Palmatine inhibits Zika virus infection by disrupting virus binding, entry, and stability

https://doi.org/10.1016/j.bbrc.2019.08.120Get rights and content

Highlights

  • Palmatine inhibits ZIKV infection by disrupting virus binding, entry, and stability.

  • Palmatine was shown to bind to restricted viruses, inhibit ZIKV infection, and resist ZIKV-induced cytopathic effects.

  • Palmatine could provide a basis for the development of novel stretages against ZIKV infection.

Abstract

Zika virus (ZIKV) is an emerging vector-borne virus that is associated with severe congenital cerebral anomalies in fetuses and paralytic Guillain-Barré syndrome in adults. In the current global health crisis, there are no vaccines or therapeutics available for the treatment of ZIKV infection. In the present study, we evaluated the efficacy of the protoberberine alkaloid, palmatine, in inhibiting ZIKV and Japanese encephalitis virus (JEV). Palmatine was shown to bind to restricted viruses, inhibit ZIKV infection, and resist ZIKV-induced cytopathic effects. Palmatine was also shown to inhibit JEV infection in multiple cell lines. Overall, the effects of palmatine in disrupting ZIKV binding, entry, and stability indicate that this small molecule would be a good starting point for the development of treatments aimed at inhibiting ZIKV infection.

Introduction

Zika virus (ZIKV) is a member of the Flaviviridae family, which includes well-known human and animal pathogens [3,4], and the Flavivirus genus [1]. ZIKV comprises 53 different species, which include non-vector, tick-borne, and mosquito-borne clusters [2], such as dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and west Nile virus (WNV). ZIKV (strain MR 766) was first isolated from rhesus monkeys in Uganda in 1947 [5]; however, it was not until 2015 that ZIKV reappeared as an epidemic in at least 33 Central and South American countries [6,7]. As of May 2019, the virus had been identified in 84 countries, territories, or subnational areas [2]. Common clinical symptoms of ZIKV infection include fever, headache, joint pain, conjunctivitis, and macular atrophy [7]. To date, there are no FDA-approved anti-ZIKV drugs, and thus most clinical treatment strategies employ analgesics and antipyretics to alleviate symptoms [8]. Evidence suggests that ZIKV can be transmitted congenitally, perinatally, and sexually [7,9] and that ZIKV infection is directly associated with neurological diseases, including Guillain–Barré syndrome and congenital microcephaly [10].

ZIKV is an enveloped RNA virus with a single-stranded positive-sense genome of approximately 10.7 kb [11]. The open reading frame of the ZIKV genome encodes a single polyprotein, which is cleaved after translation to produce (1) three structural proteins: envelope (E), capsid (C) and pre-membrane (prM) proteins, as well as (2) seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [12,13]. The three-dimensional structure of ZIKV includes NS, NS2B-NS3 protease, NS3 helicase, NS5 methyltransferase, and NS5 RNA polymerase, all of which are involved in viral polyprotein processing and RNA synthesis processes [[14], [15], [16], [17], [18], [19], [20], [21], [22], [23]], including RNA maturation, splicing, and nuclear export [24]. NS3 is a major component of the ZIKV replication complex. This non-structural protein includes an N-terminal serine protease domain (NS3Pro), a multifunctional domain which is primarily involved in polyprotein processing, and a C-terminal region which is involved in RNA replication of RNA helicase (NS3Hel) [19,23]. The N-terminal region and the cytoplasmic region of the two-component protease NS2B-NS3 constitute the ZIKV protease, which acts as a cofactor involved in positioning the catalytic triad to catalyze its substrate [25]. However, NS3Hel belongs to the helicase superfamily of nucleic acid-dependent NTPases and is capable of unraveling DNA or RNA duplex substrates, which have key functions in viral replication. These enzymes have therefore become targets in the development of antiviral drugs [26], which are desperately needed to help control future outbreaks.

Palmatine is a protoberberine alkaloid that can be isolated from plants, including Fibraurea recisa Pierre, Phellodendron Amurense, Enantia Chlorantha, Corydalis yanhusuo, and Coptis Chinensis [27,28]. The anti-inflammatory and antibacterial properties of palmatine have led to its widespread application in treating gynecological inflammation, enteritis, urinary tract infections, and conjunctivitis [29,30]. Due to its effects on the nervous and cardiovascular systems, it has also been widely used to treat hypertension, arrhythmia, and myocardial ischemia [[30], [31], [32], [33]]. Researchers have begun to explore the anti-parasitic, anti-tumor, and anti-fungal activities of palmatine as well as its potential applicability in the treatment of microbial parasitic infections [[34], [35], [36]]. Palmatine is a raw material derived from the synthesis of tetrahydropalmatine, which is an important analgesic and antiarrhythmic drug [30,37]. Palmatine has also been shown to inhibit WNV in a non-competitive manner and to inhibit DENV and YFV in a dose-dependent manner. This suggests that palmatine has the potential to be developed as a treatment for flavivirus infections as well [38].

Results from our previous study indicated that palmatine is an effective anti-ZIKV drug. These findings prompted us to investigate the mechanism by which palmatine disrupts ZIKV infection. We also examined the effectiveness of palmatine in inhibiting JEV infection in A549 and BHK-21 cells. The current study is the first report on the anti-ZIKV and anti-JEV properties of palmatine.

Section snippets

Cells, viruses, and compounds

Vero cells (ATCC® CCL-81™) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 5% fetal bovine serum (FBS), antibiotics, and L-Glutamine under 5% CO2 at 37 °C. ZIKV (ATCC® VR-1843™). JEVs (RP9 strain) were propagated in Vero cells, which were cultured in DMEM containing 0.5% FBS, antibiotics, and L-Glutamine under 5% CO2 at 37 °C. Palmatine was purchased from ChemFaces (ChemFaces, CFN98459), dissolved in 100% dimethyl sulfoxide (DMSO) as a stock of 20 mM, and maintained

Palmatine showed an ability to inhibit ZIKV infection

In these experiments (which involved seeding Vero cells in 12-well plates, infecting them with ZIKV at 400 pfu per well, and inoculating them using various concentrations of palmatine), palmatine was shown to inhibit ZIKV infection. After a 2-day incubation period, the RNA of infected cells was extracted using Trizol reagent. qRT-PCR assays revealed that palmatine (at concentrations ranging from 10 to 80 μM) was able to inhibit the production of ZIKV RNA (Fig. 1A). Supernatant collected from

Discussion

Climate change and global travel have greatly accelerated the spread of flaviviruses, such as ZIKV; however, clinically approved antiviral drugs and vaccines have yet to be developed. To effectively manage and control these infections, elucidating the mechanisms which underlie disease generation is crucial [7]. Since 2015, a number of high-throughput studies that employed whole-virus assays or protein-based assays to screen compound libraries have identified a variety of small molecules which

Conflicts of interest

None.

Acknowledgements

The authors' work was supported in part by grants from the Ministry of Science and Technology (MOST 107-2320-B-016-006- and MOST 108-2320-B-016 -011 -MY2), and the grants from Ministry of National Defense (MAB-108-061) at Taiwan.

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    Y.-J. Ho and J.-W. Lu contributed equally to this work.

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