Antiviral Activity of trans-Hexenoic Acid against Coxsackievirus B and Enterovirus A71

ABSTRACT

Enterovirus infections are life-threatening viral infections which occur mainly among children and are possible causes of viral outbreak. Until now, treatment and management of infections caused by members of the genus Enterovirus largely depended on supportive care, and no antiviral medications are currently approved for the treatment of most of these infections. The urgency of discovering new therapeutic options for the treatment of enterovirus infection is increasing. In the present study, we identified that trans-2-hexenoic acid (THA), a natural product from a dietary source, possesses antiviral activity against coxsackievirus B (CVB) and enterovirus A71 (EV-A71) in a dose-dependent manner. We found that THA possesses antiviral activity at 50% effective concentrations (EC₅₀) of 2.9 μM and 3.21 μM against CVB3 and EV-A71 infections, respectively. The time of addition assay revealed that THA inhibits both CVB3 and EV-A71 replication at the entry stage of infection. Additional results from this study further suggest that THA inhibits viral replication by blocking viral entry. Given that THA has received approval as a food additive, treatment of enterovirus infections with THA might be a safe therapeutic option or could pave the way for semisynthetic manufacturing of more antiviral drugs in the future.

INTRODUCTION

Enteroviruses (EVs) are important human pathogens that belong to the family Picornaviridae (1). Most enterovirus infections do not cause significant illness. However, infections with some human enteroviruses, in particular, polioviruses (the causative agent of acute flaccid paralysis), coxsackievirus B (CVB) (the leading pathogen of viral myocarditis), and enterovirus A71 (EV-A71) and coxsackievirus A16 (CVA16) (the viruses associated with hand, foot, and mouth disease [HFMD]), can lead to serious illness in children and young adults (2).

Members of the genus Enterovirus possess a positive single-stranded RNA (+ssRNA), which encodes four structural proteins (VP1 to VP4) and seven nonstructural proteins, including two proteases (2A^pro and 3C^pro) and an RNA-dependent RNA polymerase (3D^pol) (2). These nonstructural proteins are involved in viral replication and polyprotein processing. They are important targets for the development of antiviral agents (3). Other possible virus replication targets for drugs are virus attachment and entry, virus-cell membrane fusion/endocytosis, and uncoating. Although a number of antiviral agents targeting some points of virus replication cycles have been reported as potential candidates for the treatment of some enterovirus infections (4–6), currently, no specific effective antiviral drugs are available (7) or approved (8) for many infections caused by most enteroviruses, particularly diseases caused by CVB and EV-A71. This shows the importance of developing antiviral strategies for the treatment of these infections in preparation for a possible outbreak.

Naturally occurring substances or drugs modeled on natural products can play a fundamental role in the development of potent and effective antiviral drugs, based on their versatility and broad spectrum of action (9). Since ancient times, natural products from plants and microorganisms have provided a variety of chemical options for the development of many drugs, including the commercially available drug acyclovir, modeled on a natural product (10). Plant essential oils and their components are common and well known for their medicinal and antimicrobial properties against fungal and bacterial pathogens (11). Antiviral drug discovery tailored toward natural products may provide an alternative or play an important role in the development of more potent antivirals with more tolerable side effects (11, 12).

In an effort to identify an antiviral agent modeled on a natural product, we examined the antiviral activity of trans-2-hexenoic acid (THA) against CVB type 3 (CVB3) and EV-A71. THA is a nonnutritive, volatile acidic fraction of essential oils found in natural foods—fruits, such as apples, bananas, black currants, citrus fruit, guava fruit, star fruit, and loganberry juice, as well as Mentha oil, peanuts, and black tea (13, 14). To date, there is no available report on the antiviral activity of this compound. Interestingly, the choice to evaluate the antiviral effect of THA was supported by existing information showing that THA is not cytotoxic, genotoxic, phototoxic, or environmentally toxic (13). This compound has been evaluated for safety and received the approval of the Joint Food and Agricultural Organization of the United States and the World Health Organization Expert Committee on Food Additives.

Based on our observation, we found that THA inhibits both CVB3 and EV-A71 replication in a dose-dependent manner without noticeable cytotoxicity. Investigation of the mechanism of antiviral activity of THA using a time of addition assay revealed that THA targeted step(s) involved in viral entry, at the early stage of viral replication. Although this remains to be well elucidated, our findings present an opportunity to develop therapeutic interventions for enterovirus infection, and this could pave the way for the clinical use of THA as an antiviral agent.

