A Small-Molecule Oligosaccharyltransferase Inhibitor with Pan-flaviviral Activity

Abstract

The mosquito-borne flaviviruses include important human pathogens such as dengue, Zika, West Nile, and yellow fever viruses, which pose a serious threat for global health. Recent genetic screens identified endoplasmic reticulum (ER)-membrane multiprotein complexes, including the oligosaccharyltransferase (OST) complex, as critical flavivirus host factors. Here, we show that a chemical modulator of the OST complex termed NGI-1 has promising antiviral activity against flavivirus infections. We demonstrate that NGI-1 blocks viral RNA replication and that antiviral activity does not depend on inhibition of the N-glycosylation function of the OST. Viral mutants adapted to replicate in cells deficient of the OST complex showed resistance to NGI-1 treatment, reinforcing the on-target activity of NGI-1. Lastly, we show that NGI-1 also has strong antiviral activity in primary and disease-relevant cell types. This study provides an example for advancing from the identification of genetic determinants of infection to a host-directed antiviral compound with broad activity against flaviviruses.

Keywords: West Nile virus; Zika virus; antiviral; dengue virus; flaviviruses; host-directed therapy; oligosaccharyltransferase; yellow fever virus.

Figure 1. Targeting the OST complex with a small molecule (NGI-1) inhibits infection with mosquito-borne flaviviruses

(A) Schematic depiction of the oligosaccharyltransferase complex interacting with flaviviral replication complexes. (B) Dose-response curve of NGI-1 for DENV2 luciferase infection in HEK293 cells 48hpi. (C) Dose-response curve of NGI-1 for ZIKV-luciferase infection in HEK293 cells 48hpi. (D) Plaque-forming assay for DENV2 and ZIKV PRVABC59 harvested from infected HEK293 cells untreated and treated with 8 μM NGI-1 for 48h. (E) Immunofluorescence of DENV2- or ZIKV PRVABC59-infected Huh7 cells with or without NGI-1 treatment for 48h. Infection was assessed by fluorescence microscopy of stained viral envelope protein (green) and cell nuclei (DAPI). Scale bar is 70 μm. (F) Proliferation assay for HEK293 cells treated with different NGI-1 concentrations for 48h. (G–J) Quantitative RT-PCR for RNA of the four different DENV serotypes (DENV1-4), different ZIKV strains, YFV and WNV, and Chikungunya (CHIKV), Venezuelan equine encephalitis (VEEV) and Polio virus (PV), untreated and treated with 8 μM NGI-1 for 48h. In all figures, experiments were performed 2 or 3 times with 3 biological replicates in each experiment, and one representative is shown, Values are shown as mean ± SD in (B)–(F), and as mean ± SEM in (G)–(J). P-values are defined as ∗ p < 0.05; ∗∗ p < 0.01; *** p < 0.001.

Figure 2. NGI-1 blocks viral RNA replication and antiviral effect is independent of inhibiting the N-linked glycosylation activity of the OST complex

(A) Time course of electroporated DENV replicon expressing Renilla luciferase in untreated or NGI-1 treated HEK293 cells. (B) Quantitative RT-PCR for ZIKV PRVABC59 RNA in HAP1 cells expressing either WT or catalytically inactive STT3A, untreated or treated with NGI-1. (C) Immunoblot analysis of DENV prM, E and NS1 proteins from lysates of DENV infected cells under treatment of different NGI-1 concentrations for 48h. (D) Replication of DENV expressing Renilla luciferase in WT Huh7 cells, isogenic MAGT1 KO cells, and MAGT1 KO cells complemented with wt MAGT1, catalytically dead MAGT1, mCherry vector or empty vector. In all figures, experiments were performed 2 or 3 times with 3 biological replicates in each experiment, and one representative is shown, Values are shown as mean ± SD in (A) and (D), and as mean ± SEM in (B). P-values are defined as ∗ p < 0.05; ∗∗ p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3. DENV adaptation reveals that NGI-1 directly acts on STT3A and STT3B for its antiviral activity

(A) Sequencing of DENV passaged on STT3A- or STT3B-KO cells revealed four distinct mutations in viral genomes. 1 and 3 viruses were independently isolated for STT3B-KO and STT3A-KO adaptations, respectively. (B) Quantitative RT-PCR for DENV RNA of WT and STT3A- and STT3B-adapted viruses untreated or treated with NGI-1. The experiment was performed 3 times with 3 biological replicates each, and one representative is shown. Values are shown as mean ± SEM and p-value is defined as ∗∗ p < 0.01. (C) Replication of dengue luciferase reporter virus with WT genomic sequence or containing the four adaptive mutations in HEK293 cells treated with different concentrations of NGI-1, Tunicamycin or MK-0608. The experiment was performed 2 times with 3 biological replicates each, and one representative is shown. Values are shown as mean ± SD.

Figure 4. NGI-1 has antiviral activity against DENV and ZIKV in disease-relevant and primary cell types

(A–G) Replication of dengue or Zika luciferase virus (purple) as measured by luciferase activity and cellular proliferation (black) of Normal Human Dermis Fibroblasts (NHDF), monocyte-derived dendritic cells (MoDC), Raji DC-SIGN lymphocytes, JEG-3 placental cells and human Neural Progenitor Cells (hNPC) treated with different concentrations of NGI-1. Note that cells used in proliferation assays were uninfected, and that same proliferation data is used in Fig. 4A and 4D for NHDF, and in Fig. 4B and 4E for MoDC. (H) Immunofluorescence of ZIKV-infected JEG-3 and hNPC, and DENV-infected NHDF cultured with and without NGI-1. Infection was assessed by fluorescence microscopy of stained viral envelope protein (green) and cell nuclei (DAPI). Scale bar is 70 μm. (I) Percentage of DENV-infected Raji DC-SIGN (untreated or treated with NGI-1) as measured by flow cytometry. In all figures, experiments were performed 2 or 3 times with 3 biological replicates in each experiment; one representative is shown. Values are shown as mean ± SD and p-value is defined as ∗∗∗∗ p < 0.0001.