Normal human bronchial epithelial cells (HBE) were obtained from Lonza, Basel, Switzerland and grown in bronchial epithelial cell growth medium (BEGM) medium. Normal human dermal fibroblasts (HDF) (Lonza, Walkersville, MD, USA) were grown as adherent cultures in fibroblast basal medium supplemented with FGM SingleQuots (Lonza). Human lung adenocarcinoma cells (A549) and Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). A549 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin and maintained at 37℃ with 5% CO₂ in a humidified atmosphere. MDCK cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen) supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin.

Sodium butyrate (NaB), and nicotinamide (NAM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human interferon (IFN)-α, λ1, λ2 was purchased from R&D (Minneapolis, MI, USA) and interferon-β (IFN-β) protein was obtained from PBL Assay Science (Piscataway, NJ, USA).

Human influenza virus A/Puerto-Rico/8/34 (H1N1) PR8 and the recombinant PR8 virus expressing green fluorescent protein (GFP) (rPR8-GFP) were kindly provided by Dr. Adolfo Garcia-Sastre (Icahn School of Medicine at Mount Sinai, NY, USA) (17). Virus titers were determined by standard plaque assay in MDCK cells with few modifications. VZV strain YC01 (GenBank Accession No. KJ808816) has been described previously (18) and was cultured in HFFs, as described before (19).

Replicative cellular senescence induction models were used in this study. HBE cells were grown until 7~9 passages and HDF cells were allowed to grow until after 20~25 passages, and senescent cells were verified by their delayed population-doubling times and by using an SA-β-Gal activity assay kit (Cell Signaling Technology, Beverly, MA, USA), as described previously (20). Briefly, cells were washed twice with PBS and incubated in 1 mL of fixing solution at room temperature for 20 min. After removing the fixing solution and washing three times with PBS, the fixed cells were stained with 1 mL of freshly prepared cell staining working solution at 37℃ and protected from light for 24 h. After staining, the cells were counted for SA-β-galactosidase positive staining observed by light microscopy (Olympus, Tokyo, Japan).

Cells were stained with mitoSOX Red (Invitrogen) for 20 min and mitochondrial superoxide levels were measured by flow cytometry or confocal microscopy. For FACS analysis, cells were analyzed by a flow cytometer (Beckman Coulter, BC-FC500, Fullerton, CA, USA).

Protein lysates (30 µg total protein) were prepared using RIPA buffer (Sigma-Aldrich) and then separated by SDS-PAGE on 10~15% acrylamide gels and transferred to polyvinyldifluoride (PVDF) membranes. The membranes were then incubated in a blocking buffer comprised of 5% (w/v) BSA, 0.2 M Tris base, 1.36 M NaCl, and 0.1% Tween 20 (TBS/T) for 1 h at room temperature and washed three times with 5 mL of TBS/T for 5 min each wash. Membranes were incubated overnight with the primary antibodies against p21, STING, phospho-TBK, total TBK, p-IRF3, IRF3, β-actin (Cell Signaling Technology) at 4℃. For influenza A virus protein expression, anti-NS1 (Santa Cruz Biotechnology), and anti-NP antibodies (Sino Biological Inc., Beijing, China) were used. VZV gE antibody (Abcam, Cambridge, MA, USA) were used to measure the expression of VZV protein. After washing three times with TBS/T, the membranes were incubated with HRP-conjugated anti-rabbit or mouse IgG secondary antibody (Cell Signaling Technology) for 1 h at 25℃. After washing three times with TBS/T, membranes were incubated with Western Lumi Pico solution (ECL solution kit) (DoGen, Seoul, Korea), and exposed to film.

Total cellular RNA was prepared using TRIZOL reagent (Invitrogen). First-strand synthesis of cDNA from 1 µg of total RNA was performed using ImProm-II™ (Promega, Madison, WI, USA) according to the manufacturer's instructions. The changes in mRNA expression were calculated using the comparative Ct method as described previously (21). Data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. Primer sequences can be given upon request. Quantification of cDNA was performed by qRT-PCR using Power SYBR® Green Master Mix (Invitrogen). Cycling parameters were 95℃ for 10 min, followed by 40 cycles of 95℃ for 30 s and 60℃ for 1 min. The specificity of each reaction was validated by melt curve analysis and agarose gel electrophoresis of PCR products. Expression was normalized using the ΔCt method, in which the amount of target, normalized to an endogenous reference and relative to a calibrator, is given by 2^–ΔΔCt, where Ct is the cycle number of the detection threshold.