RESULTS

THA significantly inhibits CVB infection.

To examine the antiviral activity of the compound, the cytopathic effect (CPE)-based antiviral activity and cytotoxicity of THA were first determined in order to obtain the optimal concentration range of the compound that could be used for antiviral studies. To this end, CVB3-infected HeLa cells were treated with THA (9 μM); cell viability was determined microscopically and quantified using MTT assay. As shown in Fig. 1A, THA treatment significantly suppressed the CVB3-induced CPE at 24 h postinfection (hpi) (P = 0.0290) and 48 hpi (P = 0.0015). These initial results suggested that THA is a potential antiviral compound. Based on these data, we performed a dose-response analysis of THA antiviral activity against CVB3. The results revealed that THA antiviral activity was dose dependent. Notably, the 50% cytotoxic concentration (CC₅₀) of THA was estimated to be 33.62 μM, while the 50% effective concentration (EC₅₀) against CVB3 was estimated to be 2.9 μM, yielding a selective index (SI) value of 11.59 (Fig. 1B). Next, to confirm that the antiviral activity of THA was not limited to CVB3, we selected EV-A71 to further evaluate the antienterovirus activity of THA. Our results showed that THA consistently inhibited the CPE in EV-A71-infected cells, with an EC₅₀ of 3.21 μM. Taken together, these results indicate that THA may represent a novel antiviral agent against CVB3 and EV-A71 in vitro.

FIG 1.

THA is a potent antiviral against CVB3 replication in vitro. (A) THA inhibits virus-induced CPE. HeLa cells were infected with CVB3 or EV-A71 (MOI = 1) and cultured with medium supplemented with THA. The cells were examined microscopically (magnification, ×400), and cell viability was determined by MTT assay. (B) Effective concentration and cytotoxicity of THA. Uninfected (i), CVB3-infected (ii), and EV-A71-infected (iii) HeLa cells were treated with 5 μL of 2-fold serial dilutions of THA. CPE was measured by MTT assay at 72 hpi. EC₅₀ and CC₅₀ were calculated using nonlinear regression (GraphPad Prism 6). EC, effective concentration; CC, cytotoxic concentration; VC, virus control; NC, negative control. The error bars represent the standard deviation (SD); n = 3; Student’s t test.

Secondary confirmation of the antiviral activity of THA.

We further confirmed the above results using a virus yield reduction assay. Virus-infected cells were treated with various concentrations of THA for 24 h, and then the virus yield in the supernatant and cell lysate were quantified by calculating the 50% tissue culture infective dose (TCID₅₀). Our results showed that THA significantly reduced the titers of CVB3 in cell lysate at 8 μM (P = 0.0059), 4 μM (P = 0.0095), and 2 μM (P = 0.1653) and in culture supernatant at 8 μM (P < 0.0001), 4 μM (P = 0.0010), and 2 μM (P = 0.2589), compared to the untreated CVB3-infected cells (Fig. 2A). Similar results were obtained in EV-A71-infected cells treated with THA, suggesting that THA can effectively inhibit intra- and intercellular virus replication.

FIG 2.

THA inhibits virus replication in vitro. (A) Virus yield reduction assay. Virus-infected HeLa cells were treated with THA at the indicated concentrations. Cells were subjected to three freeze-thaw cycles, culture supernatants were collected, and quantification of virus particles was performed by TCID₅₀ assay. (B and C) Virus-infected cells were cultured in medium containing serially diluted THA, as indicated. Total RNA (B) and protein (C) were extracted 24 hpi and analyzed by RT-qPCR and Western blotting, respectively. VC, virus control. The error bars represent the SD; n = 3; Student’s t test.

Next, we examined the levels of viral RNA and protein in virus-infected cells treated with THA. We observed (Fig. 2B and C) that increasing concentrations of THA caused progressive inhibition of viral RNA levels. THA significantly reduced CVB3 viral RNA levels at 9 μM (P = 0.0003), 4.5 μM (P = 0.0052), and 2.3 μM (P = 0.0013) and 3D protein levels at 9 μM (P < 0.0001), 4.5 μM (P < 0.0001), 2.3 μM (P = 0.0052), and 1.2 μM (P = 0.0013). Similar results were obtained when EV-A71-infected cells were treated with THA. Since THA is thought to be present in a lower concentration in its natural state, we examined whether THA could inhibit virus replication at nanomolar concentrations. The results further confirmed that THA significantly decreased both RNA levels of CVB3 (360 nM [P = 0.0032] and 180 nM [P = 0.0035]) and EV-A71 (500 nM [P = 0.0158] and 250 nM [P = 0.0321]), although a significant decrease in the 3D protein levels was only achieved at concentrations of 360 nM (P = 0.0360) and 500 nM (P = 0.0085) in THA-treated CVB3- and EV-A71-infected cells, respectively.

