Honokiol Inhibits SARS-CoV-2 Replication in Cell Culture at a Post-Entry Step

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019, and the resulting pandemic has already caused the death of over 6 million people. There are currently few antivirals approved for treatment of the 2019 coronavirus disease (COVID-19), and more options would be beneficial, not only now but also to increase our preparedness for future coronavirus outbreaks. Honokiol is a small molecule from magnolia trees for which several biological effects have been reported, including anticancer and anti-inflammatory activities. Honokiol has also been shown to inhibit several viruses in cell culture. In this study, we determined that honokiol protected Vero E6 cells from SARS-CoV-2-mediated cytopathic effect, with a 50% effective concentration of 7.8 μM. In viral load reduction assays, honokiol decreased viral RNA copies as well as viral infectious progeny titers. The compound also inhibited SARS-CoV-2 replication in the more relevant human A549 cells expressing angiotensin converting enzyme 2 and transmembrane protease serine 2. Time-of-addition and other assays showed that honokiol inhibited virus replication at a post-entry step of the replication cycle. Honokiol was also effective against more recent variants of SARS-CoV-2, including Omicron, and it inhibited other human coronaviruses as well. Our study suggests that honokiol is an interesting molecule to be evaluated further in animal studies and, when successful, maybe even in clinical trials to investigate its effect on virus replication and pathogenic (inflammatory) host responses. IMPORTANCE Honokiol is a compound that shows both anti-inflammatory and antiviral effects, and therefore its effect on SARS-CoV-2 infection was assessed. This small molecule inhibited SARS-CoV-2 replication in various cell-based infection systems, with up to an ~1,000-fold reduction in virus titer. In contrast to earlier reports, our study clearly showed that honokiol acts on a postentry step of the replication cycle. Honokiol also inhibited different recent SARS-CoV-2 variants and other human coronaviruses (Middle East respiratory syndrome CoV and SARS-CoV), demonstrating its broad spectrum of antiviral activity. The anticoronavirus effect, combined with its anti-inflammatory properties, make honokiol an interesting compound to be further explored in animal coronavirus infection models.

Keywords: MERS-CoV; SARS-CoV; SARS-CoV-2; antiviral agents; coronaviruses; honokiol; inhibitor.

FIG 1

Effects of honokiol on SARS-CoV-2-mediated cytopathic effect and viral replication in Vero E6 cells. (A) Vero E6 cells were treated with increasing concentrations of HK and then infected with SARS-CoV-2 at an MOI of 0.015. After 3 days, cell viability was measured by MTS assay. The viability of noninfected cells treated with compound was determined in parallel to assess cytotoxicity of the compound. Data points represent the means ± SEM from 3 independent experiments, and analysis was done with a two-way ANOVA test, followed by Bonferroni’s post hoc test. (B and C) Vero E6 cells were treated with increasing concentrations of HK and, after 6 h, were infected with SARS-CoV-2 at an MOI of 1. Supernatant samples were harvested at 16 hpi to quantify SARS-CoV-2 RNA levels by RT-qPCR (B) and infectious progeny titers by plaque assay (C). The means ± SEM from 4 independent experiments are shown. Data were analyzed with one-way ANOVA, followed by Bonferroni’s post hoc test. (D) Vero E6 cells were treated with increasing concentrations of HK and different assays were applied to assess toxicity of the compound. Data points represent the means ± SEM from 2 independent experiments. (E) Vero E6 cells were infected with SARS-CoV-2 at an MOI of 1 in the presence or absence of 20 μM HK. After infection, treated cells were incubated for 16 h in medium with or without HK. After fixation with 3% PFA, cells were analyzed by immunofluorescence microscopy with antibodies against dsRNA and nucleocapsid protein and Hoechst to stain nuclei. Uninfected cells (mock) were included as controls.

FIG 2

HK inhibits SARS-CoV-2 replication at a postentry step of the replication cycle. (A) Assessment of the virucidal potential of HK. A SARS-CoV-2 stock was incubated in medium (control) with 50 μM HK or 70% ethanol. The remaining infectious virus titers were determined by plaque assay and normalized to that of the untreated control (100%). The means ± SEM from 3 independent experiments are shown, and data were analyzed by one-way ANOVA and Bonferroni’s post hoc test. (B) Schematic representation of the time-of-addition assay, depicting the different treatment intervals during which infected Vero E6 cells were exposed to 20 μM HK. (C) At 10 hpi, supernatants were harvested and viral load was determined by quantifying extracellular viral RNA copies by RT-qPCR. Copy numbers were normalized to the level of untreated infected cells (100%). The means ± SEM from 3 independent experiments are shown, and data were analyzed by one-way ANOVA and Bonferroni’s post hoc test.

FIG 3

Effect of HK on the early steps of the SARS-CoV-2 replication cycle. Vero E6 cells were infected at an MOI of 5 and were either left untreated (Unt) or were treated with 20 μM honokiol (HK), 100 μM suramin (SR), or 25 μM remdesivir (RDV). At 2 hpi, intracellular viral RNA levels were quantified by internally controlled multiplex RT-qPCR. Copy numbers were normalized to the levels in untreated infected cells (100%). The means ± SEM from 3 independent experiments are shown, and data were analyzed by one-way ANOVA and Bonferroni’s post hoc test.

FIG 4

Effect of HK on SARS-CoV-2 replication in A549-ACE2-TMPRSS2 cells. (A) A549 cells expressing ACE2 and TMPRSS2 were infected with SARS-CoV-2 at an MOI of 1. Treatment with HK was initiated 6 h prior to infection, and the compound remained present until medium was harvested at 16 hpi to quantify extracellular viral RNA levels by RT-qPCR. Copy numbers were normalized to the level in untreated infected cells (100%). The means ± SEM from 3 independent experiments are shown, and data were analyzed by one-way ANOVA and Bonferroni’s post hoc test. (B) Compound toxicity was assessed in parallel by MTS assay on uninfected cells treated with HK.

FIG 5

Effect of HK on SARS-CoV-2 variants of concern. Vero E6 cells were treated with 10 or 20 μM HK for 6 h and subsequently infected with a SARS-CoV-2 variant at an MOI of 1, followed by incubation for 16 h in the presence of the compound. At 16 hpi, supernatant was harvested and RT-qPCR was used to quantify the extracellular viral RNA levels. Copy numbers were normalized to the level of untreated infected cells (100%). The means ± SEM from 3 independent experiments are shown, and data were analyzed by two-way ANOVA and Bonferroni’s post hoc test.

FIG 6

Effect of HK on various human coronaviruses. (A) Vero E6 cells were infected with SARS-CoV or SARS-CoV-2, and HuH-7 cells were infected with MERS-CoV or HCoV-229E at an MOI of 1. HK was added 6 h before infection and remained present until the time of harvest at 16 hpi for SARS-CoV, SARS-CoV-2, and MERS or at 24 hpi for HCoV-229E. Medium was collected and the levels of viral RNA were quantified by RT-qPCR. Copy numbers were normalized to the level in untreated infected cells (100%). The means ± SEM from 3 independent experiments are shown, as analyzed by two-way ANOVA and Bonferroni’s post hoc test. (B) A viability assay (MTS) was done in parallel to determine the compound’s cytotoxicity in Vero E6 and HuH-7 cells. The means ± SEM from 6 independent experiments are shown.