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Development of antiviral carbon quantum dots that target the Japanese encephalitis virus envelope protein

2022; Elsevier BV; Volume: 298; Issue: 6 Linguagem: Inglês

10.1016/j.jbc.2022.101957

1083-351X

Han-Hsiang Chen, Chin‐Jung Lin, Anisha Anand, H.‐J. Lin, Hung‐Yun Lin, Ju‐Yi Mao, Peihua Wang, Yufeng Jane Tseng, Wen‐Shyong Tzou, Chih‐Ching Huang, Robert Wang,

Advanced battery technologies research

Japanese encephalitis is a mosquito-borne disease caused by the Japanese encephalitis virus (JEV) that is prevalent in Asia and the Western Pacific. Currently, there is no effective treatment for Japanese encephalitis. Curcumin (Cur) is a compound extracted from the roots of Curcuma longa, and many studies have reported its antiviral and anti-inflammatory activities. However, the high cytotoxicity and very low solubility of Cur limit its biomedical applications. In this study, Cur carbon quantum dots (Cur-CQDs) were synthesized by mild pyrolysis-induced polymerization and carbonization, leading to higher water solubility and lower cytotoxicity, as well as superior antiviral activity against JEV infection. We found that Cur-CQDs effectively bound to the E protein of JEV, preventing viral entry into the host cells. In addition, after continued treatment of JEV with Cur-CQDs, a mutant strain of JEV was evolved that did not support binding of Cur-CQDs to the JEV envelope. Using transmission electron microscopy, biolayer interferometry, and molecular docking analysis, we revealed that the S123R and K312R mutations in the E protein play a key role in binding Cur-CQDs. The S123 and K312 residues are located in structural domains II and III of the E protein, respectively, and are responsible for binding to receptors on and fusing with the cell membrane. Taken together, our results suggest that the E protein of flaviviruses represents a potential target for the development of CQD-based inhibitors to prevent or treat viral infections. Japanese encephalitis is a mosquito-borne disease caused by the Japanese encephalitis virus (JEV) that is prevalent in Asia and the Western Pacific. Currently, there is no effective treatment for Japanese encephalitis. Curcumin (Cur) is a compound extracted from the roots of Curcuma longa, and many studies have reported its antiviral and anti-inflammatory activities. However, the high cytotoxicity and very low solubility of Cur limit its biomedical applications. In this study, Cur carbon quantum dots (Cur-CQDs) were synthesized by mild pyrolysis-induced polymerization and carbonization, leading to higher water solubility and lower cytotoxicity, as well as superior antiviral activity against JEV infection. We found that Cur-CQDs effectively bound to the E protein of JEV, preventing viral entry into the host cells. In addition, after continued treatment of JEV with Cur-CQDs, a mutant strain of JEV was evolved that did not support binding of Cur-CQDs to the JEV envelope. Using transmission electron microscopy, biolayer interferometry, and molecular docking analysis, we revealed that the S123R and K312R mutations in the E protein play a key role in binding Cur-CQDs. The S123 and K312 residues are located in structural domains II and III of the E protein, respectively, and are responsible for binding to receptors on and fusing with the cell membrane. Taken together, our results suggest that the E protein of flaviviruses represents a potential target for the development of CQD-based inhibitors to prevent or treat viral infections. Viruses are responsible for more than 30% of deaths from infectious diseases (1Hansen V. Oren E. Dennis L.K. Brown H.E. Infectious disease mortality trends in the United States, 1980−2014.JAMA. 2016; 316: 2149-2151Crossref PubMed Scopus (39) Google Scholar). For example, COVID-19, an emerging infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected over 417 million people worldwide and caused more than 6 million deaths as of March 2022. Therefore, there has been a dramatic increase in the demand for antiviral agents to combat viral infections. However, the development of new antiviral drugs requires considerable time and several stages before they can be approved for human use. Therefore, the development of innovative, long-term, and highly flexible drugs with virostatic and/or virucidal properties to complement existing pharmacological approaches against viral infection and transmission is essential. Many nanomaterials have been shown to exhibit intrinsic antiviral activities (2Chakravarty M. Vora A. Nanotechnology-based antiviral therapeutics.Drug Deliv. Transl. Res. 2021; 11: 748-787Crossref PubMed Scopus (125) Google Scholar, 3Jackman J.A. Yoon B.K. Ouyang L. Wang N. Ferhan A.R. Kim J. Majima T. Cho N.J. Biomimetic nanomaterial strategies for virus targeting: Antiviral therapies and vaccines.Adv. Funct. Mater. 