Interestingly, CVA16 infection in vitro was also effectively inhibited by THA (see Fig. S1 in the supplemental material), suggesting that THA may be a broad-spectrum agent against enteroviruses.

THA possess antiviral activity against CVB3 infection in vivo.

Motivated by the results of the in vitro experiments, we examined both the cytotoxicity and antiviral activity of THA in a mouse model. To this end, newborn Kunming mice infected with CVB3 intraperitoneally were administered 15 and 30 mg/kg (body weight) of THA twice per day. Mice in the control group were treated with only phosphate-buffered saline (PBS) or infected with CVB3. The physical condition of the mice was monitored daily. As shown in Fig. 3A and B, CVB3 infection caused passive behavior and debilitating changes in the mice, and only about 25% (2/8) of the mice survived at the end of the test period (7 days postinfection [dpi]). CVB3 infection caused a dramatic decline in the body weight of the mice (Fig. 3A). All infected mice treated with 15 or 30 mg/kg of THA survived at 7 dpi (Fig. 3B). To further determine the antiviral activity of THA, the mice were euthanized at 7 dpi, and the hearts were harvested for analysis by Western blotting and reverse transcription-quantitative PCR (RT-qPCR). Our results showed that the levels of viral RNA and 3D protein were significantly reduced in the THA-treated mice (Fig. 3C and D). Consistently, massive inflammatory damage was visible in the myocardial tissue of the CVB3-infected mice, while the inflammatory injuries were significantly curbed in the myocardial tissue of the infected mice treated with 15 or 30 mg/kg of THA, demonstrating that THA effectively inhibits CVB3 replication in vivo.

FIG 3.

THA alleviates CVB3 infection in mice. CVB3-infected newborn mice were treated with THA (15 and 30 mg/kg) twice a day for 7 days. (A) General condition of the mice on day 7 postinfection. (B) Survival rates were determined and analyzed for each group of mice. (C) Protein levels were analyzed by Western blotting. (D) Total RNA levels were analyzed by RT-PCR. (E) Representative myocardial sections of various treated mice. n = 8; Error bars represent the SD; Student’s t test.

THA inhibits virus infection at the early stage of replication.

Next, we sought to gain preliminary insight into the mechanism of antiviral activity of THA. To this end, we performed a time of addition assay in order to identify the stage(s) at which THA inhibits virus replication. Briefly, THA was added to the culture medium of virus-infected cells at various time points of postinfection, as illustrated in Fig. 4A (upper diagram). As shown in Fig. 4A (lower diagram), when the test compound was added to CVB3-infected cells before and up to 1 hpi, the synthesis of viral 3D protein was completely blocked. However, when the test compound was added at 2 to 3 hpi, synthesis of 3D protein was not strongly affected. Similarly, our results also showed that treatment of EV-A71-infected cells before 2 hpi completely blocked the synthesis of viral protein, but at 3 hpi and onward, the addition of THA did not have a significant effect on virus replication. These data reveal that THA exerts its antiviral effect at the early stage of virus infection and that THA treatment of virus-infected cells after this stage leads to a gradual decrease in its antiviral activity.

FIG 4.

THA inhibits CVB3 replication in the early stages of viral infection. (A - B) THA exerts an antiviral effect in the early stages of virus infection. Virus-infected HeLa cells were cultured in medium containing THA (9 μM) at the indicated time points. Viral protein levels were determined by Western blot assay. (C - D) THA pretreatment of HeLa cells inhibits virus replication. HeLa cells were treated with THA (9 μM) for 12 h (C) or 1 h (D) before virus infection. Cells were then infected with the respective viruses and incubated in culture medium without THA. Total proteins were extracted at 12 hpi (B and C) or 12 hpi and 24 hpi (D) and analyzed by Western blotting. The virus control (VC) was not pretreated. The error bars represent the SD; n = 3; Student’s t test.