Cells were plated in 24-well plates and incubated overnight. Cells were washed twice with PBS and infected with rPR8-GFP virus at an multiplicity of infection (MOI) of 1.0 to allow for viral replication. Cells were infected with influenza in infection media followed by washing with PBS and culturing in RPMI-1640 medium with 2% FBS. After 24 h at 37℃ in 5% CO₂, nuclei were stained with 1 µg/ml of the nuclear stain 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 5 min. Coverslips were mounted on to glass slides and virus-infected cells were examined by confocal microscopy (LSM700; Carl Zeiss, Jena, Germany).

Cells were seeded in 6-well plates and allowed to grow to a confluency of 70% over 24 hrs. Transient transfections with either control scrambled or Sirt1 siRNA (Bioneer, Daejeon, Korea) were performed with Lipofectamine RNAiMAX reagent® (Invitrogen), according to the manufacturer's protocol as previously described.

Paired comparisons were performed using the Student's t-test. Differences were considered statistically significant at p<0.05. All analyses were carried out with the Prism® software program (GraphPad Software, Inc., La Jolla, CA, USA).

Replicative senescence was first described by Hayflick and Moorhead in human fibroblasts, after they observed cells undergoing extensive replication as a consequence of serial culture passages (22). Given that increased senescence-associated β-galactosidase (SA-β-gal) activity is a well-established biomarker of senescence (20), primary HBE cells were passaged up to 7~9 times until cells display senescent cell-like phenotype and positive SA-βgal staining cells were counted to validate senescence. As seen in Fig. 1A, replication-induced senescence in HBE cells resulted in increased numbers of cells positive for SA-β-gal staining, compared with that of non-senescent cells. We also observed that replicative senescence resulted in increased mitochondrial superoxide formation observed by mitoSOX staining (Fig. 1B).

We further examined whether the efficacy of influenza virus (IFV) replication differs between non-senescent (NS; passage number 2~4) and senescent (S; passage number 7~9) cell cultures. The reduced virus titers in the supernatants of IFV-infected cultures were confirmed by plaque assay. We detected increased plaque formation when analyzing the supernatants of infected cultures that had undergone replicative senescence (Fig. 1C). The protein levels of major viral proteins, nucleoprotein (NP) and nonstructural protein 1 (NS1), were also measured during senescence. Non-senescent and senescent HBE cells were infected with influenza A virus for 24 hours, and western blotting was performed. The expressions of senescence-associated markers, such as p21, were significantly higher in high passage cells than that in low passage cells. When compared with influenza-infected non-senescent cells, senescent cells had significantly elevated viral protein levels of NP and NS1 at 24 hours post-infection (hpi), suggesting that viral protein synthesis is more efficient in senescent cells than in non-senescent cells (Fig. 1D). Furthermore, Fig. 1E shows that there was delayed synthesis of viral protein NS1 in senescent cells, compared with that of non-senescent cells.

To investigate if replicative senescence has a modulating effect on other viral replication, we used human dermal fibroblasts (HDF) in culture which underwent extensive replication as a consequence of serial passaging. To confirm the senescence phenotype during replicative senescence, we compared non-senescent HDF at low passage with senescent HDF that had higher than 20 passages, following infection with VZV. Induction of senescence in highly passaged HDF was confirmed, with increased numbers of SA-β-gal-positive cells observed (Fig. 2A). Then, we also confirmed that replicative senescence of HDF cells led to increased numbers of cells with positive mitoSOX staining, regardless of VZV infection (Fig. 2B). Next, plaque assay was performed to determine whether the efficacy of VZV replication differed between non-senescent and senescent cells. Senescent HDF exhibited approximately 1.5-fold higher VZV infectivity titers than non-senescent HDF (Fig. 2C).

Given that VZV glycoprotein E (gE), encoded by ORF68, is the most abundant VZV glycoprotein and is required for viral replication in skin and T cells (23), we measured the protein synthesis of gE in VZV-infected HDF cells. At 24 hpi, gE levels were more increased in senescent HDF cells as compared to non-senescent HDF cells. Expressions of gE in VZV-infected senescent HDF cells continued to be more enhanced at 48 hpi, but were found to have decreased slightly at 72 hpi, whereas expression of gE in VZV-infected non-senescent HDF cells was the highest at 72 hpi, suggesting the delayed protein synthesis (Fig. 2D).

