 NF-κB (p-Ser276; #3037), extracellular stimulus-activated kinase (ERK) 1/2 (#9102), phosphorylated ERK1/2 (p-Thr202/Tyr204, #9101), p38 (#9212), phosphorylated p38 (p-Thr180/Tyr182, #9216), c-jun N-terminal kinase (JNK) (#9258), phosphorylated JNK (p-Thr183/Tyr185, #9251),
The effect of SeV infection on LPS-induced NO production
The effect of SeV infection on LPS-induced NO production was examined (Fig. 1). RAW 264.7 cells were infected with SeV at various multiplicity of infection (MOI) and then stimulated with LPS (100 ng/ml) for 24 h (Fig. 1A). Infection of SeV at 7 MOI markedly reduced LPS-induced NO production (% reduction, 63.6 ± 10.5, n = 9). SeV at 0.7 MOI but not 0.07 MOI significantly reduced the NO production. Next, the effect of pretreatment or post-treatment of SeV infection on LPS-induced NO production was
Discussion
In the present study we have demonstrated that SeV C protein inhibits LPS-induced NO production through reduced iNOS expression and that the inhibition is responsible for impairement of IFN-β signaling by C protein. Several lines of evidence suggest that C accessory protein exclusively impairs IFN-β signaling in response to LPS: first, wild type SeV but not C gene knockout 4C(−) SeV inhibits the phosphorylation of STAT1/2 in response to LPS; second, wild type and 4C(−) SeV does not inhibit
Acknowledgment
We thank Y. Kitagawa and T. Sugiyama for providing pCA7HA and pCA7HA-C, and piNOS-Luc and pIFN-β-Luc, respectively. This work was supported in part by a Grant-in-Aid (25460551) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan (MEXT) and a grant of MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2011-2015 (S1101027).
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