Inhibition of herpes simplex virus 1 by cepharanthine via promoting cellular autophagy through up-regulation of STING/TBK1/P62 pathway
Introduction
Herpes simplex virus type-1 (HSV-1), a member of the α-herpesviruses subfamily, replicates in epithelial cells and establishes a lifetime incubation period in neurons, causing severe disease such as herpetic meningitis and blindness among immune weakness people due to the increased frequency of reactivation of virus (Whitley and Roizman, 2001; Wilson and Mohr, 2012). Synthetic nucleoside analogs such as acyclovir and ganciclovir can inhibit the action of virus DNA polymerase and are used for treating HSV-1 infection (Kukhanova et al., 2014; Vere Hodge and Field, 2013). However, side effects and drug resistance limit their application, especially among people with low immunity, making it more crucial to search for new antiviral drugs against HSV-1 infection (Bacon et al., 2003; Florence Morfin, 2003; Prichard et al., 2011; Watson et al., 2017).
In recent years, the naturally-derived components have been increasingly captured researcher's attention for their potential use as pharmaceuticals or as lead structures for developing novel therapeutic agents. Cepharanthine (CEP) is a naturally occurring isoquinoline alkaloid extracted from plants of the genus Stephania, which belongs to the moonseed family Menispermaceae. Because of its excellent pharmacological actions such as anti-inflammatory, antitumor, antivirus, and immunity enhancing, CEP was generally combined with antitumor drugs to treat the symptoms such as immunosuppression and thrombocytopenia caused by chemotherapy and radiotherapy without obvious side effects (Bailly, 2019). Meanwhile, previous studies have been shown that CEP could inhibit infections of HIV, SARS, COVID-19, HSV-1, Ebola, and other virus and exhibit the action of immuno-regulatory, making it a promise drug to treat various autoimmune diseases and allergic reactions (Bailly, 2019; Kim et al., 2019; Moshe Rogosnitzky, 2011). CEP could be applied as a therapeutic potential broad-spectrum antiviral drug. However, no data are available for the antiviral of CEP against HSV-1, and the underlying mechanisms are elusive.
STING, also known as TMEM173, MPYS, and MITA, is an endoplasmic reticulum (ER) resident transmembrane protein crucial for innate immunity against various pathogenic microorganisms (Ishikawa et al., 2009; Kazuki Kato et al., 2017; Motwani et al., 2019). Previous studies associated with the anti-infection effect of STING have increasingly focused on STING/TBK1/IRF3 pathway, which is mediated by interferon. Nevertheless, recent studies showed that interferon-independent antiviral activities mediated by STING are indispensable against HSV-1 infections, and autophagy plays an important role in this process (Reinert et al., 2016; Wu et al., 2020; Yamashiro et al., 2020). STING could be activated by cyclic GMP-AMP (cGAMP), produced by cyclic GMP-AMP synthase (cGAS) after detection of dsDNA. The translocation of STING from the endoplasmic reticulum to Golgi apparatus enables its binding to TANK-binding kinase 1 (TBK1), which could phosphorylate the autophagic adaptor p62 on Ser-403 and promote the maturation of autophagy body into autophagy-lysosome (Gui et al., 2019; Pilli et al., 2012; Saitoh et al., 2009; Yang et al., 2019). Autophagy-lysosome exhibits antiviral actions by counteracting apoptosis in infected cells (Tovilovic et al., 2013), degrading virus particles, excess or defective proteins and organelles (Kudchodkar and Levine, 2009; Levine, 2005; Mehrbod et al., 2019), as well as activating the adaptive immune response (Dengjel et al., 2005; English et al., 2009).
During host-virus interactions, viruses exploit cellular machinery to create a suitable environment and utilize the host nucleic acids and proteins to accomplish their life cycle (Amin et al., 2019; Banerjee et al., 2020; Su et al., 2016; Zhu and Zheng, 2020). At the same time, the viruses developed multiple mechanisms to avoid degradation by autophagy-lysosome induced by STING (Cavignac and Esclatine, 2010; Liu et al., 2019; O'Connell and Liang, 2016; Verzosa et al., 2021). This study investigates the roles of CEP-mediated autophagy against HSV-1 infections. We hypothesize that autophagy-mediated by CEP may exert antiviral activity through degrading viral particles and cellular components required for viral replication and mature, and this process mainly relies on the STING/TBK1/P62 antiviral signaling pathway, which might provide novel strategies for the treatment of HSV-1 infection in the immunocompromised population.
Section snippets
Reagents, cell lines, plasmids, and virus
CEP (19052708, with a purity of 99.08 %) was purchased from Chengdu Must Biotechnology (Chengdu, Sichuan, China) and dissolved in DMSO (5 mg/mL) for preservation. Acyclovir (1411201) was purchased from QIAN JIANG Pharmaceutical (Qianjiang, Hubei, China) and dissolved in PBS (10 mg/mL) for preservation. Dulbecco's modified Eagle's medium (DMEM) and phosphatic buffer solution (PBS) were obtained from Gibco (New York, USA). Trypsin and Penicillin-streptomycin antibiotic were purchased from HyClone
Inhibitory effect of CEP on HSV-1 infection
CEP is a kind of isoquinoline alkaloid exhibiting anti-inflammatory, antivirus, and immunity enhancement (molecular structure shown in Fig. 1A). To explore its antivirus activities against HSV-1, we first test CEP's cytotoxic effects on Vero and HeLa cells. The CCK-8 results showed no obvious cytotoxicity of CEP at a concentration of less than or equal to 3 μg/mL, and the half-toxic concentration (TC50) of CEP on Vero and HeLa cells was 5.4 and 9 μg/mL, respectively (Fig. 1B). The toxicity
Discussion
Innate immunity provides the first line of defense for host cells to fight against invading microorganisms (Zheng, 2018; Zhu and Zheng, 2020). Autophagy is an important process to regulate homeostasis and cell survival, eliminating intracellular pathogens and contributing to innate immunity. Recent researches have stressed the complex interplay between autophagy and the HSV-1 virus (Cavignac and Esclatine, 2010; O'Connell and Liang, 2016). As reported, autophagy exerts dual actions in HSV-1
Conclusion
Overall, these findings have shown the potential of CEP as a promising antiviral agent against HSV-1 infection in vitro via promoting the maturation of autophagosome into autophagy-lysosome, resulting in the degradation of virus and cell components essential for the viral replication and envelope. Moreover, the results also revealed that CEP could regulate the STING/TBK1/P62 pathway without interferon induction, and the possible mechanism is proposed in Fig. 9. In addition, the reduction of HSV
Author contributions
YL and QT conceived and designed the study. YL, QT, YF, and WJL contributed to carry out the experiments. YL, QT, ZLR, and WJL contributed to data analysis. YL, FL, and XNJ wrote the manuscript. NZ and FL supervised the research. All authors read and approved the final version of the manuscript.
Funding
This work was supported by the National Natural Science Foundation of China (Grant no. 82074094), the Open Research Fund of Chengdu University of Traditional Chinese Medicine Key Laboratory of Systematic Research of Distinctive Chinese Medicine Resources in Southwest China (Grant no. 2020XSGG002), the Xinglin Scholar Research Promotion Project of Chengdu University of Traditional Chinese Medicine (Grant no. CDTD2018014), the Applied Basic Research Project of Science and Technology Department of
Declaration of interest
The authors declare that they have no competing interests.
Declaration of interests
☑ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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These authors contributed equally to this work.