Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 24 (7), 430-438

Sendai Virus C Protein Limits NO Production in Infected RAW264.7 Macrophages

Affiliations

Sendai Virus C Protein Limits NO Production in Infected RAW264.7 Macrophages

Erdenezaya Odkhuu et al. Innate Immun.

Abstract

To suppress virus multiplication, infected macrophages produce NO. However, it remains unclear how infecting viruses then overcome NO challenge. In the present study, we report the effects of accessory protein C from Sendai virus (SeV), a prototypical paramyxovirus, on NO output. We found that in RAW264.7 murine macrophages, a mutant SeV without C protein (4C(-)) significantly enhanced inducible NO synthase (iNOS) expression and subsequent NO production compared to wild type SeV (wtSeV). SeV 4C(-) infection caused marked production of IFN-β, which is involved in induction of iNOS expression via the JAK-STAT pathway. Addition of anti-IFN-β Ab, however, resulted in only marginal suppression of NO production. In contrast, NF-κB, a primarily important factor for transcription of the iNOS gene, was also activated by 4C(-) infection but not wtSeV infection. Induction of NO production and iNOS expression by 4C(-) was significantly suppressed in cells constitutively expressing influenza virus NS1 protein that can sequester double-stranded (ds)RNA, which triggers activation of signaling pathways leading to activation of NF-κB and IRF3. Therefore, C protein appears to suppress NF-κB activation to inhibit iNOS expression and subsequent NO production, possibly by limiting dsRNA generation in the context of viral infection.

Keywords: Accessory proteins; NO; double-stranded RNA; macrophages; paramyxovirus.

