Auraptene Has Antiviral Activity against Human Coronavirus OC43 in MRC-5 Cells

Auraptene (7-geranyloxycoumarin) is the abundant prenyloxycoumarin found in the fruits of Citrus spp. Auraptene has a variety of pharmacological and therapeutic functions, such as anticancer, antioxidant, immunomodulatory, and anti-inflammation activities, with excellent safety profiles. In this study, we evaluated the anticoronaviral activity of auraptene in HCoV-OC43-infected human lung fibroblast MRC-5 cells. We found that auraptene effectively inhibited HCoV-OC43-induced cytopathic effects with 4.3 μM IC50 and 6.1 μM IC90, resulting in a selectivity index (CC50/IC50) of >3.5. Auraptene treatment also decreased viral RNA levels in HCoV-OC43-infected cells, as detected through quantitative real-time PCR, and decreased the expression level of spike proteins and nucleocapsid proteins in virus-infected cells, as detected through the Western blot analysis and immunofluorescence staining. Time-of-addition analysis showed auraptene’s inhibitory effects at the post-entry stage of the virus life cycle; however, auraptene did not induce the antiviral interferon families, IFN-α1, IFN-β1, and IFN-λ1. Additionally, auraptene-treated MRC-5 cells during HCoV-OC43 infection decreased the MMP-9 mRNA levels which are usually increased due to the infection, as auraptene is a previously reported MMP-9 inhibitor. Therefore, auraptene showed antiviral activity against HCoV-OC43 infection, and we suggest that auraptene has the potential to serve as a therapeutic agent against human coronavirus.


Introduction
Coronaviruses (CoVs) are the enveloped positive-sense single-stranded RNA viruses belonging to the Nidovirales order of the Coronaviridae family, which is divided into four genera, namely Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus, along with the seven strains of human infection CoVs [1]. Among the seven human coronaviruses (HCoVs), HCoV-NL63 and HCoV-229E are included in the Alphacoronavirus genus. Severe acute respiratory syndromes, (SARS)-CoV, and SARS-CoV-2, and Middle East respiratory syndrome (MERS)-CoV, as well as HCoV-OC43 and HCoV-HKU1, are included in the Betacoronavirus genus [2]. The coronavirus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2 infection, engendered a global health crisis that led to 770 million confirmed cases and 7 million deaths over three years as of the end of May 2023 [3]. Despite the development of several highly effective COVID-19 vaccines, many people are still vulnerable to virus infection and developing the disease due to the high rate of transmissibility and the frequent occurrence of concerning variants of SARS-CoV-2. The Food and Drug Administration approved Veklury ® (remdesivir) as the first antiviral treatment for COVID- 19 and Lagevrio™ (molnupiravir) and Paxlovid™ (ritonavir-boosted nirmatrelvir) for emergency use authorization during the COVID-19 pandemic, but additional antiviral therapeutics to

HCoV-OC43 Viral RNA Copy Number Analysis
Viral RNAs were harvested from the culture media supernatant of HCoV-OC43-infected MRC-5 cells using a QIAamp viral RNA mini kit (Qiagen N.V., Hilden, Germany), and viral RNAs from cell lysate of HCoV-OC43-infected MRC-5 cells were isolated using the RNeasy ® Mini Kit (Qiagen), following manufacturer's instructions. These RNAs were used to synthesize and amplify cDNA using a One Step TB Green ® PrimeScript™ RT-PCR kit (Takara Bio Inc., Kusatsu, Japan) and CFX Opus 96 Real-time PCR system (Bio-Rad Lab., Hercules, CA, USA) according to manufacturer's instructions with the following primers for the HCoV-OC43 Nucleocapsid protein: (sense primer) 5 -AGCAACCAGGCTGATGTCAATACC-3 and (antisense primer) 5 -AGCAGACCTTCCTGAGCCTTCAAT. A standard curve of HCoV-OC43 viral RNA was used to calculate the viral RNA copy number, as previously described [15].

Western Blot Assay
Cells were lysed in Glo lysis buffer (Promega), total cell lysates were separated on a 10% SDS-PAGE gel (Bio-Rad Lab), and proteins were transferred to a PVDF membrane (Bio-Rad Lab). The transferred membranes were incubated with 5% skim milk in TBST (TBS with 0.5% Tween20) for 30 min at 25 • C and washed three times with cold TBST. Thereafter, the anti-HCoV-OC43 spike protein antibody (Catalog Number CSB-PA336163EA01HIY, Cusabio, Houston, TX, USA), anti-HCoV-OC43 nucleocapsid protein antibody (Cat. No. MAB9012, Merck & Co., Inc., Rahway, NJ, USA), or anti-β-actin antibody (Cat. No. 3700S, Cell Signaling Technology, Danvers, MA, USA) was incubated at 4 • C overnight. The membranes were then washed with cold TBST and interacted with secondary antibodies conjugated with horseradish peroxidase (Abcam PLC, Cambridge, UK) for 1 h at 25 • C. Protein bands were observed using a Clarity Max Western ECL substrate (Bio-Rad) and the Chemidoc™ MP Gel Imaging System (Bio-Rad).