Motivated by the results of the time of addition assay, we sought to determine the time course of different stages of viral replication. To this end, virus-infected cells were treated with THA, and viral protein was extracted at different time intervals. Untreated virus-infected cells were used as the control. As shown in Fig. 4B, viral protein levels were almost undetectable between 0 and 3 hpi. Arguably, viral attachment, entry, and uncoating occur at this time point (0 to 3 hpi), before viral protein expression is detected (3 to 6 hpi). Taken together, our results clearly show that THA inhibits virus replication at the early stage of infection, possibly at the stage(s) of virus internalization and uncoating.

To identify the specific step(s) in the early stage of virus replication that THA targets, cells were pretreated with THA (9 μM) for 12 h as described by Lee et al., with slight modifications (15). The pretreated cells were infected with CVB3 or EV-A71 and were cultured in a medium without THA. Total protein was extracted at 12 hpi, and the viral protein level was determined by Western blotting. As shown in Fig. 4C, the levels of viral protein in CVB3-infected (VP1, P = 0.0140; 3D, P = 0.0019) and EV-A71-infected (VP1, P = 0.0071; 3D, P = 0.0013) cells were significantly reduced in the THA-pretreated cells compared to the cells that were not pretreated. Furthermore, to rule out the possibility of toxicity because of the long duration of cell pretreatment, the experiment was repeated. This time, HeLa cells were pretreated with THA (9 μM) for 1 h and were infected with the virus afterward. Similar results were observed, showing that THA substantially reduced the CVB3 viral protein levels at 12 hpi (VP1, P = 0.0007; 3D, P = 0.0002) and 24 hpi (VP1, P = 0.0003; 3D, P < 0.0001) and the EV-A71 viral protein levels at 12 hpi (VP1, P = 0.0034; 3D, P = 0.0022) and 24 hpi (VP1, P = 0.0014; 3D, P = 0.0003) (Fig. 4D). These data suggest that THA pretreatment of cells effectively inhibits virus infection, possibly at the stage of virus attachment and entry.

THA inhibits virus entry by targeting the viral capsid protein VP1.

To predict possible interactions of THA with the viral structural capsid protein, we docked THA into the active site of VP1, and other nonstructural and structural proteins of seven different families and genera of RNA viruses: Enterovirus, Coronavirus, Lentivirus, Arenavirus, Herpesvirus, Flavivirus, and Orthomyxovirus. The binding energy was calculated for each and ranked accordingly, as shown in Table 1. The docking score was used to determine the strength of the protein-ligand interaction. A more negative binding score represents a favorable interaction between protein and ligand. Our findings showed that the VP1 of CVB and nonstructural proteins of other viruses are potential targets of THA. Representative diagrams are presented in Fig. 5A.

TABLE 1.

Docking scores of THA with various viral proteins

Family or genus	Virus	Target proteina	PDB accession no.	Docking score
Enterovirus	Poliovirus 3	VP1	1PIV	−5.5
Enterovirus	Poliovirus 2	VP1	1EAH	−5.4
Lentivirus	HIV-1 group M subtype B	Reverse transcriptase	1DTQ	−5.4
Arenavirus	Lassa virus	Nucleoprotein	3MX2	−5.3
Enterovirus	Poliovirus 1	VP1	1PO1	−5.0
Herpesvirus	Herpes virus	Thymidine kinase	1E2N	−4.8
Coronavirus	SARS coronavirus	Replicase 1a	3MJ5	−4.7
Flavivirus	Hepatitis C virus genotype 1b	RdRp	3QGE	−4.3
Orthomyxovirus	Influenza virus	Neuraminidase enzyme	1A4Q	−4.3
Enterovirus	Rhinovirus 16	RdRp	1TP7	−4.2

^aRdRp, RNA-dependent RNA polymerase; VP1, viral protein 1.

FIG 5.

