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. 2015 Sep 15;54(36):5589-604.
doi: 10.1021/acs.biochem.5b00623. Epub 2015 Sep 1.

Replication-Competent Influenza Virus and Respiratory Syncytial Virus Luciferase Reporter Strains Engineered for Co-Infections Identify Antiviral Compounds in Combination Screens

Dan Yan  1 , Marco Weisshaar  1 , Kristen Lamb  1 , Hokyung K Chung  2 , Michael Z Lin  3   4 , Richard K Plemper  1
Affiliations

Replication-Competent Influenza Virus and Respiratory Syncytial Virus Luciferase Reporter Strains Engineered for Co-Infections Identify Antiviral Compounds in Combination Screens

Dan Yan et al. Biochemistry. .

Abstract

Myxoviruses such as influenza A virus (IAV) and respiratory syncytial virus (RSV) are major human pathogens, mandating the development of novel therapeutics. To establish a high-throughput screening protocol for the simultaneous identification of pathogen- and host-targeted hit candidates against either pathogen or both, we have attempted co-infection of cells with IAV and RSV. However, viral replication kinetics were incompatible, RSV signal window was low, and an IAV-driven minireplicon reporter assay used in initial screens narrowed the host cell range and restricted the assay to single-cycle infections. To overcome these limitations, we developed an RSV strain carrying firefly luciferase fused to an innovative universal small-molecule assisted shut-off domain, which boosted assay signal window, and a hyperactive fusion protein that synchronized IAV and RSV reporter expression kinetics and suppressed the identification of RSV entry inhibitors sensitive to a recently reported RSV pan-resistance mechanism. Combined with a replication-competent recombinant IAV strain harboring nanoluciferase, the assay performed well on a human respiratory cell line and supports multicycle infections. Miniaturized to 384-well format, the protocol was validated through screening of a set of the National Institutes of Health Clinical Collection (NCC) in quadruplicate. These test screens demonstrated favorable assay parameters and reproducibility. Application to a LOPAC library of bioactive compounds in a proof-of-concept campaign detected licensed antimyxovirus therapeutics, ribavirin and the neuraminidase inhibitor zanamivir, and identified two unexpected RSV-specific hit candidates, Fenretinide and the opioid receptor antagonist BNTX-7. Hits were evaluated in direct and orthogonal dose-response counterscreens using a standard recRSV reporter strain expressing Renilla luciferase.