Immunofluorescence Staining Assay
MRC-5 cells were grown on poly-L-lysine-coated coverslip, fixed with 4% paraformaldehyde for 10 min and rinsed with cold PBS. Then, cells were permeabilized in 0.2% Triton X-100-contained PBS for 10 min and blocked with 3% BSA in permeabilizing buffer. Cells were incubated with anti-HCoV-OC43 spike protein antibody (CusaBio), or anti-HCoV-OC43 nucleocapsid protein antibody (Merck) at 4 • C overnight. Cells were washed with cold PBS three times and incubated with AlexaFluor488-conjugated goat anti-rabbit IgG antibody (Cat. No. A11001, Thermo Fisher Scientific Inc.) or AlexaFluor555-conjugated goat anti-mouse IgG antibody (Cat. No. A21428, Thermo Fisher, Waltham, MA, USA) for 1 h at room temperature. These cells were washed with PBS and mounted using SlowFade™ Gold Antifade Mountant with DAPI (Invitrogen, Waltham, MA, USA) and visualized using an Olympus IX71 fluorescence microscope (Olympus Corporation, Tokyo, Japan) and the cellSens program (Olympus).

Time-of-Addition Assay
MRC-5 cells were grown overnight at a density of 1 × 10 4 cells/96-well culture plate. As pretreatment, the cells were pretreated with the indicated concentration of auraptene for 4 h before HCoV-OC43 infection and then the compound was removed. Cells were infected with the virus and incubated for four days. For cotreatment, the compound was added with the virus for 4 h; then, cells were washed and incubated with the virus for four days. As posttreatment, cells were infected with the virus and after 4 h, the compound was added with the virus during four days. At 4 days postinfection, a cytopathic effect reduction assay was performed using an MTS assay kit (Promega).

Auraptene Inhibits the HCoV-OC43-Induced Cytopathic Effects
To investigate the anticoronaviral activity of auraptene ( Figure 1A), we examined its effect on HCoV-OC43-induced cytopathic effects in a human lung cell line, MRC-5 cells. Auraptene did not induce cytotoxic effects under 10 µM, although approximately 10% of cell death was induced by 15 µM of auraptene, suggesting 50% of cytotoxic concentration (CC 50 ) of >15 µM. When MRC-5 cells were infected with 10 4.5 TCID 50 /mL of HCoV-OC43, virus infection induced around 89% of cell death due to cytopathic effects at 4 days postinfection. However, treatment with serially diluted concentrations of auraptene reduced the HCoV-OC43-induced cytopathic effects dose-dependently ( Figure 1B). The 50% and 90% inhibitory concentration (IC 50 ) and (IC 90 ) values were 4.3 µM and 6.1 µM, respectively, as obtained via nonlinear regression analysis, resulting in a selectivity index (CC 50 /IC 50 ) of >3.5. Thus, we used up to 10 µM of auraptene for further experiments. In addition, we confirmed the complete protection of 10 µM auraptene from the HCoV-OC4-induced cytopathic effects based on cellular morphology. The data revealed that treatment with 10 µM auraptene effectively prevented cell death caused by HcoV-OC43-induced cytopathic effects ( Figure 1C, the third image, HCoV-OC43). Thus, the morphology of virus-infected cells treated with auraptene ( Figure 1C, the fourth image, HCoV w/ Auraptene) remained intact, resembling the morphology of non-infected cells ( Figure 1C, the first image, MOCK) and auraptene-treated cells ( Figure 1C, the second image, Auraptene). Therefore, these data showed that auraptene efficiently protected from HCoV-OC43-induced cytopathic effects.

Auraptene Inhibits HCoV-OC43 Viral RNA Replication and Viral RNA Expression
To examine the effect of auraptene on viral replication, intracellular and extracellular viral RNA levels were measured in HCoV-OC43-infected and 10 μM auraptene-treated MRC5 cells using qRT-PCR. The level of extracellular viral RNA increased up to 5 × 10 7 viral RNA copy number/μL, however, auraptene treatment decreased it to 8 × 10 6 viral RNA copy number/μL at 4 days postinfection. In addition, the intracellular viral RNA level peaked at 1.4 × 10 10 viral RNA copy number/μL at 2 days postinfection, and decreased with auraptene treatment to 1.2 × 10 9 viral RNA copy number/μL at 2 days postinfection ( Figure 2A). Moreover, the Western blot analysis showed that viral proteins spike (S, 145 kDa) and nucleocapsid protein (N, 50.4 kDa) could be detected in HCoV-OC43-infected cells but not in 10 μM auraptene-treated MRC5 cells between 2 and 4 days postinfection ( Figure 2B). Immunofluorescence staining analysis also showed that spike (S) and nucleocapsid protein (N) could be detected in HCoV-OC43-infected cells between 1 and 3 days postinfection but not in 10 μM auraptene-treated MRC5 cells (Figure 2C). Therefore, these data showed that auraptene inhibited viral replication and the expression level of viral proteins, suggesting the antiviral activity of auraptene against HCoV-OC43 infection.