THA inhibits virus replication by blocking viral entry. Viral structural proteins are the potential targets of THA. (A) Docking sites of THA on different virus proteins, as indicated. Docking results were generated using the MedusaDock online program. (B and C) THA pretreatment of virus inhibits virus replication. Virus was pretreated with favipiravir (52 μM), CHX (0.03 μM), and THA (9 μM) in culture medium without serum for 1 h at 37°C. HeLa cells were infected with the pretreated viruses and incubated in culture medium without an inhibitor. The virus-induced CPE was examined microscopically, and total protein was extracted and analyzed by Western blotting at 12 and 24 hpi, respectively. (D) THA interferes with viral entry, thus inhibiting virus replication. HeLa cells were infected with THA-pretreated virus and allowed to attach for 1 h. The infected cells were washed thrice with PBS and incubated in culture medium without THA. Total protein was extracted and analyzed by Western blotting at 24 hpi. (E) Chloroform treatment of a virus-THA mixture attenuates THA inhibition of virus entry. Chloroform was added to a THA-virus mixture (1:200, vol/vol) (previously incubated for 1 h at 37°C under 5% CO₂), and the mixture was vortexed at low speed for 1 min at room temperature. The aqueous phase that contained the virus was used to infect HeLa cells. Total protein was extracted and analyzed by Western blotting at 24 hpi. CHX, cycloheximide; VC, virus control. The error bars represent the SD; n = 3; Student’s t test.

The viral capsid protein VP1 is involved in receptor recognition and binding. These processes are vital for virus attachment and entry, which are critical steps in the early stage of the virus replication cycle. Since the protein-ligand interaction profile predicts that THA may bind to VP1, we hypothesize that this interaction might perturb virus entry into a susceptible host. To determine this, CVB3 was pretreated with THA (9 μM) at 37°C for 1 h before infection. Cycloheximide (CHX; a compound that interferes with eukaryotic protein synthesis) (16) and favipiravir (a nucleotide analog) (17) were used as the controls. HeLa cells were infected with the pretreated viruses and were incubated in separate culture media without THA, CHX, and favipiravir. Microscopic examination showed that the CVB3-induced CPE was alleviated following infection of HeLa cells with THA-pretreated viruses (Fig. 5B). Likewise, THA pretreatment of CVB3 inhibited virus replication and significantly decreased the VP1 levels at 12 hpi (P = 0.0025) and 24 hpi (P = 0.0018). Pretreatment of CVB3 with CHX at 12 hpi (P = 0.0674) and 24 hpi (P = 0.0777) and with favipiravir at 12 hpi (P = 0.5246) and 24 hpi (P = 0.3055) had no significant effect on CVB replication (Fig. 5C). Similar results that showed that the pretreatment of EVA71 with THA significantly inhibited virus replication were also obtained. These data signify the possible ability of THA to inhibit virus entry and suggest that this ability is not limited to the strain of CVB3 used for this study.

To validate the above results, cells were infected with THA-pretreated viruses and were incubated for 1 h for virus attachment; then, the cells were washed with PBS twice to remove unbound virus (18). Afterwards, the cells were incubated in a medium without inhibitors for 24 h. Prevention of virus binding was accessed by evaluating viral replication (19). Our result suggest that THA pretreatment of a virus inhibits its replication. Viral protein levels in CVB3-infected (VP1, P = 0.0004; 3D, P = 0.0008) and EV-A71-infected (VP1, P = 0.0001; 3D, P = 0.0003) cells were significantly reduced as a result of virus pretreatment (Fig. 5D).

Since THA is a fatty acid (FA) that is soluble following chloroform treatment, next, we examined whether chloroform treatment of a THA-virus mixture would reverse the binding between THA and the virus (20, 21). To this end, chloroform was added to a THA-virus mixture (1:200, vol/vol) (previously incubated for 1 h at 37°C and 5% CO₂) and vortexed at low speed for 1 min at room temperature. After that, the aqueous phase that contained the virus was used to infect HeLa cells. The results showed that the antiviral activity of THA was reversed with chloroform treatment, as demonstrated by the inability of THA pretreatment to inhibit CVB3 and EV-A71 replication after chloroform treatment (Fig. 5E), suggesting that THA binds to a specific site on the outer capsid protein of CVB3 and EV-A71. Collectively, our data suggest that THA pretreatment reduces enterovirus attachment or entry into cells.