2021; 31: 2008352Crossref Scopus (20) Google Scholar, 4Zhou J. Krishnan N. Jiang Y. Fang R.H. Zhang L. Nanotechnology for virus treatment.Nano Today. 2021; 36: 101031Crossref PubMed Scopus (45) Google Scholar, 5Yoon B.K. Jeon W.-Y. Sut T.N. Cho N.-J. Jackman J.A. 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Sangam S. Kochhar D. Pahari S. Kar C. Mukherjee M. Exploring the role of triazole functionalized heteroatom Co-doped carbon quantum dots against human coronaviruses.Nano Today. 2020; 35: 101001Crossref PubMed Scopus (42) Google Scholar, 26Iannazzo D. Pistone A. Ferro S. De Luca L. Monforte A.M. Romeo R. Buemi M.R. Pannecouque C. Graphene quantum dots based systems as HIV inhibitors.Bioconjug. Chem. 2018; 29: 3084-3093Crossref PubMed Scopus (99) Google Scholar, 27Tong T. Hu H. Zhou J. Deng S. Zhang X. Tang W. Fang L. Xiao S. Liang J. Glycyrrhizic-acid-based carbon dots with high antiviral activity by multisite inhibition mechanisms.Small. 2020; 16: 1906206Crossref Scopus (143) Google Scholar). Compared to other antiviral nanomaterials, the highly flexible CQDs from different precursors confer their ability to fight various viruses. The precursor molecules residing on CQDs are mainly responsible for their antiviral effects. Although many antiviral mechanisms have been proposed for CQDs, such as preventing viral particles from attaching to host cells, inhibiting viral replication, and stimulating innate immune response, most reported CQDs interfere with the attachment and penetration steps through direct contact between CQDs and viral membranes (13Lin C.J. Chang L. Chu H.W. Lin H.J. Chang P.C. Wang R.Y. Unnikrishnan B. Mao J.Y. Chen S.Y. Huang C.C. High amplification of the antiviral activity of curcumin through transformation into carbon quantum dots.Small. 2019; 15e1902641Google Scholar, 23Barras A. Pagneux Q. Sane F. Wang Q. Boukherroub R. Hober D. Szunerits S. High efficiency of functional carbon nanodots as entry inhibitors of herpes simplex virus type 1.ACS Appl. Mater. Inter. 2016; 8: 9004-9013Crossref PubMed Scopus (93) Google Scholar, 24Du T. Liang J. Dong N. Liu L. Fang L. Xiao S. Han H. Carbon dots as inhibitors of virus by activation of type I interferon response.Carbon. 2016; 110: 278-285Crossref Scopus (105) Google Scholar, 25Garg P. Sangam S. Kochhar D. Pahari S. Kar C. Mukherjee M. Exploring the role of triazole functionalized heteroatom Co-doped carbon quantum dots against human coronaviruses.Nano Today. 2020; 35: 101001Crossref PubMed Scopus (42) Google Scholar, 26Iannazzo D. Pistone A. Ferro S. De Luca L. Monforte A.M. Romeo R. Buemi M.R. Pannecouque C. Graphene quantum dots based systems as HIV inhibitors.Bioconjug. Chem. 2018; 29: 3084-3093Crossref PubMed Scopus (99) Google Scholar, 27Tong T. Hu H. Zhou J. Deng S. Zhang X. Tang W. Fang L. Xiao S. Liang J. Glycyrrhizic-acid-based carbon dots with high antiviral activity by multisite inhibition mechanisms.Small. 2020; 16: 1906206Crossref Scopus (143) Google Scholar). Even so, the mechanism of binding of CQDs to viral particles is still far from being fully understood. We previously reported that CQDs derived from curcumin (Cur) exhibited superior antiviral activity against enterovirus 71 (13Lin C.J. Chang L. Chu H.W. Lin H.J. Chang P.C. Wang R.Y. Unnikrishnan B. Mao J.Y. Chen S.Y. Huang C.C. High amplification of the antiviral activity of curcumin through transformation into carbon quantum dots.Small. 2019; 15e1902641Google Scholar). However, the antiviral mechanism of Cur-CQDs is unclear, even though we proposed the CQDs bind mainly to virions and prevent them from attaching to host cells. In this work, we sought to achieve antiviral potency of Cur-CQDs against another virus, Japanese encephalitis virus (JEV), and to provide direct insight into the interaction between CQDs and virions. Japanese encephalitis has become one of the serious health problems in tropical and subtropical regions of the world, with high mortality and neurological complications in patients even after recovery (29Turtle L. Solomon T. Japanese Encephalitis−The prospects for new treatments.Nat. Rev. Neurol. 