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Figures

Figure 1
Figure 1
Generation of a recIAV WSN-NanoLuc reporter strain. A) Schematic of the WSN PB2-NanoLuc or PB2-Gaussia genome segment (PS*: downstream packaging signal that was inactivated through silent mutagenesis; 2A PTV-derived cleavage site; NanoLuc or Gaussia: luciferase ORF; KDEL: ER retention signal; and PS: engineered packaging signal. Grey shading specifies the reading frame of the engineered segment. Individual segments are not drawn to scale. B) Reporter expression profile of IAV WSN-Gaussia on A549 cells (N = 3; means ± SD are shown). Instrument gain 250; RLU (relative luciferase unit) C) Signal window of the recIAV WSN-Gaussia and analogous recIAV WSN-NanoLuc reporter strains. A549 cells were exposed at infection to the potent inhibitor 5-iodotubercidin at 10 μM or the vehicle (DMSO) volume equivalent. RLUs were determined 48 hours post-infection. Values were normalized for vehicle controls (N = 3; means ± SD are shown); numbers above the columns show raw data means ± SD. Z’ and S/B values are specified below the graph. D) Reporter expression profiles of recIAV WSN-NanoLuc as in (B) after infection of A549 cells at two different MOIs (N = 3; means ± SD are shown). Instrument gain 135 for recIAV WSN-NanoLuc. The IAV WSN-Gaussia profile was added for comparison. E) recIAV WSN-NanoLuc and WSN-Gaussia are genetically stable over several passages. Progeny viral titers were determined by plaque assay (left panel) and TCID50 titration using luciferase activity as the read-out (right panel) after each passage (N = 3; means ± SD are shown).
Figure 2
Figure 2
Development of second-generation recRSV reporter strains. A) Schematic of the recRSV-L19FD489E-firefly and renilla luciferase genomes. B) Reporter expression profile after infection with recRSV-L19F-renilla or newly generated recRSV-L19FD489E-firefly or recRSV-L19FD489E-renilla (MOI 0.3 each; instrument gain 200). Values represent cell-associated luciferase activities and were normalized to the highest signal of each series (N ≥ 3; means ± SD are shown). Purification of recRSV-L19FD489E-firefly and recRSV-L 19FD489E-renilla progeny virions through different techniques. RLUs in virus stocks before and after purification were determined and background clearance (RLUbefore/RLUafter) calculated (N = 3; means ± SD are shown; 2-tailed t-test, *: p < 0.05). D) Signal window of the recRSV reporter strains. A549 cells were exposed at infection to 10 μM KUC109767, an inhibitor of RSV RdRp activity , or the vehicle (DMSO) volume equivalent. RLUs were determined 44 hours post-infection and values normalized for vehicle controls (N = 3; means ± SD are shown); numbers above the columns show raw data means ± SD.
Figure 3
Figure 3
recRSV-L19FD489E-fireSMASh allows induced reporter degradation. A) Schematic of the fireSMASh cassette inserted into the recRSV-L19FD489E genome (cleav: HCV NS3 cleavage site). B) Immunodetection of firefly luciferase after infection of cells with the specified recRSV-L19FD489E strain in the presence or absence of the NS3 inhibitor asunaprevir (ASV) and SDS-PAGE of cell lysates. Cellular GAPDH levels were determined as loading controls. C) Peak recRSV-L19FD489E-fireSMASh progeny titers after incubation in the presence of 3 μM ASV or vehicle (DMSO). (N = 3; means ± SD are shown). D) Immunodetection of firefly luciferase after serial passaging of recRSV-L19FD489E-fireSMASh and reinfection of cells in the presence or absence of 3 μM ASV. Passage 2 (P2) and passage 5 (P5) are shown, GAPDH levels were determined as loading controls. E) Firefly activity after growth of recRSV-L19FD489E-fireSMASh in the presence or absence of 3 μM ASV. Cells were infected at the specified MOIs and harvested 44 hours post-infection (N = 3; means ± SD are shown; 2-tailed t-test, **: p < 0.01; ***: p < 0.001). F) Signal window of the recRSV-L19FD489E-fireSMASh reporter strain was calculated as described in figure 2D N = 3; means ± SD are shown); numbers above the columns show raw data means ± SD. Z’ and S/B values are specified below the graph. G) Fold-change of contaminating firefly luciferase after gradient purification of recRSV-L19FD489E-firefly and recRSV-L19FD489E-fireSMASh preparation to unpurified recRSV-L19FD489E-firefly (N = 3; means ± SD are shown; 2-tailed t-test; **: p < 0.01).
Figure 4
Figure 4
Infection conditions for synchronized RSV and IAV reporter expression. A and B) Luciferase activities in three different human respiratory host cell lines 44 hours post-infection at the specified MOIs with recRSV-L19FD489E-fireSMASh (A) or recIAV WSN-NanoLuc (B; N = 4; means ± SD are shown). Two-way ANOVA with Tukey’s multiple comparison post-tests were carried out to assess statistical significance of sample divergence. Results are shown for MOI 0.1 (A) and 0.04 (B); *: p < 0.05; ***: p < 0.01. C-E) Reporter activity profiles after infection of BEAS-2B cells singly with recRSV-L19FD489E-fireSMASh (C) or recIAV WSN-NanoLuc (D), or after co-infection with both strains at an MOI of 0.1 (RSV) and 0.02 (IAV), respectively (E). Values represent cell-associated luciferase activities and were normalized to the highest signal of each series (N = 3; means ± SD are shown); grey shaded area in (D) marks the time window post-infection when signal intensities of both luciferase reporters are ≥80% of max.
Figure 5
Figure 5
Assay miniaturization and validation. A) Co-infection of BEAS-2B cells with recRSV-L19FD489E-fireSMASh and recIAV WSN-NanoLuc as specified in figure 4E in a 96-well plate format. Known RSV-specific (KUC109767 (10 μM) , GPAR3710 (10 μM) , and BMS-433771 (10 μM) ) IAV-specific (5-iodotubercidin (10 μM) ), and MeV-specific inhibitors (ERDRP-0519 (10 μM) , AS-48 (40 μM) ), broad-spectrum antivirals (ribavirin (40 μM) and JMN3-003 (10 μM) ), and cytotoxic cycloheximide (100 μg/ml) were used for assay validation (N = 5; means ± SD are shown). B) Co-infection assay parameters obtained in 96-well (manual; one plate each; N = 5; means ± SD are shown) and 384-well (automated; four plates each; N = 128; means ± SD are shown) format. The broad-spectrum myxovirus inhibitor JMN3-003 was used as a reference compound. AVR dynamic range: mean dynamic range across all replicate plates; ND: not determined. C) Z-score profiles of automated dual-pathogen pilot screens of the NCC collection in 384-well plate format in four replicates. Symbols mark Z-scores of individual replicate screens, solid black lines represent the assay Z-score mean, and dashed black lines show the hit cut-off (assay mean + 2.5 × (assay Z-score SD)). Final screening concentration was 5 μM. D) Individual Z-scores of the replicate (repl. I-IV) screens shown in (C) are plotted as a function of the mean %-inhibition for each compound. Dashed horizontal and vertical black lines show hit cut-offs based on Z-score (assay mean + 2.5 × (assay Z-score SD)) and biological effect (mean inhibition >75%), respectively. Numbers represent Pearson correlation coefficients (r) and 95% confidence intervals.
Figure 6
Figure 6
Test screen of a 1280-compound LOPAC library of known bioactives (overall Z’ = 0.31). A) Z-score profiles of the automated proof-of-concept screen of the LOPAC library in 384-well format. Solid green lines show Z-score means, dashed black lines hit cut-offs (assay mean + 2.0 × (assay Z-score SD) for recRSV A2-L19FD489E-fireSMASh, assay mean + 2.5 × (assay Z-score SD) for rec IAV WSN-NanoLuc). Final screening concentration was 5 μM. B) Dose-response assays of hit candidates in a concentration (conc.) range of 10-0.078 or 10-0.0006 μM. Only hits with CC50 concentrations ≥10 μM and confirmed inhibition of at least one primary target virus are shown. Values were normalized (norm.) for vehicle (DMSO)-treated infections and represent mean % inhibition or % cell viability (viab.) of three replicates ± SD. Regressions curves for antiviral (black) or cytotoxic (grey) activities are based on four-parameter modeling where applicable.
Figure 7
Figure 7
Overview of the replication-competent IAV and RSV reporter strain-based next-generation dual pathogen HTS protocol for the simultaneous identification of IAV-specific, RSV-specific, and broad spectrum inhibitors. The assay is validated for human respiratory BEAS-2B cells, but adaptable to all cell lines that are permissive for either virus strain. Infection at high MOI will predominantly identify inhibitors of viral entry and polymerase, while low MOI multi-cycle infections allow interrogation of all stages of the viral live cycle. Counterscreens are required to distinguish between hit candidates or reporter interfering compounds (specific antiviral activity), and hit candidates, reporter interfering compounds, cytotoxic compounds, or promiscuous pan-assay interfering (PAIN) compounds (broad spectrum activity).
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