DISCUSSION

Nonpolio enteroviruses such as CVB and EV-A71 affect millions of people annually and are possible causes of large outbreaks worldwide, especially in Asia (22). Despite the possible severity of some of these viruses, like the life-threatening inflammatory cardiomyopathy caused by CVB and encephalitis by EV-A71, there is no approved therapy for the treatment for most of these viral diseases. Although several attempts have been made to develop enterovirus inhibitors (4, 5) and prevention agents (6) in order to address their disease burden, for many years, these attempts have faced setbacks due to safety concerns, cytotoxicity issues, or insufficient therapeutic outcomes. Therefore, this study aimed to identify a potent antiviral compound that is generally well-tolerated and cost-effective, with improved antiviral potency and minimum potential side effects. To this end, we proposed that (i) a natural product from a dietary source may be well-tolerated and considered safe for long-term therapeutic use (23), and (ii) the use of antiviral strategies that directly target viral-host factors but have reduced cytotoxicity (unlike most synthetic antivirals targeting host-dependent virus replication proteins) could provide a better option to combat viral infections. As a result, we investigated the antiviral activity of THA, a volatile compound found in several fruits and food products, since evidence shows that THA is not cytotoxic, genotoxic, phototoxic, or environmentally toxic (13).

We first screened the antiviral activity of THA in a cell-based CPE inhibition assay. Our results revealed that THA elicited potent antiviral activity against infection with CVB3 and EV-A71 at concentrations (EC₅₀) as low as 2.9 and 3.21 μM, respectively, yielding an SI value higher than 10. Since compounds with an SI value of >10 are generally considered safe (24), this suggests that THA could possibly be a clinically useful, safe inhibitor of enterovirus infections. Conversely, THA is an unsaturated short-chain FA, and increased excessive accumulation of some FA can lead to cellular dysfunction and eventual cell death, termed lipotoxicity (25). However, based on the cell viability assay in this study, there was no evidence of THA-induced lipotoxicity at the highest concentration used in this study. The antiviral dose of THA used for this study had no significant adverse effect on cell viability, proliferation, or cytotoxicity based on MTT assay (26).

In addition, we evaluated the ability of THA to reduce viral yield and inhibit viral RNA and protein expression in vitro. Our results further show that treatment of infected cells with THA at a concentration range below their EC₅₀ for 24 h reduced the virus titer to about 2-fold of the initial titer in a cell culture. At a lower concentration (nanomolar), THA was able to decrease the respective viral RNA and protein levels. Furthermore, as proof of the antiviral activity of THA, we examined its antiviral activity in an animal model. We found that THA treatment improved the survival rate of CVB3-infected mice. THA treatment reduced the viral load, which directly relates to reduced severity and alleviation of CVB infection-related pathological features in the myocardial tissues.

To examine the possible mechanism of the antiviral activity of THA against the selected enteroviruses, we performed a time of addition assay in order to determine the time point at which THA inhibits virus replication. Our results show that THA inhibits virus replication between 0 and 2 h after virus infection, suggesting that it exerts its antiviral activity at the early stage of infection. The inhibition of virus replication at the early stage of infection could be the result of the interference of the compound with any of the steps involved in the early stage of the virus replication cycle. The early stages of the enterovirus replication cycle are initiated by the binding of the viral capsid protein to the host cell surface receptors and entrance to the host cell through endocytosis. Following this step, the virus undergoes uncoating and the release of genomic RNA into the cytoplasm. The released RNA serves as a template for RNA replication and the translation of viral polyprotein. Since the early stages of enterovirus replication involve several steps, we sought to determine the specific step(s) targeted by THA by examining the time course for different early stages of events. The results from this study reveal that the synthesis of viral protein was completely unnoticeable until between 3 and 6 hpi; arguably, virus attachment, entry, and uncoating, which are the first steps of virus replication, have occurred before this time point. Overall, the time of addition experiment indicated that THA exerts its antiviral effects earlier in the virus replication cycle, possibly at the viral absorption (attachment and entry) or uncoating stages. Thus, in this study, we experimentally examined the effects of THA on these stages.

Recent studies have shown that some FAs possess antiviral activity against a wide range of enveloped viruses for their ability to inhibit viral entry of the majority of lipid-containing mammalian enveloped viruses. Entry of influenza virus (an enveloped virus) has been shown to be inhibited following exposure to palmitoleic acid (27). Similarly, a report by Goc et al. shows that the pretreatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with polyunsaturated omega-3 fatty acids inhibited virus binding and entry (28). One possible reason for this is that FAs could alter the lipid composition of the viral envelope, which may inhibit viral entry by influencing virus-receptor interactions or causing conformational changes to receptors (15, 29). Unlike enveloped viruses, FAs are thought to have no effect on naked viruses (30). This is because naked viruses lack an envelope, which is associated with FA disintegration or inactivation (30). In contrast to this general belief, in this study, we demonstrated that first, a brief exposure of cells (1 h) to THA, at nontoxic levels, promptly induced a prolonged (up to 24 hours) antiviral state within the cell. A possible explanation is that THA blocks virus-to-cell attachment or induces another early host antiviral response (15).