2018; 14: 298-313Crossref PubMed Scopus (156) Google Scholar). JEV, an enveloped flavivirus, is the leading cause of viral encephalitis in Asia, with an estimated 70,000 clinical cases per year (30Mansfield K.L. Hernández-Triana L.M. Banyard A.C. Fooks A.R. Johnson N. Japanese encephalitis virus infection, diagnosis and control in domestic animals.Vet. Microbiol. 2017; 201: 85-92Crossref PubMed Scopus (101) Google Scholar). Currently, there is no effective antiviral treatment for JEV, and patients can only be provided with supportive therapy to relieve symptoms. As with other flaviviruses, once JEV enters the host cell via endocytosis, the surface envelope (E) protein of the virion plays a central role in mediating the process of fusion of the viral envelope with the endosomal membrane at low pH. As a result, the genome is released into the cytoplasm, which then completes the steps of replication, assembly, and egress of the JEV. In this study, the antiviral Cur-CQDs were obtained by pyrolysis of Cur at 180 °C for 2 h. Cur-CQDs exhibited superior solubility (50 mg ml−1) in an aqueous solution compared to Cur ( 100 μg ml−1; incubation of CQDs and BHK-21 cells was performed at 4 °C), further supporting the effect of Cur-CQDs on JEV (Fig. S4A, Supporting information). Meanwhile, addition of Cur-CQDs (100 μg ml–1) immediately prior to infection of BHK-21 cells with JEV (MOI=1) without removal of Cur-CQDs showed similar antiviral activity as in the cotreatment experiments (premixed JEV and Cur-CQDs, followed by infection of BHK-21 cells). The IC50 of Cur-CQDs to inhibit the virus in a cotreatment manner was much lower than most reported antiviral carbon-based nanomaterials (24Du T. Liang J. Dong N. Liu L. Fang L. Xiao S. Han H. Carbon dots as inhibitors of virus by activation of type I interferon response.Carbon. 2016; 110: 278-285Crossref Scopus (105) Google Scholar, 26Iannazzo D. Pistone A. Ferro S. De Luca L. Monforte A.M. Romeo R. Buemi M.R. Pannecouque C. Graphene quantum dots based systems as HIV inhibitors.Bioconjug. Chem. 2018; 29: 3084-3093Crossref PubMed Scopus (99) Google Scholar, 43Du T. Lu J. Liu L. Dong N. Fang L. Xiao S. Han H. Antiviral activity of graphene oxide–silver nanocomposites by preventing viral entry and activation of the antiviral innate immune response.ACS Appl. Bio. Mater. 2018; 1: 1286-1293Crossref PubMed Scopus (78) Google Scholar, 44Fahmi M. Sukmayani W. Khairunisa S.Q. Witaningrum A. Indriati D. Matondang M. Chang J.-Y. Kotaki T. Kameoka M. Design of boronic acid–attributed carbon dots on inhibits HIV-1 entry.RSC Adv. 2016; 6: 92996-93002Crossref Google Scholar, 45Ziem B. Azab W. Gholami M. Rabe J.P. Osterrieder N. Haag R. Size-dependent inhibition of herpesvirus cellular entry by polyvalent nanoarchitectures.Nanoscale. 2017; 9: 3774-3783Crossref PubMed Google Scholar). This suggests that the antiviral activity of Cur-CQDs is mainly through the inhibition of the early steps of the viral infection, which may be the mechanism of viral attachment and entry. Since the antiviral activity of Cur-CQDs is due to the inhibition of early viral infection stages, we further elucidated that the antiviral effect is due to the binding of Cur-CQDs to viral particles or host cell membranes. The pretreatment assay was performed at 37 °C or 4 °C to understand the binding between Cur-CQDs and host cells or viruses. Cur-CQDs were added to cell cultures and incubated at 37 °C or 4 °C for 2 h, followed by infection with JEV for 8 h or 24 h. The results of RT-qPCR analysis showed a reduction in viral RNA under pretreatment at 37 °C and after 8 h or 24 h of infection (Fig. 3A). However, a more significant reduction of about 40% in viral RNA was observed with pretreatment at 4 °C (Fig. 3B). Therefore, we hypothesized that Cur-CQDs would attach to the cell membrane at 4 °C and prevent viral particles from binding to the cell, thereby inhibiting virus infection. However, cotreatment exhibited >95% inhibition (Fig. 3C), indicating that Cur-CQDs bound to JEV contributed significantly to viral inhibition. The interaction between Cur-CQDs and JEV was supp