Biochemical and Biophysical Research Communications
Palmatine inhibits Zika virus infection by disrupting virus binding, entry, and stability
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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2022, Biophysical ChemistryCitation Excerpt :It is the major component of herbal preparations used in traditional medicine Chinese, Korean and Indian for its properties such as anti-malarial, anti-oxidant, anti-inflammatory and low toxicity on human cells [18–20]. In anti-cancer therapy, as well as berberine [21], its analogue palmatine seems to inhibit the telomerase activity effectively [9,22]. In aqueous solutions palmatine shows a positive charge, which would favour the interaction with negatively charged phosphates within DNA structure.
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2021, Antiviral ResearchCitation Excerpt :Thereafter, cells were washed twice with PBS again before being added to fresh medium. After a 48 h incubation period, the levels of intracellular viral RNA and virus protein were determined by qRT-PCR and western blotting, respectively (Ho et al., 2015, 2019). The cells were infected with ZIKV (MOI = 0.05) at 4 °C for 1 h, and then incubated at 37 °C for another hour.
Cephalotaxine inhibits Zika infection by impeding viral replication and stability
2020, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Briefly, confluent Vero cells seeded in 12-well microplates were infected with FFUs of DENV1, DENV2, DENV3, or DENV4 and then incubated with indicated concentrations of CET for 48 h. Following this, dose-dependent viral RNA reduction assays were performed using qRT-PCR to determine whether CET was effective in inhibiting viral replication [23]. Statistical analysis was performed using GraphPad Prism software, and data were expressed as the mean ± standard deviation of triplicate experiments, The statistical significance of data was assessed using a two-tailed Student’s t-test, in which a p value of <0.05 was considered significant [22–24]. Earlier research found that CET was able to inhibit HBV infection [16]; however, the anti-viral effects of CET against other viruses have not been previously investigated.
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Y.-J. Ho and J.-W. Lu contributed equally to this work.