Antiviral Action of Tryptanthrin Isolated from Strobilanthes cusia Leaf against Human Coronavirus NL63

Abstract

Strobilanthes cusia (Nees) Kuntze is a Chinese herbal medicine used in the treatment of respiratory virus infections. The methanol extract of S. cusia leaf contains chemical components such as β-sitosterol, indirubin, tryptanthrin, betulin, indigodole A, and indigodole B that have diverse biological activities. However, the antiviral action of S. cusia leaf and its components against human coronavirus remains to be elucidated. Human coronavirus NL63 infection is frequent among immunocompromised individuals, young children, and in the elderly. This study investigated the anti-Human coronavirus NL63 (HCoV-NL63) activity of the methanol extract of S. cusia leaf and its major components. The methanol extract of S. cusia leaf effectively inhibited the cytopathic effect (CPE) and virus yield (IC₅₀ = 0.64 μg/mL) in HCoV-NL63-infected cells. Moreover, this extract potently inhibited the HCoV-NL63 infection in a concentration-dependent manner. Among the six components identified in the methanol extract of S. cusia leaf, tryptanthrin and indigodole B (5aR-ethyltryptanthrin) exhibited potent antiviral activity in reducing the CPE and progeny virus production. The IC₅₀ values against virus yield were 1.52 μM and 2.60 μM for tryptanthrin and indigodole B, respectively. Different modes of time-of-addition/removal assay indicated that tryptanthrin prevented the early and late stages of HCoV-NL63 replication, particularly by blocking viral RNA genome synthesis and papain-like protease 2 activity. Notably, tryptanthrin (IC₅₀ = 0.06 μM) and indigodole B (IC₅₀ = 2.09 μM) exhibited strong virucidal activity as well. This study identified tryptanthrin as the key active component of S. cusia leaf methanol extract that acted against HCoV-NL63 in a cell-type independent manner. The results specify that tryptanthrin possesses antiviral potential against HCoV-NL63 infection.

Keywords: Strobilanthes cusia; antiviral; human coronavirus NL63; indigodole B; tryptanthrin; virucidal.

Figure 1

Inhibitory effects of S. cusia leaf methanol extract on viral cytopathicity and progeny virus production in HCoV-NL63-infected cells. LLC-MK-2 cells were infected with HCoV-NL63 at 0.01 multiplicity of infection (MOI) and simultaneously treated with the extract. Virus-induced cytopathic effects were imaged 36 h post-infection (hpi) by microscopy (A). The supernatant from treated infected cells was harvested 36 hpi and serially diluted for determining the HCoV-NL63 yield in the supernatant using the plaque assay (B, right y axis). The rate of virus yield inhibition was calculated based on the ratio of the loss in the titer of the treated group to mock-treated group (B, left y axis). +, add; −, not add; *, p value < 0.05; ***, p value < 0.001 compared with mock-treated infected cells. Scale bar, 100 μM.

Figure 2

Inhibition of HCoV-NL63 infectivity by S. cusia leaf methanol extract. LLC-MK2 cells were infected with HCoV-NL63 and immediately treated with the extract for 36 h at 37 °C, and then subjected to immunofluorescence staining with anti-HCoV-NL63 immunized sera and secondary antibody Alexa Fluor anti-mouse IgG (A, top); total cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (A, bottom). Infectivity inhibition was determined according to the decrease in the ratio of HCoV-NL63-positive cells to total cells (B). +, add; −, not add; ***, p value < 0.001 compared with untreated infected cells. Scale bar, 100 μM.

Figure 3

Inhibitory effects of S. cusia extract components on viral cytopathicity and HCoV-NL63 infectivity. LLC-MK2 cells were infected with HCoV-NL63 and immediately treated with 40 μM of the indicated components for 36 h at 37 °C, photographed for evaluating the relative cytopathic effect (CPE) levels (A), and the residual HCoV-NL63 infectivity was determined using immunofluorescence staining with anti-HCoV-NL63 antibodies (B, top) and DAPI (B, bottom). The ratio of HCoV-NL63-positive cells was calculated as the percentage of positive cells to total cells (C). +, add; −, not add; ***, p value < 0.001 compared with mock-treatment group. Scale bar, 100 μM.

Figure 4

Inhibition of viral cytopathicity and virus yield by tryptanthrin and indigodole B. The structure of tryptanthrin and indigodole B is shown in (A). Images of CPE reduction by tryptanthrin and indigodole B were captured 36 hpi (B,C). The inhibitory activity of tryptanthrin and indigodole B on virus yield was calculated based on the ratio of titer loss in the treated group to that in the mock-treated group (D,E). +, add; −, not add; **, p value < 0.01; ***, p value < 0.001 compared with untreated infected cells. Scale bar, 100 μM.

Figure 5

Tryptanthrin inhibited HCoV-NL63 infectivity in human airway epithelial cells. Calu-3 cells were infected with HCoV-NL63 and immediately treated with tryptanthrin for 36 h at 32 °C. Images of relative CPE levels in each group were captured (A, top). In addition, the cells were subjected to immunofluorescence staining with anti-HCoV-NL63 antibodies plus secondary antibody Alexa Fluor anti-mouse IgG (A, middle) and DAPI (A, bottom). Infectivity inhibition activity was determined based on the change in the percentage of HCoV-NL63-positive cells (B). +, add; −, not add; **, p value < 0.01; ***, p value < 0.001 compared with untreated infected group. Scale bar, 100 μM.

