Viruses harness YxxØ motif to interact with host AP2M1 for replication: A vulnerable broad-spectrum antiviral target

Abstract

Targeting a universal host protein exploited by most viruses would be a game-changing strategy that offers broad-spectrum solution and rapid pandemic control including the current COVID-19. Here, we found a common YxxØ-motif of multiple viruses that exploits host AP2M1 for intracellular trafficking. A library chemical, N-(p-amylcinnamoyl)anthranilic acid (ACA), was identified to interrupt AP2M1-virus interaction and exhibit potent antiviral efficacy against a number of viruses in vitro and in vivo, including the influenza A viruses (IAVs), Zika virus (ZIKV), human immunodeficiency virus, and coronaviruses including MERS-CoV and SARS-CoV-2. YxxØ mutation, AP2M1 depletion, or disruption by ACA causes incorrect localization of viral proteins, which is exemplified by the failure of nuclear import of IAV nucleoprotein and diminished endoplasmic reticulum localization of ZIKV-NS3 and enterovirus-A71-2C proteins, thereby suppressing viral replication. Our study reveals an evolutionarily conserved mechanism of protein-protein interaction between host and virus that can serve as a broad-spectrum antiviral target.

Fig. 1. Intracellular trafficking pathways are vulnerable and druggable for antiviral intervention.

(A) Time-course responses of cell fate determination pathways upon infection of different viruses. Luciferase reporter gene assays reflecting the activity of Wnt, TGF-β, Notch, and PI3K/Akt signaling were performed on the pdmH1N1-infected HBTEC, ZIKV-infected human fibroblasts, and EV-A71–infected human neural progenitor cells, respectively. The results are shown as a heatmap. (B) RNA interference (RNAi) screening identified genes essential for pdmH1N1 virus replication. Left: Experimental workflow that was performed in human lung epithelial A549 cells with 0.1 MOI virus infection. Right: Among 142 membrane and cellular trafficking genes, individually knockdown of 17 genes (black dots) can suppress virus replication for >1-log magnitude when comparing with the scramble–small interfering RNA (siRNA) control. (C) Small-molecule compound library screening identified ACA as a broad-spectrum antiviral in vitro. A membrane transporter/cellular trafficking library was primarily screened in pdmH1N1-infected Madin-Darby canine kidney (MDCK) cells (0.01 MOI and 48 hpi) through cell protection assays (box 1, blue dots indicated >90% cell viability), followed by secondary screening using viral load reduction assays (box 2, magenta dots indicated >3 logs of viral load reduction applying 10 μM drug concentration) and tertiary screening (box 3, yellow dots indicated >2 logs of viral load reduction using 1 μM drug concentration). ACA was prioritized due to its broad-spectrum antiviral efficacy. Shown in the last panel are the antiviral effects against six different viruses as indicated. Shown are the plaque-forming unit (PFU) or 50% tissue culture infectious dose (TCID₅₀) or OD₄₅₀ (optical density at 450 nm) value of indicated concentrations relative to controls in the absence of compound (%).

Fig. 2. Anti–influenza activity of ACA in vitro and in vivo.

(A) ACA reduced influenza A NP–positive MDCK cells as quantitated by flow cytometry. (B) ACA exhibited cross-subtype anti–influenza activity including influenza A H5N1, H7N7, H7N9, and H9N2 viruses. **P < 0.01 by one-way analysis of variance (ANOVA). (C) ACA suppressed pdmH1N1 replication in HBTECs (0.01 MOI). One-way ANOVA was used for comparison with the dimethyl sulfoxide (DMSO)–treated group (0 μM). *P < 0.05. (D) ACA (10 μM) showed anti–influenza activity in differentiated human AOs. Shown is the intracellular viral gene copies normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). ***P < 0.001 by Student’s t test. (E) Representative 2D images of human AOs, after immunofluorescence staining for viral NP (green), 4′,6-diamidino-2-phenylindole (DAPI) (blue, for nucleus), and Phalloidin (magenta, for cellular membrane), were examined with confocal microscopy. Scale bars, 20 μm. (F to I) ACA provided full protection against lethal challenge with influenza A H1N1 (black lines) or H7N9 (red lines) viruses. The mice were treated with intranasal ACA (open triangles), zanamivir (open circle), or 0.5% DMSO (closed squares) for one dose starting 1 hour after challenge. (F) Survival and clinical disease were monitored for 14 days or until death. ***P < 0.001 by log-rank (Mantel-Cox) tests. (G) Daily body weights of surviving mice. (H) Lung tissues (n = 5 per group) were collected for detection of viral titers using plaque assay at 2 and 4 dpi, respectively. A value of 10 to 15 PFU/ml was assigned for any titer below the detection limit (the dotted line). (I) Representative histological sections of lung tissues (4 dpi) from the indicated groups with hematoxylin and eosin (H&E) staining. Greater alveolar damage and interstitial inflammatory infiltration were present in the DMSO group. Magnification: ×200.