Auraptene Inhibits HCoV-OC43 Viral RNA Replication and Viral RNA Expression
To examine the effect of auraptene on viral replication, intracellular and extracellular viral RNA levels were measured in HCoV-OC43-infected and 10 µM auraptene-treated MRC5 cells using qRT-PCR. The level of extracellular viral RNA increased up to 5 × 10 7 viral RNA copy number/µL, however, auraptene treatment decreased it to 8 × 10 6 viral RNA copy number/µL at 4 days postinfection. In addition, the intracellular viral RNA level peaked at 1.4 × 10 10 viral RNA copy number/µL at 2 days postinfection, and decreased with auraptene treatment to 1.2 × 10 9 viral RNA copy number/µL at 2 days postinfection ( Figure 2A). Moreover, the Western blot analysis showed that viral proteins spike (S, 145 kDa) and nucleocapsid protein (N, 50.4 kDa) could be detected in HCoV-OC43-infected cells but not in 10 µM auraptene-treated MRC5 cells between 2 and 4 days postinfection ( Figure 2B). Immunofluorescence staining analysis also showed that spike (S) and nucleocapsid protein (N) could be detected in HCoV-OC43-infected cells between 1 and 3 days postinfection but not in 10 µM auraptene-treated MRC5 cells ( Figure 2C). Therefore, these data showed that auraptene inhibited viral replication and the expression level of viral proteins, suggesting the antiviral activity of auraptene against HCoV-OC43 infection.

Auraptene Inhibited HCoV-OC43 Infection at the Post-Entry Infection Stage
To define the mechanism of auraptene's antiviral effects, a time-of-addition assay was conducted ( Figure 3A). In the pretreatment experiment, MRC-5 cells were pretreated with 10 µM auraptene for 4 h, washed, and then infected with HCoV-OC43 for 4 days. In the cotreatment experiment, MRC-5 cells were cotreated with 10 µM auraptene and HCoV-OC43 infection for 4 h. At 4 days postinfection, auraptene did not inhibit HCoV-OC43 infection based on cytopathic effects reduction data in the pre-and cotreatment experiments ( Figure 3B,C). In the posttreatment assay, MRC-5 cells were infected with HCoV-OC43 and then treated with 10 µM auraptene at 4 h postinfection. Auraptene was found to inhibit virus-induced cytopathic effects in the posttreatment assay ( Figure 3D). These data suggested that auraptene inhibited HCoV-OC43 infection after 4 h of infection, which corresponds to the post-entry stage.

Auraptene Inhibited HCoV-OC43 Infection at the Post-entry Infection Stage
To define the mechanism of auraptene's antiviral effects, a time-of-addition assay was conducted ( Figure 3A). In the pretreatment experiment, MRC-5 cells were pretreated with 10 μM auraptene for 4 h, washed, and then infected with HCoV-OC43 for 4 days. In the cotreatment experiment, MRC-5 cells were cotreated with 10 μM auraptene and HCoV-OC43 infection for 4 h. At 4 days postinfection, auraptene did not inhibit HCoV-OC43 infection based on cytopathic effects reduction data in the pre-and cotreatment experiments ( Figure 3B,C). In the posttreatment assay, MRC-5 cells were infected with HCoV-OC43 and then treated with 10 μM auraptene at 4 h postinfection. Auraptene was found to inhibit virus-induced cytopathic effects in the posttreatment assay ( Figure 3D). These data suggested that auraptene inhibited HCoV-OC43 infection after 4 h of infection, which corresponds to the post-entry stage.