Our previous data suggested that Stimulator of Interferon Genes (STING) is important for host defense against VZV infection. Thus, expression levels of STING and its downstream signaling pathways, TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), were measured after VZV infection in HDF cells. Although mock-infected senescent cells showed higher basal expression levels of STING and p-IRF3 than mock-infected non-senescent cells, the induction levels of these proteins following VZV infection differed. Expression level of STING and phosphorylation of TBK-1 and IRF3 in non-senescent HDF cells appeared to be highly up-regulated after VZV infection at indicated times, compared with that in senescent HDF cells. These data suggest that viral infection-mediated host antiviral signaling pathways can be affected by replicative senescence (Fig. 2D).

To further characterize whether replicative senescence would have any effect on host antiviral gene expression patterns, we compared mRNA fold induction levels of IFN-β and interferon-associated genes, such as IFN-γ-Inducible Protein 10 (IP-10) during IFV infection and interferon-stimulated gene 15 (ISG15) in response to VZV infection, in non-senescent and senescent cells. There were approximately ~5 and ~7 fold increases in IFN-β and IP-10 mRNA levels, respectively, at early timepoints (8 hpi) in non-senescent HBE cells, whereas its fold induction level was lower in senescent HBE cells (Fig. 3A). Similar to IFV infection, we also measured transcriptional levels of IFN-β and ISG15 in VZV-infected cells. In comparison with VZV-infected non-senescent cells, we observed significantly elevated mRNA levels of VZV ORF14 in VZV-infected senescent cells at 24 hpi. (data not shown). Replicative senescence in HDF cells drove a significant down-regulation of IFN-β and ISG15 gene induction, compared with that of VZV-infected non-senescent cells (Fig. 3B). These findings suggest that the induction patterns of antiviral genes following viral infection differ between non-senescent and senescent primary human cells.

Following viral infection, cells begin to synthesize and secrete a range of antiviral molecules, including type I and III interferons, that are important for the recruitment of immune effector cells, activation of adaptive immunity, and attenuation of localized spread of virus infection by producing interferon stimulated genes (ISGs) (24). Most viruses are highly sensitive to interferon signaling and thus, the pre-treatment of cells with recombinant IFN proteins can result in the suppression of plaque formation. Recombinant human interferons α and β were used to represent type I IFNs, whereas interferons λ1 (IL-29) and λ2 (IL-28A) represented type III IFNs. Antiviral activities of these IFNs were measured by plaque assays, shown in Fig. 3C and D. Both type I and III IFN pre-treatments in non-senescent cells led to a significant reduction in plaque formation for IFV and VZV, compared with control-treated cells. Interestingly, antiviral effect of IFN-α and –β treatment resulted in greater reduction in plaque formation that that of IFN-λ1 or λ2 treatment in IFV-infected non-senescent cells. Moreover, type III IFN-mediated antiviral effect against IFV became attenuated in senescent cells, contributing to higher numbers of cells with plaque formation. For VZV infection, both type I and III IFN treatment led to 40~60% decrease in viral plaque formation, regardless of senescence levels of HDF cells. These data suggest that type I IFN-mediated antiviral responsiveness in senescent cells is not significantly affected by replicative senescence.

SIRT1 can be considered as the most well-known anti-aging gene known so far but the effect of SIRT1 during viral infection has not been fully investigated. Thus, we investigated whether modulation of SIRT1 activity would have an impact on influenza viral replication. Nicotinamide (NAM) was used as a SIRT1-specific inhibitor and sodium butyrate (NaB) was used as an inhibitor of histone deacetylation in cultured cells. Among SIRT family members 1~7, SIRT1 expression was specifically induced upon influenza virus infection at 24 hpi (Data not shown). Influenza-infected cells were exposed to the SIRT1 inhibitor NAM and class I HDAC inhibitor NaB and cell viability was measured. At 1mM concentration of NAM and NaB, no cellular toxicity was found in A549 cells (Fig. 4A). Exposure of cells to NaB and NAM following influenza infection resulted in increased numbers of viral plaques as well as viral replication measured by rPR8-GFP-expressing virus infection model (Fig. 4B and C). Furthermore, we examined the effect of SIRT1 knockdown on influenza hemagluttinin (HA) gene expression by qRT-PCR. SIRT1 expression was efficiently inhibited by siRNA treatment, and there was a significant increase in the mRNA expression of influenza HA upon SIRT1 knockdown (Fig. 4D). Consistent with these findings, the knockdown of SIRT1 also led to a significant increase in viral plaque formation (Fig. 4E). Thus, these results suggest that SIRT1 can contribute to antiviral effect against influenza virus infection.