Fig. 3. ACA shows broad-spectrum antiviral activity in vivo.

(A to D) ACA conferred protection against lethal challenge of ZIKV. Four- to 6-week-old A129 mice were inoculated subcutaneously with 1 × 10⁶ PFU of ZIKV-PR under anesthesia. Each mouse received one dose of subcutaneously administered ACA (1 mg/kg) or 0.5% DMSO in PBS at 1 hpi. The mice were monitored daily for (A) survival rate and (B) body weight change. (C) Five mice in each group were euthanized at 6 dpi, and brain tissues were harvested for vial titer determination by plaque assay. The dotted line indicates the lower detection limit of plaque assay (***P < 0.001; for the purpose of statistical analysis and clarity, a value of 10 to 15 PFU/ml was assigned for any titer below the detection limit). (D) Histopathologic and immunohistochemistry (IHC) analyses of the brain samples at 6 dpi indicated less severe meningitis (by H&E, ×200) and less virus infected cells as indicated by ZIKV NS1 antigen staining (red arrows, by IHC, ×200 magnification) after ACA treatment. (E to H) ACA protected human dipeptidyl peptidase 4 (hDPP4) transgenic mice from MERS-CoV infection. The hDPP4 mice were intranasally inoculated with 500 PFU of MERS-CoV and intranasally treated by ACA or 0.5% DMSO for one dose starting 1 hpi. Shown are (E) survival rate, (F) mean body weight, (G) lung viral titer at 2 dpi (n = 5 per group), and (H) representative lung tissues stained by H&E and anti–MERS-CoV-NP immunofluorescence. The staining suggested less inflammatory cell filtration (by H&E, ×200 magnification) and less virus infected cell antigens (by immunofluorescence (IF) staining, green fluorescence) as detected in ACA-treated mouse lungs. Results are presented as mean values ± SD. Differences in survival rates were compared using log-rank (Mantel-Cox) tests and viral titer by Student’s t test. ***P < 0.001, **P < 0.01, *P < 0.05.

Fig. 4. ACA targets host AP2M1 protein.

(A) ACA blocked the initiation of pdmH1N1 virus replication cycle. Cell lysate were collected for virus mRNA (blue lines), vRNA (magenta lines), and cRNA (black lines) quantification as indicated. Student’s t test for each RNA type and corresponding time point. (B) ACA inhibited nuclear import of the IAV vRNA. Synchronized H1N1 infection were performed on MDCK cells (50 MOI). Cells were fixed at the indicated time points and hybridized with RNA probes against the IAV negative-stranded NP vRNA (red) and stained for DNA (blue), examined by confocal microscopy. Images are representative of three independent experiments. Scale bars, 10 μm. (C and D) Click chemistry/WaterLOGSY/protein ID (CWID) platform for identification of drug-binding targets. (C) Click-chemistry: chemical structure of azido-ACA showing the location of azido group (green circle) on ACA. Cellular distribution of azido-ACA is shown (green), whereas ACA was used as a negative control due to the lack of phosphine-reactive azido group. Scale bars, 50 μm. (D) WaterLOGSY-guided cellular fractionation was subjected to analysis for ACA-featured NMR spectra. “*” and “***” indicate mild and strong binding signals, respectively. The native polyacrylamide gel electrophoresis gel photo shows the selected cell fraction as detected by a fluorescent image analyzer. Red arrow indicates the specific azido-ACA–binding fragment. (E) Mutagenesis analysis of AP2M1 to rescue pdmH1N1 virus replication against ACA. Full-length AP2M1 (full), longin-like domain (LLD), MHD, and mutant AP2M1 were transfected to MDCK cells before virus infection and ACA treatment. One-way ANOVA. **P < 0.01; n.s, not significant. (F) Partial sequence alignment of human, mouse, and dog AP2M1 is shown. N217 and K410, the key residues for ACA binding, are highlighted with a box. The predicted interaction surfaces on AP2M1 (red) are shown, while ACA (green) is displayed in stick and mesh representation.

Fig. 5. Host AP2M1 is exploited by multiple viruses via the viral protein YxxØ motif.

(A) A summary of virus protein YxxØ motif interacting with host AP2M1 proteins. (B) Competitive ELISA showing the blockade of AP2M1 and biotin-YxxØ peptide after ACA addition. The low binding affinity mutant D176A was taken as a control. One-way ANOVA. *P < 0.05 and **P < 0.01. (C) A known AP2M1-YxxØ blocker Tyrphostin A23 exhibited broad-spectrum antiviral activity. Shown are the antiviral effects against four different viruses as indicated. One-way ANOVA when compared with the 0 μM group (0.1% DMSO). (D) CRISPR knockout of AP2M1 reduced pdmH1N1, EV-A71, ZIKV, and MERS-CoV replication. Viral load in the cell lysate (n = 3) was evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Student’s t test. (E) AP2M1^−/− and WT 293T cells were treated with CHX before virus infection (10 MOI). Nuclear (Nuc) and cytoplasmic (Cyto) fractions were separated and detected at 2 hpi by Western blotting. (F) A549 cells transfected with GFP influenza–NP and mCherry-AP2M1 were incubated with DMSO or ACA for 24 hours. Live cell imaging was performed, and motile AP2M1/NP puncta were tracked (movies S1 and S2). Shown is the average velocity of trackable puncta within the overall distance traveled. Student’s t test. (G) AP2M1 facilitates the viral protein localization. Synchronized infections were used throughout the experiments. Colocalization was quantified using ImageJ (JACoP) colocalization software and Manders’ colocalization coefficients (MCCs). Bar charts indicate mean MCC values represented as percent colocalization (the fraction of green intensity that coincides with blue intensity in the case of IAV-NP/nucleus and the fraction of green intensity that coincides with red intensity in the case of EV-A71-2C/ER and ZIKV-NS3/ER) ± SD (error bars, n = 10 to 15). Scale bars, 10 μm. ***P < 0.001 by Student’s t test.

Fig. 6. Host AP2M1-mediated virus trafficking is essential to initiate their replication cycles.

(A) Effects of virus NP-YxxØ substitutions on virus growth and replication. Recombinant viruses were subject to multiple-cycle replication assays in A549 cells . N.D indicates failed rescue in three independent experiments. (B) Effects of virus NP-YxxØ substitutions on IAV NP nucleus import. A549 cells were treated with CHX before infection with WT and mutant viruses (10 MOI). Nuclear (Nuc) and cytoplasmic (Cyto) fractions were separated at 2 hpi for Western blotting to quantify the NP amount. Human β-actin and Lamin A were used for normalization of Cyto and Nuc, respectively. (C) Disruption of host AP2M1/ virus NP binding abolished the replication and transcriptional activity of influenza A polymerase. Shown are the relative polymerase activities with AP2M1 knockout (magenta bars) or NP mutants on YxxØ sites (blue bars). AP2M1 KD efficiency and NP overexpression were detected by Western blotting. Student’s t test . (D to F) Growth of WT and mutant (Y296A) GFP virus in a mouse model. BALB/c mice were intranasally infected with 10⁵ or 10⁴ PFU of the indicated viruses. Shown are the (D) survival rate and (E) body weight change. (F) Three mice from each 10⁵ PFU-infected group were euthanized on 1, 3, and 5 dpi for analysis of in vivo dynamics after GFP virus infection. (G) Proposed model for host AP2M1-mediated intracellular trafficking of different viruses. Various viral proteins as indicated are commonly recruited by the mu subunit of host membrane trafficking AP2 adaptor complex (i.e., AP2M1 or μ2) through recognizing the viral YxxØ motif, while ACA disrupts these distinct steps of the viral life cycle.