Conflict of interest statement

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Effect of NO on SeV multiplication. Following treatment with the indicated concentrations (a, b) or 30 μM (c) SNAP, one h before infection (b, upper panel, and c) or one h after infection (b, lower panel), Vero cells in a 96-well plate were mock-infected or infected with wtSeV at MOI 0.001 and incubated in 3 μg/ml trypsin. Culture media were collected 24 h postinfection and assayed for nitrite (a). Cells were also stained 72 h postinfection with 0.5% w/v amido black 10B dissolved in 20% ethanol and 10% acetic acid (b) or immunoblotted with rabbit serum against SeV (c). Viral proteins P, N, and M are marked in (c). SeV: Sendai virus; SNAP: S-nitroso-N-acetyl-DL-penicillamine; wt: wild type.
Figure 2.
Figure 2.
NO and iNOS production in RAW264.7 cells infected with SeV 4C(–). (a–d) Cells were mock-infected or infected with SeV strains at MOI 5 (a, c, d), or as indicated (b). Culture media were collected 24 h postinfection (a, b) or at the indicated time points (c) and assayed for nitrite. Cells were also harvested at the indicated time points and immunoblotted with anti-iNOS (d). (e) Cells were transfected with pCA7HA vector with (CSeV) or without (Empty) C protein, along with pRL-TK or piNOS-Luc, a firefly luciferase reporter plasmid driven by the iNOS promoter. At 24 h posttransfection, cells were mock-infected or infected with SeV 4C(–) at MOI 5, harvested eight h thereafter, and assayed by a dual-luciferase assay system to evaluate activation of the iNOS promoter. *P < 0.01 vs infection with wtSeV (a), vs mock infection (b,c), or vs pCA7HA empty vector.iNOS: inducible NO synthase; SeV: Sendai virus; TK: thymidine kinase; wt: wild type.
Figure 3.
Figure 3.
Role of IFN-β in NO and iNOS production in RAW264.7 cells infected with SeV 4C(–). (a–c) Cells were mock-infected or infected with wt or SeV 4C(–) at MOI 5. Culture media were collected 24 h postinfection and assayed for IFN-β (a), whereas cells were harvested at five h (b) or 24 h postinfection (c) and immunoblotted with Abs to unphosphorylated and phosphorylated STAT1 and STAT2. (d) Cells were infected with SeV 4C(–) at MOI 5 and incubated for 24 h in the presence or absence of neutralizing Abs to IFN-β. Culture media were then assayed for nitrite. Cells were transfected with pRL-TK or pISRE-Luc, a firefly luciferase reporter plasmid driven by the ISRE promoter. At 24 h posttransfection, cells were mock-infected or infected with SeV 4C(–) at MOI 5, harvested after 8 h, and assayed by a dual-luciferase assay system to evaluate activation of the iNOS promoter. (e, f) Cells were mock-infected or infected with wtSeV at MOI 5. Cells were then mock-treated or treated with 103 or 104 IU/ml IFN-β for 20 h, beginning at 4 h postinfection. Culture media were then assayed for nitrite (e), whereas cells were immunoblotted with anti-iNOS (f). *P < 0.01 vs mock treatment with a-IFN-B antibody (d) or vs treatment with 104 IU/ml IFN-B after mock infection (e).iNOS: inducible NO synthase; ISRE: IFN-stimulated response element; SeV: Sendai virus; TK: thymidine kinase; wt: wild type.
Figure 4.
Figure 4.
Activation of NF-κB in RAW264.7 cells infected with SeV 4C(–). (a, b) Cells were mock-infected or infected with wt or SeV 4C(–) at MOI 5. Culture media were collected 5 h (a) and 24 h (b) thereafter and assayed for TNF-α and IL-6, whereas cells were harvested at 5 h (d) and 24 h (c) and immunoblotted with Abs to phosphorylated p65, p65, and COX-2. (e) Cells were mock-treated or treated with Bay 11-7082. (Bay) for 30 min and then infected with SeV 4C(–) at MOI 5 for 24 h. Culture media were then assayed for nitrite (upper panel), whereas cells were immunoblotted with rabbit serum against SeV (lower panel). Viral proteins P, N, and M are marked in the lower panel. *P < 0.01 vs infection with wtSeV (a, b) or vs mock treatment with Bay (e). COX-2: cyclooxygenase-2; SeV: Sendai virus; wt: wild type.
Figure 5.
Figure 5.
Effect of influenza virus NS1 on NO production induced by SeV 4C(–). (a) RAW264.7 cells were mock-infected or infected with wt or SeV 4C(–) at MOI 5. At 4 h postinfection, cells were mock-treated or treated with 100 ng/ml LPS, harvested 24 h (a) thereafter, and immunoblotted using Abs to COX-2. (b) RAW264.7 cells stably expressing Flag-tagged NS1 (RAW-NS1) were immunoblotted with anti-Flag Ab. (c, d) RAW264.7 cells stably transfected with the empty vector (RAW-empty) or the vector expressing NS1 protein (RAW-NS1) were mock-infected or infected with SeV 4C(–) at MOI 5. Culture media (c) and cells (d) were collected 24 h postinfection and assayed for nitrite and iNOS, respectively. *P < 0.01 vs cells stably transfected with the empty vector (RAW-empty). COX-2: cyclooxygenase-2; NS1: nonstructural protein 1; SeV: Sendai virus; wt: wild type.

Similar articles

See all similar articles

References

    1. Vareille M, Kieninger E, Edwards MR, et al. The airway epithelium: Soldier in the fight against respiratory viruses. Clin Microbiol Rev 2011; 24: 210–229. - PMC - PubMed
    1. Villalón-Letelier F, Brooks AG, Saunders PM, et al. Host cell restriction factors that limit influenza A infection. Viruses 2017; 9. - PMC - PubMed
    1. Cline TD, Beck D, Bianchini E. Influenza virus replication in macrophages: Balancing protection and pathogenesis. J Gen Virol 2017; 98: 2401–2412. - PMC - PubMed
    1. Regev-Shoshani G, Vimalanathan S, McMullin B, et al. Gaseous nitric oxide reduces influenza infectivity in vitro. Nitric Oxide 2013; 31: 48–53. - PubMed
    1. Abdul-Cader MS, Amarasinghe A, Abdul-Careem MF. Activation of toll-like receptor signaling pathways leading to nitric oxide-mediated antiviral responses. Arch Virol 2016; 161: 2075–2086. - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback