A cell-based screening system for anti-influenza A virus agents

Emerging of drug resistant influenza A virus (IAV) has been a big challenge for anti-IAV therapy. In this study, we describe a relatively easy and safe cell-based screening system for anti-IAV replication inhibitors using a non-replicative strain of IAV. A nickel (II) complex of polyhydroxybenzaldehyde N4-thiosemicarbazone (NiPT5) was recently found to exhibit anti-inflammatory activity in vivo and in vitro. NiPT5 impedes the signaling cascades that lead to the activation of NF-κB in response to different stimuli, such as LPS and TNFα. Using our cell-based screening system, we report that pretreating cells with NiPT5 protects cells from influenza A virus (IAV) and vesicular stomatitis virus (VSV) infection. Furthermore, NiPT5 inhibits replication of IAV by inhibiting transcription and translation of vRNAs of IAV. Additionally, NiPT5 reduces IAV-induced type I interferon response and cytokines production. Moreover, NiPT5 prevents activation of NF-κB, and IRF3 in response to IAV infection. These results demonstrate that NiPT5 is a potent antiviral agent that inhibits the early phase of IAV replication.

replication inhibitors. A non-replicative PR8 strain IAV carrying eGFP in the place of the PB1 gene (PR8-PB1flank-eGFP) (Fig. 1a) was used to infect A549 cells that stably expressing PB1 protein (A549-PB1). The eGFP reporter gene was flanked by minimum sequences required for efficient and stable packaging into virions 10 . Upon PR8-PB1flank-eGFP viral infection and replication, infected A549-PB1 cells will express the eGFP reporter. Thus the IAV infection can be easily monitored and measured by fluorometry (Fig. 1b), fluorescent microscopy ( Fig. 1c), and fluorescentactivated cell sorting (FACS) (Fig. 1d). A549-PB1 cells were stained with ER-tracker as an internal control for fluorometry measurement (Fig. 1b). In all cases, the fluorescent signal correlates very well with the amount of PR8-PB1flank-eGFP virus we used for infection.
Identification of NiPT5 as an antiviral agent. We previously showed that NiPT5 inhibits TNF-and LPS-induced activation of NF-kB 9 . Induction of IFNb upon virus infection requires the activation of NF-kB 11 . Thus, NiTP5 may render cells sensitive to virus infection and is an ideal compound to test our cell-based screening system. Prior to understand the effect of NiPT5 on IAV infection, we determined the cytotoxic effect of NiPT5 in A549-PB1  cells with a standard MTT assay. After two days incubation, the IC 50 of NiPT5 was 45.3 mM in A549-PB1 cells (Fig. 2a).
To test if NiPT5 renders cells sensitive to virus infection, we pretreated A549-PB1 cells with different concentrations of NiPT5 for four hours and infected the cells with PR8-PB1flank-eGFP virus at a multiplicity of infection (MOI) of 1.0. Pre-treating the A549-PB1 cells with NiPT5 did not render the cells more sensitive to IAV infection ( Fig. 2b and 2c). In contrary, NiPT5 protected the cells from IAV infection in a dose dependent manner. Pretreating A549-PB1 cells with 5 mM of NiPT5 reduced GFP positive cells (Fig. 2b) and significantly decreased the total fluorescent signal by 23% (Fig. 2c p 5 0.036). IAV infection was completely abolished at 15 mM or higher concentration of NiPT5. The antiviral activity of NiPT5 was not due to the cytotoxicity of NiPT5, since cell proliferation was unaffected at 5 mM and only slightly decreased at 10 mM ( Fig. 2a and S1a). Furthermore, we showed that NiPT5 was working in A549-PB1 cells since it inhibited TNF-induced activation of NF-kB in A549-PB1 cells (Fig. S1b). Pretreating A549-PB1 cells with  NiPT5 dramatically inhibited the induction of several NF-kB target genes, including IP10, CCL5 and IL1b in response to TNFa stimulation. These results suggest that NiPT5 shows antiviral activity against IAV and our cell-based provides a quick and easy way to screen for anti-IAV agents.