Second, we showed that the pretreatment of CVB3 and also EV-A71 with THA significantly reduced virus replication by about 2-fold (as signified by the reduction of the protein levels of viral 3D and VP1). We proposed that this is possibly due to the inability of THA-bound viruses to attach to susceptible cells or undergo cell-mediated virus uncoating due to the interaction of THA with the hydrophobic pockets within VP1. The surface of these nonenvelope enteroviruses is formed by the subunits of the viral capsid proteins VP1 to VP4. Of importance to the development of antiviral drugs is the hydrophobic pocket factor within VP1 (31). This factor plays a crucial role in the stability of the virus particle and binding of the virus to its receptor, CAR, in a susceptible host in order to permit virus attachment and entry (32, 33). Consistent with the above postulation, a study on bovine enterovirus, a pathogen in the Picornaviridae family, showed that the interaction between FAs and a specific site on the viral capsid protein caused the failure of virus to undergo cell-mediated attachment and uncoating (21). According to electron density and uncoating studies, the “pocket factors” within VP1 outer protein when exposed to short-chain FAs can accommodate lipophilic interactions that could possibly affect viral attachment (20, 21, 31). Although the binding of THA with VP1 was not demonstrated in our study, we showed that the antiviral effect of THA pretreatment of CVB3 and EV-A71 was reduced following chloroform treatment. These data further support our assumption that THA binds to a specific site on the outer capsid protein of the viral particle and suggest that the THA-virus interaction at least partly inhibits virus entry. At present, most of the reported antienterovirus agents work on the later phases, including the steps of biosynthesis, maturation, and release, by targeting the key viral proteins 2A^pro, 3C^pro, and 3D^pol (34), THA is of potential prophylactic and therapeutic importance.

The effect of THA on CVA16 infection was also briefly evaluated under in vitro conditions. A similar outcome was observed, suggesting that THA may be a broad-spectrum agent against enteroviruses. However, an in vivo evaluation of THA against EV-A71 and CVA16 was not performed, partly because the pathogenesis of both viruses differs from that of CVB. The antienterovirus spectrum of THA needs further verification with in vivo evidence. Enteroviruses enter cells mainly through receptor-mediated endocytosis and macropinocytosis (35, 36). Given that THA is a fatty acid and a broad-spectrum antienterovirus agent, it is interesting to know whether THA has the capability of modulating endocytosis and macropinocytosis.

In conclusion, our findings demonstrate that THA is a potent antiviral compound against enteroviruses, particularly CVB3 and EV-A71. THA suppresses enterovirus replication by targeting virus infection at the early stages of replication, possibly by blocking virus entry. However, we have not demonstrated the direct binding of THA with viral capsid protein; thus, the exact underlying mechanism of enterovirus inhibition by THA needs further clarification. After further investigation, THA might be a safe natural therapeutic compound for the treatment of virus infection or a promising strategy for the development of semisynthetic antiviral drugs.

MATERIALS AND METHODS

Compound, cells, and viruses.

The compound THA (Aladdin, Shanghai, China), supplied in semisolid form, was dissolved in dimethyl sulfoxide (DMSO) and stored at room temperature. THA was diluted in the culture media to the required concentration just before usage. A monolayer cell culture of HeLa cells was maintained in Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C under 5% CO₂. CVB3 strain Woodruff and EV-A71 strain BrCr were used for the antiviral experiments.

Virus infection and virus yield reduction assay.

For the in vitro virus infection experiments, cultured cells at 60% to 70% confluence were washed thrice with phosphate-buffered saline (PBS) and infected with virus in DMEM without serum. The virus was allowed to attach for 1 h before treatment. Cells were infected at a multiplicity of infection (MOI) of 1, except where otherwise indicated.

To test the antiviral activity of THA, a virus yield reduction assay was performed. Briefly, HeLa cells were seeded in 6-well plates and maintained at 37°C under 5% CO₂ for 18 h. After viral infection, the cells were cultured in medium supplemented with 2-fold serial dilutions of THA (2 to 8 μM) for 24 h postinfection (hpi). No inhibitor was added to the virus control well. The culture supernatant and cell lysate (subjected to 3 freeze-thaw cycles) were collected at 24 hpi. The amount of virus particles was quantified by median tissue culture infectious dose (TCID₅₀) assay.