Figure 6

Time-of-addition and removal assay for analyzing antiviral action of tryptanthrin against HCoV-NL63. The cell monolayer was infected with HCoV-NL63 and treated with tryptanthrin simultaneously (A, early stage), or 1 hpi (C, late stage). After a 2 h of incubation, the virus/tryptanthrin mixture was removed; the cell monolayer was washed with PBS and cultured for an additional 36 h with incubation at 37 °C, and then subjected to immunofluorescence staining using anti-HCoV-NL63 antibodies plus secondary antibody Alexa Fluor anti-mouse IgG (A,C, top) and DAPI (A,C, bottom). Infectivity was determined according to the percentage of HCoV-NL63-positive cells (B,D). +, add;−, not add; ***, p value < 0.001 compared with untreated infected group. Scale bar, 100 μM.

Figure 7

Time-of-addition and removal assay for examining the effect of tryptanthrin on the early and late stages of HCoV-NL63 replication. The cell monolayer was infected with HCoV-NL63 and treated with tryptanthrin simultaneously (A, early stage), or 1 hpi (B, late stage). After 2 h of incubation, the virus/tryptanthrin mixture was removed; the cell monolayer was washed with PBS and cultured for an additional 36 h with incubation at 37 °C. The extracellular virus yield in the supernatant was determined using the plaque assay; the inhibition rate was analyzed based on the loss in the ratio of virus titer in treated group to that in the mock-treated group. **, p value < 0.01; ***, p value < 0.001 compared with mock-treated infected cells.

Figure 8

Inhibitory effect of tryptanthrin on the synthesis of viral RNA genome in the early and late stages of HCoV-NL63 replication. The cell monolayer was infected with HCoV-NL63 and treated with tryptanthrin simultaneously (A, early stage), or 1 hpi (B, late stage). After 2 h of incubation, the virus/tryptanthrin mixture was removed; the cell monolayer was washed with PBS and cultured for an additional 24 h with incubation at 37 °C. Total RNA from treated and transfected cells was extracted and reverse transcribed with HCoV-NL63-specific primers. Relative viral RNA genomes were measured by quantitative PCR and normalized by β-actin mRNA. **, p value < 0.01; ***, p value < 0.001 compared with mock-treated infected cells.

Figure 9

Inhibition of in vitro trans-cleavage activity of recombinant HCoV-NL63 papain-like protease 2 by tryptanthrin. The purified recombinant HCoV-NL63 papain-like protease 2 (PLP2) was analyzed by 10% SDS-PAGE with Coomassie blue staining (A, right) and Western Blotting with anti-His tag antibodies (A, left). The trans-cleavage activity of HCoV-NL63 PLP2 was evaluated using the ELISA with horseradish peroxidase (HRP) containing an LXGG motif as the substrate. After 2 h of incubation at 37 °C, the enzyme activity of the residual HRP (in intact form) was detected using a chromogen reagent (ABTS/H₂O₂). The ELISA product was measured at A_{405 nm} (B). Tryptanthrin at indicated concentrations was added into the mixture of HCoV-NL63 PLP2 and HRP, and then incubated for 2 h at 37 °C. Lastly, the enzyme activity of the residual HRP (in intact form) was determined using the chromogen reagent (ABTS/H₂O₂), calculated as 1−(OD405 with PLP2)/(OD405 without PLP2) (C). Moreover, the relative inhibition of the cleavage activity of PLP2 by tryptanthrin was estimated as 1−(OD405 without PLP2−OD405 with PLP2 plus tryptanthrin)/(OD405 without PLP2−OD405 with PLP2) (D). **, p value < 0.01; ***, p value < 0.001 compared with the mock-treated group.

Figure 10

Virucidal activity of tryptanthrin, indigodole B, and S. cusia extract. Tryptanthrin (A), indigodole B (B), or the extract (C) at the indicated concentrations was mixed with HCoV-NL63 (10⁵ pfu), and incubated at 37 °C for 1 h. The 1000-fold dilution of the compound/virus mixture was added into the LLC-MK2 cell monolayer in 6-well plates for examining the residual infectivity by plaque assay. Virucidal activity was calculated based on the percentage of residual plaques in each treated group. ***, p value < 0.001 compared with untreated infected cells.

Figure 11

Dot-blotting assay of the HCoV-NL63/tryptanthrin mixture incubated at 37 °C for 1 h. Tryptanthrin at indicated concentrations was mixed with HCoV-NL63 (10⁵ pfu), and then incubated at 37 °C for 1 h. Fifteen microliters of the compound/virus mixture was added onto the nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% skim milk in Tris-Buffered Saline plus 0.1% Tween-20 (TBST) for 2 h, incubated with anti-HCoV-NL63 antibodies overnight, and then reacted with HRP-conjugated anti-mouse IgG antibodies. After washing with TBST, immunoreactive spots of HCoV-NL63 were developed with ECLTM Western Blotting Detection Reagents, and then imaged by the Multi-function Gel Image System. +, add; −, not add;