To test if NiPT5 exerts antiviral activity against other virus, we pretreated A549-PB1 cells with NiPT5 for four hours and infected A549-PB1 cells with a vesicular stomatitis virus carrying a GFP (VSV-GFP) at a MOI of 1.0. In the DMSO treated control A549-PB1 cells, VSV-GFP infection resulted in greater than 90% of GFP positive cells (Fig. 2d). NiPT5 reduced GFP positive cells ( Fig. 2d and S1c) and the total fluorescent signal (Fig. 2e) in a dose-dependent manner. This finding suggests that NiPT5 protects cells from VSV-GFP infection.
NiPT5 reduces IAV replication. To test whether NiPT5 inhibits viral replication, we investigated IAV polymerase activity by measuring the expression of viral RNAs (vRNAs) with RT-qPCR. A549-PB1 cells were pretreated with NiPT5 at various concentrations for four hours and were then infected with PR8-PB1flank-eGFP virus at a MOI of 1.0. The expression of influenza vRNAs, NP, NS and M1, were measured with RT-qPCR twelve hours post-infection. The expression of NP, NS and M1 was readily detected in PR8-PB1flank-eGFP virus infected DMSO-treated control A549-PB1 cells (Fig. 3a). On the other hand, NiPT5 inhibited the transcription of NP, NS and M1 in a dose-dependent manner. For example, at the concentration of 10 mM of NiPT5, the expression of NP, NS and M1 was reduced by 98%. To determine whether NiPT5 affects the influenza virus protein level, whole cell extracts were prepared twelve hours post-infection and the NP protein level was measured by immunoblotting with antibody against NP protein. NP protein was detected in PR8-PB1flank-eGFP virus infected A549-PB1 cells but not detected in the presence of 10 mM of NiPT5 (Fig. 3b). This result indicates that NiPT5 blocks viral protein synthesis.
To explore whether NiPT5 influences the replication of virus, we measured viral titer in the culture media collected from PR8-PB1flank-eGFP virus infected A549-PB1 cells in the presence or absence of NiPT5. We quantified infected cells by sorting the virus-infected, eGFP-positive cells using FACS. Consistent with fluorescent microscopy analysis (Fig. 2b), NiPT5 considerably inhibited IAV infection (Fig. 3c, left panel). Next, supernatants from infected A549-PB1 cells were collected and subjected to virus titering assay using FACS. We found that there was about 20-fold reduction of new virus generated in the presence of NiPT5 (Fig. 3c, right panel). Taken together these results suggested that NiPT5 inhibits IAV replication.

NiPT5 inhibits IAV-induced interferon and cytokine expression.
Upon viral infection, host cells will mount the type I interferon response and produce cytokines to induce innate immunity against virus. To test if NiPT5 inhibits virus infection through enhancement of antiviral responses in host cells, we measured the expression of type I interferon response genes (ISGs) and cytokines upon IAV infection with or without pretreating A549-PB1 cells with NiPT5. First we checked the gene expression kinetics of several vRNAs, ISGs and cytokines under our experiment setting. We infected A549-PB1 cells with PR8-PB1flank-eGFP virus at a MOI 1.0 and measured gene expression with RT-qPCR at the indicated time points. The expression of vRNAs was detected as early as 1 hour post-infection (Fig. 4a). Most of the ISGs and cytokines, including IFNb, IFIT2, TNFa, and IL6 were detected two hours post-infection. The expression of late genes, IP10 and CCL5, was detected twelve hours post-infection. The expression of ISGs and cytokines at twelve hours post-infection was substantially reduced in the presence of 10 mM of NiPT5 and was abolished in 15 mM of NiPT5 treated cells (Fig. 4b). These results imply that NiPT5 does not protect cells from IAV infection by enhancing antiviral responses in the host cells.
