A Marine Natural Product, Harzianopyridone, as an Anti-ZIKV Agent by Targeting RNA-Dependent RNA Polymerase

Abstract

The Zika virus (ZIKV) is a mosquito-borne virus that already poses a danger to worldwide human health. Patients infected with ZIKV generally have mild symptoms like a low-grade fever and joint pain. However, severe symptoms can also occur, such as Guillain-Barré syndrome, neuropathy, and myelitis. Pregnant women infected with ZIKV may also cause microcephaly in newborns. To date, we still lack conventional antiviral drugs to treat ZIKV infections. Marine natural products have novel structures and diverse biological activities. They have been discovered to have antibacterial, antiviral, anticancer, and other therapeutic effects. Therefore, marine products are important resources for compounds for innovative medicines. In this study, we identified a marine natural product, harzianopyridone (HAR), that could inhibit ZIKV replication with EC₅₀ values from 0.46 to 2.63 µM while not showing obvious cytotoxicity in multiple cellular models (CC₅₀ > 45 µM). Further, it also reduced the expression of viral proteins and protected cells from viral infection. More importantly, we found that HAR directly bound to the ZIKV RNA-dependent RNA polymerase (RdRp) and suppressed its polymerase activity. Collectively, our findings provide HAR as an option for the development of anti-ZIKV drugs.

Keywords: RdRp; ZIKV; antiviral drugs; harzianopyridone; marine natural products.

Figure 1

The structure of harzianopyridone (HAR).

Figure 2

The antiviral effects of HAR in SNB19, Vero, and A549 cells. (A) The ZIKV titers in SNB19, Vero, and A549 cells were quantified by real-time PCR (MOI = 0.2). The supernatant was obtained from cells treated with HAR at the indicated concentrations at 48 h post-infection (hpi). (B) Representative images of the plaque-forming assay. On Vero cells, the plaque-forming assay was used to confirm the ZIKV titers in the supernatant. The supernatant was obtained from SNB19, Vero, and A549 cells treated with HAR at the indicated concentrations at 48 h post-infection (hpi).

Figure 3

HAR inhibits the expression of ZIKV proteins. Western blotting analysis of protein expression of ZIKV NS5 and E in the cell lysates of SNB19, Vero, and A549 for the anti-ZIKV activity of HAR or DMSO at the indicated concentrations at 48 h post-infection (hpi) (MOI = 0.2).

Figure 4

HAR protects cells against ZIKV infection. (A) Immunofluorescence staining for ZIKV-E in ZIKV-infected cells (MOI = 0.2) under treatment by DMSO or 7.5 µM HAR. DAPI staining (blue) is used to color the cell nuclei. ZIKV-E protein is indicated in red (scale bar, 100 µm). (B) Quantification of ZIKV+ cells relative to mock infection is exhibited in the histogram counted by Image J software (1.50i NIH, Bethesda, MD, USA). * p < 0.05.

Figure 5

HAR directly targets the ZIKV RdRp. (A) HAR inhibited the activity of the ZIKV NS5 protein. 293T cells were co-transfected with the Gluc reporter and the ZIKV NS5 plasmid, as well as treated with different concentrations of HAR for 24 h. The culture medium was collected for luminescence examination. The transfection efficiency of NS5 was detected by a Western blotting assay. Used GAPDH as a loading control. * p < 0.05, *** p < 0.001. The symbol “+” means inclusion, and “−” means exclusion. (B) The effects of HAR on Gluc activity. 293T cells were transfected with Gluc reporter plasmid, followed by treatment with HAR (12.5, 25 or 50 µM) or 1% DMSO as a control. Determined the Gluc activities after transfection at about 24 h. “ns” indicates not significant. Experiments were performed in triplicate. (C,D) The surface plasmon resonance biosensor (SPR) assay was performed to examine the interaction between ZIKV NS5 (RdRp) protein and HAR (C). Ribavirin (D) was used as a negative control. BIAcore T100 analysis software (BIAevaluation Version 3.1) was used to analyze the K_d values.