1. Introduction
Enterovirus 71 (EV71) is a positive single-strand RNA virus belonging to the genus Enterovirus of the family Picornaviridae .[ 1 ] EV71 is spread through contact with virus-containing body fluids, respiratory droplets, and feces, and it usually causes hand, foot, and mouth disease (HFMD) in children younger than 5 years. Most of the clinical symptoms of EV71 viral infection are mild and self-limited, a few EV71 infections are often associated with neurological diseases, and severe EV71 infection can lead to death.[ 2,3 ]
Enterovirus 71 was first isolated from patients in California in 1969, and many large-scale outbreaks have occurred worldwide in the following decades.[ 4–6 ] In 2008, the first large-scale EV71 infection broke out in China, and HFMD caused by EV71 infection has become a serious threat to children’s health in China.[ 7 ] However, no effective antiviral drugs are available for the prevention and treatment of HFMD at present. Since 2016, Chinese children have been vaccinated with inactivated EV71 vaccine, and the number of cases and deaths of HFMD decreased significantly in the following years, indicating that the inactivated EV71 vaccine has protected children from EV71 infection.[ 8 ] However, the current EV71 inactivated vaccine coverage rate is not very high. In addition, vaccines do not replace antiviral drugs for the treatment of viral diseases. Therefore, drugs with anti-EV71 activity should be urgently developed.
At present, most of the approved antiviral drugs are direct-acting antivirals (DAAs) with good antiviral activities, which targets the proteins encoded by viruses. In addition to specific virus, DAAs may have activities against similar related viruses. Corresponding to DAAs are host-targeting agents. As obligate intracellular parasites, viruses depend on host-cell machinery for replication. Therefore, some cellular proteins that participate in viral replication can be used as new targets for antiviral drug development. In addition, enhancing some existing host-limiting factors that inhibit viral replication and prevent viral transspecies transmission may also become new targets of antiviral drugs.[ 9–11 ]
Direct-acting antivirals have strong antiviral activities and good specificities, but their antiviral spectrum is narrow, and DAAs are prone to drug resistance. Previous DAAs developed for enterovirus infection, such as pleconaril, have not been marketed because of their efficacy and drug resistance.[ 12–14 ] Host-targeting agents, such as interferon (IFN) and ribavirin, have a weak antiviral effect, but have a broad spectrum antiviral effect, and are not easy to be resistant.[ 15 ]
Apolipoprotein B messenger RNA (mRNA)–editing enzyme catalytic polypeptide-like 3G (A3G, APOBEC3G) is a cellular restriction factor of EV71 and a mediator of the antiviral activity of a N -phenylbenzamide derivative IMB-Z.[ 16 ] After further structural modification, IMB-0523 [Figure 1 A], a chemical modification product of IMB-Z with better properties, developed good anti–hepatitis B virus (HBV) effects by upregulating the expression of A3G.[ 17 ] However, the mechanism by which IMB-0523 upregulates A3G expression remains unclear. Here, we mainly explored the effect and mechanism of IMB-0523 against EV71.
Figure 1: Cytotoxicity of IMB-0523 was determined by CCK assay. (A) The chemical structure of IMB-0523 . (B) Cytotoxicity of IMB-0523 to multiple cell lines were determined by CCK assay. CCK: cell counting kit.
2. Materials and methods
2.1. Cells and virus
African green monkey kidney (Vero) cells were obtained from the American Type Culture Collection (ATCC) and cultured in Modified Eagle’s Medium (MEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% inactivated fetal bovine serum (FBS; Gibco, Grand Island, USA) and 1% penicillin-streptomycin (Invitrogen). Human colon cancer (HCT-8) cells were obtained from the Cell Culture Center of Peking Union Medical College or Chinese Academy of Sciences and cultured in DMEM (Invitrogen) supplemented with 10% FBS and 1% penicillin-streptomycin (Invitrogen) at 37°C in an incubator with 5% CO2 .
The EV71 strain H (VR-1432) and strain BrCr (VR-1775) were obtained from ATCC. The EV71 strain JS-52 was provided by Dr Xiangzhong Ye, Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. Coxsackieviruses of group B1 (CVB1, strain Conn-5), CVB2 (strain Ohio-1), CVB3 (strain Nancy), CVB4 (strain J.V.B.), CVB5 (strain Faulkner), and CVB6 (strain Schmitt) were obtained from the ATCC. All viral strains were propagated in Vero cells.
2.2. Compounds and siRNAs
IMB-0523 was synthesized in the Medicinal Chemistry Laboratory of the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, with purity of more than 98.0%. Stock solutions (40 mmol/L) of IMB-0523 were prepared in dimethyl sulfoxide (DMSO). IMB-0523 was diluted to final working solutions as indicated in experiments. Recombinant human IFN-α2b injection (3 × 106 IU/mL, batch number: 20191205) provided by Anhui Anke Biotechnology (Group) Co., Ltd, was diluted to an appropriate concentration in the medium. Polyriboinosinic acid–polyribocytidylic acid, Poly (I:C), was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. Stattic was obtained from MedChemExpress (Monmouth Junction, NJ, USA).
JAK1 siRNA (si-JAK1), TYK2 siRNA (si-TYK2), STAT3 siRNA (si-STAT3 ), and control siRNA (si-NC) were obtained from Santa Cruz Biotechnology (Dallas, CA, USA).
2.3. Cytotoxicity assay
The cytotoxic effect of IMB-0523 was assayed using CCK (TransGen Biotech Beijing, china).[ 18 ] The 50% toxicity concentration (TC50 ) of IMB-0523 was determined using the Reed-Muench method.
2.4. Transfection experiment
HCT-8 cells were transfected with 100 nmol/L si-NC, si-JAK1, si-TYK2, or si-STAT3 by using Lipofectamine RNAiMAX (Invitrogen). After 24 hours, the cells were infected with EV71 (multiplicity of infection (MOI) = 0.1) for 1 hour and treated with DMSO or IMB-0523 10 μmol/L for 24 hours. Cells were lysed, and proteins were extracted for Western blot assay.
2.5. Real-time qRT-PCR
Total RNA was isolated from cells using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) and analyzed with TransScript II Green One-Step qRT-PCR SuperMix (TransGen Biotech) by using the ABI 7500 Fast real-time PCR system (Applied Biosystems, Foster City, CA, USA).[ 16 ] Oligonucleotide primer pairs are shown in Supplementary Table 1, available at https://links.lww.com/IDI/A14
2.6. Western blot assay
Western blot assay was performed as described previously.[ 16 ] The primary antibodies used in this study included antibodies against β-actin, P-JAK, JAK1, P-TYK2, TYK2, P-STAT1, STAT1, P-STAT2, STAT2, P-STAT3 , STAT3 , TRIF, MX1, ISG15, IFIT1, IFITM1, IFITM3, and OAS1 from Cell Signaling Technology (Beverly, MA, USA), EV71 VP1 from GeneTex (California, USA), and A3G from Abcam (Cambridge, MA, USA). The goat antirabbit and antimouse Horseradish Peroxidase (HRP)-labeled antibodies were obtained from Cell Signaling Technology.
2.7. Cytopathic effect (CPE) inhibition assay for antienteroviruses
The antienteroviruses activities of IMB-0523 were evaluated using CPE assay as described previously.[ 18 ] Briefly, Vero cells were infected with 100TCID50 (50% tissue culture infective doses) enteroviruses (EV71, CVA16, CVB1, CVB2, CVB3, CVB4, CVB5, CVB6) for 1 hour at 37°C. Then, DMSO or various concentrations of IMB-0523 were added for 48 hours. The 50% inhibitory concentration (IC50 ) was determined using the Reed and Muench method. The selectivity index was calculated as the ratio of TC50 /IC50 .
2.8. Statistics
Statistical analyses were performed using GraphPad Prism 7.0 software. Results are expressed as mean ± SD. Statistical significance was assessed using 1-way analysis of variance (ANOVA) with Holm-Sidak multiple comparisons test or 2-way ANOVA with Holm-Sidak multiple comparisons test. A P value less than 0.05 was considered significant.
3. Results
3.1. IMB-0523 inhibits EV71 replication
To ensure that the antiviral effect of IMB-0523 is not caused by cytotoxicity to cells, we first determined the cytotoxicity of IMB-0523 by CCK assay. Compared with DMSO-treated cells, the maximum nontoxic concentration (TC0 ) of IMB-0523 was approximately 200 μM [Figure 1 B]. At nontoxic concentrations, IMB-0523 treatment decreased the levels of viral VP1 protein in a dose-dependent manner in Vero and HCT-8 cells [Figure 2 A]. Moreover, IMB-0523 treatment can dose-dependently reduce the VP1 RNA level of EV71 in HCT-8 and Vero cells [Figure 2 B], while 40 IU/mL of IFNα2b also has a certain inhibitory effect on the levels of viral VP1 protein and RNA [Figure 2 A and 2B]. Therefore, IMB-0523 inhibits EV71 replication.
Figure 2: IMB-0523 inhibited EV71 replication in Vero and HCT-8 cells. HCT-8 or Vero cells were infected with EV71 (H, MOI = 0.1) for 1 hour, and then cells were treated with DMSO or IMB-0523 or IFN-α2b for 24 hours. Cells were lysed and proteins were extracted for Western blot (A) and RNA were extracted for qRT-PCR assay (B) (n = 3). *P < 0.05 and ***P < 0.001 by 1-way ANOVA with Holm-Sidak multiple comparisons test, compared with DMSO. DMSO: dimethyl sulfoxide; EV71-VP1: enterovirus 71 virus protein 1; IFN-α2b: interferon alpha 2b; qRT-PCR: real-time quantitative polymerase chain reaction.
3.2. IMB-0523 enhances the expression of ISGs
Interferon molecules bind to cell surface receptors to initiate a series of signal transduction that ultimately leads to the transcriptional regulation of IFN-stimulating genes (ISGs ), such as A3G.[ 19 ] Considering that IMB-0523 can significantly increase the expression of A3G, we explored whether IMB-0523 regulates the expression of A3G or has a regulatory effect on other ISGs . As shown in Figure 3 A, compared with DMSO-treated cells, both IMB-0523 and IFNα2b treatment could both significantly upregulate the expression of various ISGs , including A3G, IFN stimulated gene 15 (ISG15), 2′-5′-oligoadenylate synthetase 1 (OAS1), IFN-induced transmembrane protein 3 (IFITM3), and TIR domain-containing adaptor inducing IFN-β (TRIF). Moreover, IMB-0523 can upregulate the expression of various ISGs in a time-dependent manner [Figure 3 B]. Therefore, IMB-0523 may have a similar function to IFN and play an antiviral role by enhancing ISGs expression.
Figure 3: IMB-0523 upregulated the expression of ISGs . (A) HCT-8 cells were treated with DMSO or 40 μmol/L IMB-0523 or 40 IU/mL IFN-α2b for 12 hours, and then cells were lysed and proteins were extracted for Western blot. (B) HCT-8 cells were treated with 40 μmol/L IMB-0523 for different time, and then cells were lysed and proteins were extracted for Western blot. A3G: Apolipoprotein B messenger RNA [mRNA]-editing enzyme catalytic polypeptide-like 3G; DMSO: dimethyl sulfoxide; IFIT1: tetratricopeptide repeats 1; IFITM1: IFN-induced transmembrane protein 1; IFITM3: IFN-induced transmembrane protein 3; IFN-α2b: Interferon alpha 2b; ISGs : interferon-stimulated genes; MX1: myxovirus protein 1; TRIF: TIR domain-containing adaptor inducing interferon-β; ISG15: interferon-stimulated gene 15; OAS1: 2′-5′-oligoadenylate synthetase 1.
3.3. IMB-0523 has no upregulation effect on IFNs
IMB-0523 can upregulate the expression of various ISGs , and this IFN-like function is suspected to promote the transcription of ISGs gene by upregulating the expression of IFN. Therefore, we studied the effect of IMB-0523 treatment on IFN expression. IMB-0523 did not upregulate the mRNA levels of IFN-α, IFN-β, and IFN-γ in HCT-8 cells [Figure 4 A and 4B], while IFN inducer Poly (I:C) significantly upregulated the mRNA levels of IFN-α and IFN-β in HCT-8 cells [Figure 4 B]. Therefore, the antiviral effect of IMB-0523 is independent of the regulation of IFNs. In addition, Vero cells are deficient in IFN-α and IFN-β genes and can not express IFN-α and IFN-β,[ 20 ] but IMB-0523 still has an anti-EV71 effect in Vero cells, indicating that the antiviral effect of IMB-0523 does not depend on the regulation of IFNs.
Figure 4: IMB-0523 did not affect the expression of IFNs. (A) HCT-8 cells were treated with DMSO or 40 μmol/L IMB-0523 for different time, and then cells were lysed and RNA was extracted, intracellular IFN-α, IFN-β, and IFN-γ mRNA were determined by qRT-PCR assay (n = 3). (B) HCT-8 cells were treated with DMSO or 40 μmol/L IMB-0523 or 50 μg/mL Poly (I:C) for 6 hours, and then cells were lysed and RNA were extracted, intracellular IFN-α and IFN-β mRNA were determined by qRT-PCR assay (n = 3). **P < 0.01 vs. control. **P < 0.01 by 1-way ANOVA with Holm-Sidak multiple comparisons test, compared with DMSO. DMSO: dimethyl sulfoxide; IFN: interferon; Poly (I:C): polyriboinosinic acid–polyribocytidylic acid.
3.4. IMB-0523 activates STAT3 signaling
Interferons bind to cell surface receptors to activate the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway, leading to the transcription of ISGs .[ 21 ] Although IMB-0523 did not affect the expression of IFNs, and IMB-0523 can upregulate the expression of ISGs . Therefore, IMB-0523 may directly activate IFN downstream signaling pathways to promote ISG transcription. Accordingly, we further examined whether IMB-0523 could activate the JAK/STAT signaling pathway. HCT-8 cells were treated with IFNα2b and IMB-0523 for 15 minutes, and then proteins were extracted to detect the effects of IFNα2b and IMB-0523 on JAK/STAT signaling pathway. As shown in Figure 5 A, compared with DMSO-treated cells, 40 IU/mL IFNα2b significantly activated the phosphorylation of JAK1/TYK2/STAT1/2/3 protein, while IMB-0523 only activated the phosphorylation of JAK1/TYK2/STAT3 proteins rather than STAT1 and STAT2. Notably, the reduction of endogenous JAK1 or TYK2 by siRNA interference had no effect on the antiviral activity of IMB-0523 against EV71 [Figure 5 B], but reducing the endogenous STAT3 by siRNA interference in HCT-8 cells attenuated the antiviral activity of IMB-0523 against EV71 [Figure 5 B]. Moreover, stattic, a nonpeptide STAT3 small-molecule inhibitor that effectively inhibits STAT3 activation and nuclear translocation[ 22,23 ] also attenuated the antiviral activity of IMB-0523 against EV71 [Figure 5 C]. Therefore, IMB-0523 can phosphorylate STAT3 by activating JAK1/TYK2 or directly activate STAT3 to initiate downstream signal transduction to inhibit EV71 replication [Figure 6 ].
Figure 5: IMB-0523 activated STAT3 signaling. (A) HCT-8 cells were treated with DMSO or 40 μmol/L IMB-0523 or 40 IU/mL IFN-α2b for 15 minutes, and then cells were lysed and proteins were extracted for Western blot. (B) HCT-8 cells were transfected with control siRNA or JAK1 siRNA or TYK2 siRNA or STAT3 siRNA for 24 hour and then infected with EV71 (H, MOI = 0.1) for 1 hour and treated with DMSO or IMB-0523 10 μmol/L for 24 hours. Cells were lysed and proteins were extracted for Western blot. (C) HCT-8 cells were infected with EV71 (H, MOI = 0.1) for 1 hour and treated with DMSO or IMB-0523 10 μmol/L or static 10 μmol/L for 24 hours. Cells were lysed and proteins were extracted for Western blot. DMSO: dimethyl sulfoxide; EV71: enterovirus 71 ; EV71-VP1: enterovirus 71 virus protein 1; JAK1: Janus kinase-1; P-JAK1: phosphorylated JAK1; P-TYK2: phosphorylated TYK2; P-STAT1; phosphorylated STAT1; P-STAT2: phosphorylated STAT2; STAT1: signal transducer and activator of transcription 1; STAT2: signal transducer and activator of transcription 2; STAT3 : signal transducer and activator of transcription 3; P-STAT3 : phosphorylated STAT3 ; TYK2: tyrosine kinase 2.
3.5. IMB-0523 inhibits the replication of enteroviruses
Considering that IMB-0523 can upregulate the expression of ISGs , it should have broad-spectrum antiviral effects. In the present study, CPE assay was used to study the effect of IMB-0523 on 4 EV71 strains, CVA16, and Coxsackievirus B1–B6. As shown in Table 1 , IMB-0523 has good inhibitory activities against all strains tested. In addition, IMB-0523 has an anti-HBV effect,[ 17 ] suggesting that IMB-0523 may be used as a lead compound in the development of broad-spectrum antiviral drugs.
Table 1 -
Antiviral activities of
IMB-0523 against enteroviruses in Vero cells
Strain
IMB-0523
TC50 (μmol/L)
IC50 (μmol/L)
SI
EV71 (SZ-98)
>400
4.64 ± 1.41
>86.21
EV71 (JS-52)
>400
3.65 ± 0.76
>109.59
EV71 (H)
>400
4.64 ± 1.10
>86.21
EV71 (BrCr)
>400
3.27 ± 0.45
>122.32
CVB1
>400
18.16 ± 7.33
>22.03
CVB2
>400
38.52 ± 11.38
>10.38
CVB3
>400
15.51 ± 4.30
>25.79
CVB4
>400
31.78 ± 29.48
>12.59
CVB5
>400
12.28 ± 14.48
>32.57
CVB6
>400
27.17 ± 8.63
>14.72
CVA16
>400
33.34 ± 6.02
>12.00
EV71: enterovirus 71 ; CVB: coxsackievirus B; CVA16: coxsackievirus A16; TC50 : 50% toxicity concentration; IC50 : 50% inhibitory concentration; SI: selectivity index.