NiPT5 inhibits IAV induced NF-kB and IRF3 activation. NF-kB and IRF3 pathways mainly regulate the production of virus-induced ISGs and cytokines. To examine if NiPT5 inhibits IAV induced NF-kB and IRF3 activation, we measured the phosphorylation of IkBa (p-IkBa) and the phosphorylation of IRF3 (p-IRF3), a biochemical hallmark of NF-kB and IRF3 activation respectively. The p-IkBa was detected at 30 minutes post-infection (Fig. 4c), while the p-IRF3 was detected twelve hours post-infection ( Fig. 4d and S2). Pretreating A549-PB1 cells with NiPT5 greatly reduced IAV-induced phosphorylation of IkBa (Fig. 4c). Furthermore, IAV-induced phosphorylation of IRF3 was inhibited by NiPT5 in a dose-dependent manner (Fig. 4d). These results suggest that NiPT5 inhibits IAV-induced activations of NF-kB and IRF3.

Discussion
NiPT5 was first characterized as a potent anti-inflammation agent by inhibiting activation of NF-kB induced by TNFa and LPS stimulations 9 . In this study, NiPT5 shows antiviral activity against IAV and VSV (Fig. 2). NiPT5 inhibits the transcription of IAV vRNAs and the expression of IAV NP protein (Fig. 3). Moreover, NiPT5 does not protect the A549-PB1 cells from virus infection by enhancing the intrinsic antiviral response in the host cells. NiPT5 however inhibits virus-induced activation of NF-kB and IRF3 (Fig. 4).
Type I interferon response is the key intrinsic antiviral pathway blocking type I interferon response will render cells susceptible to virus infection 12

. We observed that NiPT5 inhibits type I interferon response but does not sensitize cells to virus infection suggesting that NiPT5 may block viral replication at a step before the detection of virus infection by the host cells. IAV is detected by host cells through
TLRs, RIG-I, and NLRP3 pathways 13 . Its genomic ssRNA is detected by TLR7/8 in the lumen of endosome 14,15 , and by RIG-I in the cytosol 16 . NLRP3 is activated through influenza M2 mediated disturbances of intracellular ionic concentrations 17 . A549 cells do not express TLR7/8 18 and NLRP3 does not induce type I interferon response 19 . As a result, detection of influenza virus infection by A549-PB1 cells mainly relies on the RIG-I pathway. The activation of RIG-I requires the release of influenza genomic ssRNA from endosome to cytoplasm. Thus NiPT5 may inhibit IAV replication at or before the release of influenza genomic ssRNA from the endosome, including attachment of virus particle to cell surface receptor or internalization through virus-host cell fusion. However, we cannot rule out the possibility that NiPT5 inhibits both IAV replication as well as IAV-induced IRF3 and NF-kB activation. Further experiments are required to understand the molecular mechanism of NiPT5mediated antiviral infection.
In conclusion, we succeeded in using a nonreplicative PR8 strain of influenza virus carrying eGFP in the place of the PB1 gene (PR8-PB1flank-eGFP) and A549-PB1 cells to demonstrate the antiviral activity of NiPT5. The PR8-PB1flank-eGFP virus is lacking the PB1 gene and thus will not replicate in normal cells. With the incorporation of an eGFP reporter, the infected cells can be quantified either using FACS or a fluorometer. This system is suitable for screening inhibitors that block IAV replication from any step of the viral life cycle. Moreover, this system can be adopted for high throughput screening for anti-influenza virus agents with the incorporation of an automated liquid handling system together with a fluorometer or an automated fluorescent microscope or a FACS.
Cell proliferation assay. A549-PB1 cells were treated with or without various concentrations of NiPT5 in a 96 wells format. After incubation for two days, 10 ml of 2 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in DMEM medium was added and cells were further incubated for three hours at 37uC in a CO 2 incubator. Cells were spun down at 2500 rpm for 5 minutes and the medium was carefully removed. One hundred and fifty microliters of DMSO was added to each well. After pipetting up and down several times, the absorbance was measured with a M200 PRO microplate reader (Tecan) at the wavelength of 540 nm. IC 50 was calculated with GraphPad Prism 6.
RT-PCR. A549-PB1 cells pretreated with or without NiPT5 for 4 hours were infected with PR8-PB1flank-eGFP virus at a multiplicity of infection (MOI) of 1.0 for 12 hours. Total RNAs were isolated with the Thermo Scientific GeneJET RNA Purification Kit. Complementary DNAs were synthesized and Quantitative PCR was performed with 2X SYBR Green PCR Master mix (Thermo Scientific) and run on a Bio-Rad Connect Real-Time PCR System. All data were then normalized to L32. The sequences of the primers are listed in table S1.