Discussion
Auraptene is the most abundant prenyloxycoumarin in nature and commonly exists in the fruits of Citrus spp. [9]. Auraptene exhibits a variety of pharmacological and therapeutic properties, mediating host signaling pathways and inhibiting the functions of target proteins [10]. In this study, we showed auraptene's antiviral activity against HCoV-OC43 infection in human lung fibroblast cells, MRC-5, by effectively inhibiting virus-induced cytopathic effects and the expression level of viral RNA and viral proteins during HCoV-OC43 infection.
Previously, auraptene was shown to have antiviral activity against influenza virus A, H1N1 [13]. Although auraptene has previously been shown to inhibit enterovirus 71 infection at the attachment and entry step of the viral life cycle by targeting capsid proteins, VP1 and VP2, using a reverse genetic system [14], the time-of-addition assay of auraptene against HCoV-OC43 in our study showed that auraptene inhibited HCoV-OC43 infection at the post-entry stage. These results suggest that auraptene exhibits distinct modes of action in different viral infections.
Among the well-known modes of action of auraptene as an anti-tumor agent, it suppressed the expression of MMP-2, MMP-7, and MMP-9 proteins in colon cancer cells and colonic mucosa from colitis mice [17] and also repressed MMP-2 and -9 activity to inhibit the migration and invasion of cervical and ovarian cancer cells [18]. Moreover, auraptene inhibited the Porphyromonas gingivalis bacterial infection which causes periodontal diseases by reducing the secretion and activity of MMP-8 and MMP-9 [19]. Similar to these previous studies, we found that auraptene treatment suppressed MMP-9 expression in HCoV-OC43infected MRC-5 cells, and this suppression occurred in a dose-dependent manner, despite the induction of MMP-9 by HCoV-OC43 infection in a time-dependent manner.
In our study, we showed for the first time that HCoV-OC43 infection induced MMP-9 but not MMP-2 mRNA expression in MRC-5 cells. Virus infections have been shown to modulate MMP-9 expression. For instance, MMP-9 protein was increased by West Nile virus infection in the circulation and brain and was involved in its entry into the central nervous system (CNS) [20]. Similarly, human immunodeficiency virus Tat protein was found to upregulate MMP-9 expression, which can accelerate the trafficking of leukocytes into the CNS, resulting in the acquired immune deficiency syndrome dementia complex [21]. Dengue virus and Japanese encephalitis virus were both found to increase MMP-2 and MMP-9 expression and increase blood-brain barrier permeability, resulting in brain damage [22,23]. The upregulated MMP9 expression by Zika virus promotes virus entry into the testes by disrupting the blood-testis barrier [24]. Epstein-Barr virus oncoprotein induces MMP-9 expression, which may contribute to tumor invasion and metastasis [25].
In addition, MMP-9 is reported to modulate virus replication. Respiratory syncytial virus (RSV) infection induces MMP-9 expression in vitro and in vivo, whereas MMP-9 deletion decreases RSV replication [26]; however, MMP-9 also recruited neutrophiles resulting in the RSV clearance as antiviral activity in vivo [27]. A higher level of MMP-9 has been reported in influenza A virus-infected murine lung, whereas virus-infected MMP-9 deficient mice showed lower viral titer, being protected from lung disease by an effective immune response [28]. These data indicate that MMP-9 could be involved in HCoV-OC43 infection and replication. Moreover, Hepatitis B virus infection induces MMP-9 expression in immune cells, thereby evading host immunity by binding MMP-9 to IFN receptor I and blocking IFN signaling. This mechanism enables the virus to maintain a persistent infection [29]. Based on these results, it is possible that HCoV-OC43 infection also induces the expression of type I interferon genes, but the virus-induced MMP-9 proteins could potentially block the IFN signal, thereby evading the host's antiviral response in MRC-5 cells, which could be blocked by auraptene treatment. However, to validate this hypothesis, further studies are required.
Recently, it was reported that SARS-CoV-2 infection induced MMP proteins. SARS-CoV-2 infection in K18-hACE2-transgenic mice (K18-hACE2) increased the expression of MMP-8, MMP-9, and MMP-14 proteins in the lung, suggesting that SARS-CoV-2 infection is associated with the activity of the MMP family which may cause tissue damage [30]. Furthermore, MMP-9 levels increased while MMP-2 levels decreased in COVID-19 patients, which is consistent with HCoV-OC43 infection in MRC-5 cells. Moreover, the increased serum MMP proteins levels in COVID-19 patients may be useful biomarkers of severe COVID-19 [31,32]. Therefore, HCoV-OC43 infection may induce the expression of MMP-9 proteins, which can modulate virus replication and pathophysiology. However, the treatment with auraptene has the potential to inhibit these effects, making it a promising therapeutic agent.

Conclusions
In this study, we presented the antiviral effect of a natural prenyloxycoumarin, auraptene, against human coronavirus HCoV-OC43 infection by inhibiting virus-induced cytopathic effects, viral RNA replication, and viral protein expression. A time-of-addition assay suggested that auraptene inhibited HCoV-OC43 infection at the post-entry stage of the virus life cycle. Furthermore, auraptene decreased the induced MMP-9 expression caused by HCoV-OC43 infection in human lung fibroblast cells. Thus, auraptene could be a future and potential anticoronaviral therapeutic agent. Further research is needed to clarify the molecular mechanism of auraptene in detail to allow its development into a therapeutic treatment with an excellent safety profile.