4. Discussion
The large-scale outbreak of HFMD, especially in Asia, has seriously affected the stability of social development. Researchers around the world continuously study the pathogenesis of EV71 and develop anti-EV71 drugs and vaccines. Inactivated EV71 vaccines were first administered to Chinese children in 2016.[ 24,25 ] The number of cases of HFMD has decreased since vaccination, indicating that the vaccine provides protection against EV71 infection.[ 25,26 ] However, whether the vaccine can prevent EV71 infection in all subtypes remains unclear. Moreover, it has no cross-protection against other enteroviruses, such as CVA16, which also causes HFMD.[ 27 ] However, no effective drugs are available for the prevention and treatment of HFMD.
The results of the present study show that IMB-0523 can play an anti-EV71 role by activating STAT3 signaling to upregulate ISG expression [Figure 6 ]. Interestingly, our study found that IMB-0523 had no effect on the expression of IFNs, indicating that the activation of IMB-0523 on the JAK/STAT signaling pathway is independent of the binding of IFNs and IFN receptors [Figures 4 and 5 ]. CDM-3008, a selective type I IFN receptor agonist, can directly activate the JAK/STAT pathway through binding to IFNα/β receptor 2.[ 28 ] However, our current study could not determine whether IMB-0523 binds to IFN receptors to activate the JAK/STAT signaling pathway or whether IMB-0523 has other mechanisms that regulate the JAK/STAT signaling pathway. Moreover, IMB-0523 directly activates STAT3 in a manner independent of JAK1/TYK2 signal transduction, suggesting that IMB-0523 may act as a STAT3 agonist, which needs to be further confirmed. Our subsequent research aims to confirm the targets of IMB-0523 to facilitate its development as an antiviral drug.
Figure 6: IMB-0523 inhibited EV71 replication by activating STAT3 signaling to upregulate the expression of ISGs . A3G: apolipoprotein B messenger RNA (mRNA)–editing enzyme catalytic polypeptide-like 3G; EV71: enterovirus 71 ; JAK1: Janus kinase-1; MX1: myxovirus protein 1; OAS1: 2′-5′-oligoadenylate synthetase 1; STAT3 : signal transducer and activator of transcription 3; TYK2: Tyrosine kinase 2; ISGs : interferon-stimulated genes.
IMB-0523 upregulated the expression of various ISGs , suggesting that it may have broad-spectrum antiviral effects [Figure 3 ]. In the present study, IMB-0523 indeed inhibited the replication of different EV71 strains and CVBs [Table 1 ]. Moreover, IMB-0523 inhibited the replication of CVA16 [Table 1 ]. Our previous study also confirmed that IMB-0523 has anti-HBV effect, indicating that IMB-0523 has broad-spectrum antiviral effects.[ 17 ] Considering the targeting of host proteins, long-term administration of host-targeting antiviral drugs may have toxic adverse effects. IMB-0523 showed low acute toxicity (LD50 , 448 mg/kg) in mice.[ 17 ] Therefore, although IMB-0523 has a similar effect of upregulating ISGs as IFNs, its toxic adverse effects may be far less than those of IFNs. Moreover, the short-term administration of acute viral diseases may not cause significant adverse effects. Therefore, IMB-0523 may be more suitable for the treatment of acute viral diseases.
In conclusion, this study proved that IMB-0523 upregulates ISG expression by activating STAT3 signaling to inhibit EV71 and CVBs replication. The IFN-like activity and low toxicity of IMB-0523 suggest that IMB-0523 may be a lead compound with good potential for the development of broad-spectrum antiviral drugs.
Funding
The work was supported by CAMS Innovation Fund for Medical Sciences (2021-I2M-1-030) and the National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (2018ZX09711003).
Author Contributions
Yuhuan Li configured this study. Huiqiang Wang and Miao Ge carried out most of the assay, analyzed data, and wrote the manuscript. Yanping Li was responsible for the synthesis of IMB-0523 . Zhuorong Li and Yuhuan Li contributed to the critical revision of the manuscript. All authors reviewed and approved the final version.
Conflicts of Interest
None.
Data Availability Statement
The data sets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
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