Immune Netw.  2016 Oct;16(5):286-295. 10.4110/in.2016.16.5.286.

Enhanced Viral Replication by Cellular Replicative Senescence

• Kim JA
• Seong RK
• Shin OS

• KMID: 2355022
• DOI: http://doi.org/10.4110/in.2016.16.5.286

Abstract

Cellular replicative senescence is a major contributing factor to aging and to the development and progression of aging-associated diseases. In this study, we sought to determine viral replication efficiency of influenza virus (IFV) and Varicella Zoster Virus (VZV) infection in senescent cells. Primary human bronchial epithelial cells (HBE) or human dermal fibroblasts (HDF) were allowed to undergo numbers of passages to induce replicative senescence. Induction of replicative senescence in cells was validated by positive senescence-associated β-galactosidase staining. Increased susceptibility to both IFV and VZV infection was observed in senescent HBE and HDF cells, respectively, resulting in higher numbers of plaque formation, along with the upregulation of major viral antigen expression than that in the non-senescent cells. Interestingly, mRNA fold induction level of virus-induced type I interferon (IFN) was attenuated by senescence, whereas IFN-mediated antiviral effect remained robust and potent in virus-infected senescent cells. Additionally, we show that a longevity-promoting gene, sirtuin 1 (SIRT1), has antiviral role against influenza virus infection. In conclusion, our data indicate that enhanced viral replication by cellular senescence could be due to senescence-mediated reduction of virus-induced type I IFN expression.

Keyword

Senescence; Influenza; VZV; SIRT1

MeSH Terms

Aging
Cell Aging*
Epithelial Cells
Fibroblasts
Herpesvirus 3, Human
Humans
Influenza, Human
Interferon Type I
Orthomyxoviridae
RNA, Messenger
Sirtuin 1
Up-Regulation
Interferon Type I
RNA, Messenger
Sirtuin 1

Figure

• 

  Figure 1 Replicative senescence of primary human bronchial epithelial cells (HBE) is associated with dysregulated influenza virus replication. Induction of replicative (passage number–associated) senescence was confirmed by SA-β-galactosidase (gal) staining. (A) SA-β-gal staining was increased in senescent (S) HBE cells (right panel) relative to that of non-senescent (NS) cells (left panel). Magnification X200, Relative % SA-β-gal positive cells were counted and represented in the graph. (B) Assessment of mitochondrial superoxide generation was performed by mitoSOX RED staining. (C) NS and S cells were infected with the influenza virus strain PR8 at an MOI of 0.1 for 24 h. Progeny viral titers were calculated and expressed as % viral titers. Data are shown as means±SEM of three different experiments and are presented as the percentage of viral titer normalized to 100% in NS cells. Statistical analysis: *p<0.05 compared with 100% viral titers in NS cells. (D) Western blot analysis was performed in cell lysates of NS and S cells infected with PR8 virus at an MOI of 0.1 for 24 hours to assess the expression of senescence-associated p21 and influenza protein nucleoprotein (NP), nonstructural protein 1 (NS1) and actin. The blot shown is representative of three independent experiments. (E) NS and S cells were mock-infected or infected with PR8 for indicated length of time and western blotting was performed, similar to figure 1D.

• 

  Figure 2 Varicella Zoster Virus (VZV) replication efficiency in replication-induced senescent human dermal fibroblasts (HDF) cells. (A) SA-β-gal staining was increased in senescent (S) HDF cells (right panel) relative to that of non-senescent (NS) cells (left panel). Magnification X200, Relative % SA-β-gal positive cells were counted and represented in the graph. (B) Assessment of mitochondrial superoxide formation was performed by flow cytometry. MitoSOX Red stain revealed increased numbers of cells with superoxide in senescent HDF. (C) VZV progeny viral titers were calculated and expressed as plaque forming units (PFU)/mL. Virus titers in NS cells were normalized to 100% and relative virus titers were determined in S cells. Statistical analysis: ***p<0.001 compared with virus-infected NS cells. (D) HDF cells were mock-infected (m) or infected with the VZV for 24, 48 and 72 h. Protein levels of STING, pTBK-1, TBK-1 and p-IRF3 and IRF3 were analyzed by western blotting. Anti-actin monoclonal antibody was used as a loading control. VZV gE expression was also measured. The blot shown is representative of three independent experiments.

• 

  Figure 3 The effect of senescence on viral infection-induced IFN expression and IFN-mediated antiviral activities. Host antiviral gene expression was assessed by qRT-PCR. The expression of IFN-β and IFN-γ-Inducible Protein 10 (IP-10) in response to IFV infection (A) and IFN-β and interferon-stimulated gene 15 (ISG15) in response to VZV infection (B) was normalized to GAPDH and shown as fold induction level relative to mock (m) control. The results are representative of three independent experiments. Statistical analysis: *p<0.05, **p<0.01 compared with virus-infected NS cells. (C, D) Recombinant human IFN-α (10 ng/mL), IFN-β (10 ng/mL), IFN-λ1 (10 ng/mL) and IFN-λ2 (10 ng/mL) were added to cells. Next day, cells were infected with IFV or VZV and plaque assays were performed. Progeny viral titers were calculated and expressed as plaque forming units (PFU)/mL. Control-treated cells were normalized to 100% viral titers and data are presented as the percentage of control treated samples. Data are shown as means±SEM of two independent experiments.

• 

  Figure 4 Antiviral effect of anti-senescence gene SIRT1. (A) A549 cells were treated with 3 concentrations (1, 5, and 10 mM) of SIRT1 inhibitor nicotinamide (NAM) and class I HDAC inhibitor sodium butyrate (NaB) for 24 h, after which cell viability was measured using the MTT assay. Data are shown as a percentage (%) of the cell viability of the control cells. The average of 3 independent experiments is shown. Statistical analysis: *p< 0.05 vs. DMSO control-treated group. (B) Cells were treated with 1 mM NAM and 1mM NaB, infected with influenza A PR8 virus, and plaque assay was performed. (C) A549 cells were infected with rPR8-GFP virus (MOI of 1) for 1 h, after which drugs were added in the media for 24 h and GFP expression was monitored by confocal microscopy. Scale bar=20 µM(D) Control or SIRT1-specific siRNA was transfected in A549 cells and the knockdown efficiency was measured by realtime qRT-PCR. SIRT1 mRNA level was greatly reduced upon the mRNA silencing, whereas knockdown of SIRT1 led to upregulation of influenza A hemagluttinin (HA) mRNA expression. Transcript expression levels were calculated in relation to the expression level of GAPDH. Statistical analysis: *p<0.05 compared with virus-infected control siRNA-transfected cells. (E) Plaque assays were performed following SIRT1-specific siRNA transfection in A549 cells. The average of two independent experiments is shown. Statistical analysis: *p <0.05 vs. the control siRNA-transfected cell.

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