Virus amplification. PR8-PB1flank-eGFP virus was amplified by infecting 239T-PB1 cells. Prior to virus infection, 293T-PB1 cells were washed once with PBS and then changed into influenza growth media (RPMI supplemented with 0.2% bovine serum albumin, 0.01% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 1 mM calcium chloride) containing 2 mg/ml TPCKtreated trypsin. After 48 hours the supernatant was harvested through a 0.45 mm filter. The virus titer was determined by FACS (see below).
Virus infection. Ten thousand A549-PB1 cells per well were seeded in a 96-well flat plate. After overnight incubation, cells were washed once with PBS and then changed into influenza growth media containing 0.2 mg/ml TPCK-treated trypsin. PR8-PB1flank-eGFP virus was added at a MOI of 1.0. After 24 hours, cells were analyzed with an Olympus IX73 inverted microscope at 2003 final magnification and photographed using the Olympus DP73 digital camera and Cellsens standard software. The total fluorescent signal was measured with a Tecan Infinite F200 Pro fluorometer.
Ten thousand A549-PB1 cells per well were seeded in a 96-well flat plate. After overnight incubation, the cells were pre-treated with different concentrations of NiPT5 as indicated for 4 hours and then infected with vesicular stomatitis virus (VSV) carrying a GFP (VSV-GFP) at a MOI of 1.0 for 14 hours.
Cells were also stained with 5 mM ER-tracker Red (Life technology) according to manufacturer's protocol. The total fluorescent signal was measured with a Tecan Infinite F200 Pro fluorometer, and used as an internal control to normalize eGFP or GFP fluorescent readings.
Virus titering. Virus titer was determined as previous described with slight modification 10 . Briefly, A549-PB1 cells were pretreated with or without 10 mM NiPT5 for 4 hours and then infected with PR8-PB1flank-eGFP virus at a MOI of 0.5 in influenza growth media plus 2 mg/ml TPCK-treated trypsin. Twenty-four hours later the supernatants were collected and filtered with 0.45 mm filters. The viruses in the collected supernatants were tittered by infecting fresh A549-PB1 cells in influenza growth media plus 0.2 mg/ml TPCK-treated trypsin. Low concentration of TPCK-  www.nature.com/scientificreports treated trypsin was used to ensure no infectious IAV was produced to avoid any secondary infection ( Figure S3). After 24 hours, cells were washed with PBS and fixed with 0.1% formaldehyde to inactivate virus. Fixed cells were analyzed using a MACSQuant Analyzer (Miltenyi Biotec) to quantified eGFP positive cells. Data were further analyzed with FlowJo software. Total infectious virus particle (IP) was calculated based on the following formula: IP/ml 5 (% of eGFP positive cell) X (total cell number)/(total volume of supernatant used to infect the cells, in ml). One IP per cell was used when infecting cells at MOI of 1.0.
Immunoblotting. Two hundred thousand A549-PB1 cells were seeded. After overnight incubation, culture media were removed and changed into IAV infection media. The cells were pre-treated with different concentration of NiPT5 for 4 hours followed by PR8-PB1flank-eGFP virus infection at a MOI of 1.0 for 12 hours. Whole cell lysate extracts were prepared from treated cells using a kinase lysis buffer (20 mM Tris pH7.5, 120 mM NaCl, 10% glycerol, 1% Triton X-100, 25 mM bglycerophosphate, 1 mM sodium orthovanadate, 1 mM DTT, and 1 mM PMSF). After 30 minutes incubation on ice, lysates were collected by centrifugation at 15 k rpm 4uC for 10 minutes. Immunoblotting was performed using anti-p-p38, anti-NP, anti-p-IRF3, and anti-HSP90a antibodies. Western blot images were captured using ChemiDoc TM MP System (Bio-Rad). Images were processed using BioRad Image Lab version 5.0 and Adobe Photoshop CS4.
Statistical analysis. Data were analyzed by Microsoft Excel and presented as the mean 6 SD. Data are representative of three or more independent experiments. Statistical significance was assessed by use of the Student's t-test.