Cytotoxicity and effective concentration assay.

To determine the cytotoxic concentration (CC) of THA, HeLa cells were seeded into the 96-well plate and incubated for 18 h prior to the addition of THA. Twofold serial dilutions of THA (5 μL) were added to each well. In the cell control well, only DMEM was added. Immediately after the addition of THA, 45 μL of DMEM with 10% serum was added to each well. The total volume in each well was 100 μL. After 72 hpi, cell viability was determined using MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] (Meilunbio Technology, Suzhou, China) following the manufacturer’s instructions. The absorbance was read using a SpectraMax microplate reader (Molecular Devices, San Jose, CA) at 542 nm. The 50% cytotoxic concentration (CC₅₀) of THA was defined as the concentration of the compound that resulted in 50% inhibition of cell viability. To determine the effective concentration (EC) of THA, a cytopathic effect (CPE) inhibition assay was performed in parallel with a cytotoxicity assay, with the addition of virus. The 50% effective concentration (EC₅₀) was defined as the concentration of the inhibitor required to achieve a half-maximal inhibition effect. The selective index (SI) of THA was calculated as SI = CC₅₀/EC₅₀.

Time of addition assay.

A time of addition assay was performed to examine the mechanism of antiviral activity of THA. HeLa cells were seeded in a 12-well plate and maintained at 37°C under 5% CO₂ for 18 h. Virus-infected cells were treated with THA (9 μM) at hourly intervals from -1 to 5 hpi. No inhibitors were added to the virus control well. At 12 hpi, total protein was extracted and analyzed by Western blotting.

THA in vivo treatment.

Newborn Kunming mice were purchased and housed in a well-regulated and pathogen-free environment. Mice were allowed to access food and water ad libitum. All procedures related to the care and handling of the mice were carried out in strict accordance with the regulations on the use and care of laboratory animals for research as approved by the Ethics Committee of Harbin Medical University, China.

To demonstrate the in vivo antiviral activity of THA, newborn mice (4 to 5 days after birth) were infected with 1.5 × 10⁶ TCID₅₀ of CVB3 by intraperitoneal injection once, 12 h before treatments. No treatment was administered to the virus control group, while PBS was administered to the sham group. All treated mice were administered 15 or 30 mg/kg (body weight) of THA, 12 hpi and subsequently twice per day (for 7 days) by intraperitoneal injection. The total compound volume administered was 40 μL. Mice were euthanized at day 7 postinfection, and the hearts were harvested for analysis.

SDS-PAGE and Western blotting.

The total protein, extracted using radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific, IL, USA) containing 1% phenylmethanesulfonyl fluoride (PMSF) (Beyotime, Shanghai, China), was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were incubated at 4°C overnight with primary antibody, washed with 0.1% Tween 20 in PBS, and incubated with anti-rabbit IgG for 1 h at room temperature. The blots were viewed using a FluorChem M charge-coupled-device (CCD) camera (ProteinSimple, San Jose, CA).

PCRs.

To perform RT-qPCR, 1 μg RNA was reverse transcribed to cDNA. Then, 2 μL of the amplified cDNA was mixed with 10 μL SYBR premix Ex Taq II (TaKaRa, Dalian, China), 1 μL of reverse and forward primer, and 8 μL of double-distilled water (ddH₂O) to a total volume of 20 μL. The RT-qPCR was performed using the LightCycler system (Roche, Basel, Switzerland). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA was used as the internal control.

Virtual screening of THA.

The dimeric structures of the structural and nonstructural proteins of different virus groups retrieved from the Protein Data Bank (PDB) were docked with THA. The position constraint was set at a distance between 2 and 3 Ậ, and the energy bonus was -1 Kcal/mol. The receptor residue was set at different points. The cutoff and number of rounds were set at 10 and 1, respectively. Docking scores generated using the MedusaDock online program were ranked. The Mcule drug discovery platform (https://mcule.com/apps/1-click-docking/) was used for confirmation. Docking was set to run with default settings except for some slight changes.

Statistical analysis.

Data are presented as the mean ± standard deviation (SD) of three independent experiments. Statistical significance (P < 0.05) was determined by Student’s t test using GraphPad Prism version 6.02.