Ca2+ signaling is required for the entry of (+)ss RNA virus into host cells. It has been reported by a number of groups that Ca2+ signaling is essential for virus infection 22, 23, 24, 25, 26, 27, 28, 29, 30. Here, we added to this accumulating evidence by demonstrating that chelation of intracellular Ca2+ with BAPTA-AM markedly suppressed the JEV-induced expression of JEV envelope protein (JEV-E) (Fig. 1A). Since it has been reported that a Ca2+-dependent signaling pathway regulates the entry of the influenza A virus 27, 28, we subsequently studied if BAPTA-AM inhibits internalization of JEV. We performed in situ RNA hybridization to detect the positive strand RNA of JEV, the viral genome (vRNA), and showed that JEV was internalized as early as 20 min after infection, and a stronger vRNA signal was detected by 80 min after viral infection. However, when cells were treated with BAPTA-AM, no JEV vRNA was detected inside cells (Fig. 1B). We also examined whether the removal of extracellular Ca2+ might affect ZIKV infection of host cells by performing both in situ RNA hybridization assay to detect the vRNA of ZIKV, and immunocytochemistry to investigate the localization of ZIKV envelope protein (ZIKV-E). We showed that at 90 min after viral infection, in the presence of extracellular Ca2+ ZIKV vRNA was detected inside host cells (middle panel in Fig. 1C), but in the absence of extracellular Ca2+, no ZIKV vRNA was detected inside these cells (right panel in Fig. 1C). Moreover, in the absence of extracellular Ca2+, ZIKV virions, (manifested by ZIKV-E immunostaining), were detected at the plasma membrane of the host cells (right panel in Fig. 1C). These results indicate that Ca2+ signaling is required for the entry of (+)ss RNA viruses into host cells.
Berbamine inhibits JEV, ZIKV, DENV, SARS-CoV-2, and MERS-CoV infection in vitro. Double stranded RNAs (dsRNAs) are an intermediate in viral genome replication. They are generated by viral RNA polymerases following the infection of the (+)ss RNA viruses. These dsRNAs can be detected by performing immunofluorescence staining with an anti-dsRNA antibody 33, 34, 35. Thus, we used a combination of in situ RNA hybridization for the negative strand viral RNA ((-)RNA), and dsRNA immunostaining in the host cells to investigate the infection of the HeLa cells with EV-A71 or JEV. We found that 7h after virus infection, EV-A71 (-)RNA exhibited strong co-localization with dsRNA (Fig. S1A). DsRNA puncta were also detected in cells infected with JEV, and these puncta exhibited weak co-localization with EEA1 (an early endosome marker) or LAMP1 (a late endosome and lysosome marker) (Fig. S1B). This weak co- localization between dsRNA and endosomal or lysosomal markers suggests that for JEV, viral RNA replication does not occur at either the endosomes or lysosomes. Nevertheless, these results confirm that dsRNA immunostaining can be used to detect (+)ss RNA virus infection in host cells.
We, subsequently, developed a high-content image detection platform to detect and quantify the dsRNA immunostaining in virus-infected cells by automated fluorescence microscopy (Fig. 2A). We then applied this high-content image platform to assess the anti-viral activity of various compounds reported in the literature as being modulators of Ca2+ channels or Ca2+ signaling. Thus, A549 cells were seeded in 96-well plates, pretreated with different concentrations of various compounds for 1 h, and then infected with JEV, ZIKV or DENV. After 48 h of infection, the cells were fixed and subjected to dsRNA immunostaining and DAPI staining, after which images were captured with the CellInsight CX7 high-content screening platform and analyzed in HCS Studio™ 3.0 to quantify the percentage of infected cells (Fig. 2A).
By applying this high content image platform, we found that berbamine, a bis- benzylisoquinoline alkaloid isolated from the traditional Chinese medicine, Berberis, (which has reported effects on Ca2+ signaling), significantly inhibited the infection of JEV (Fig. 2B), ZIKV (Fig. 2C), and DENV (Fig. 2D). In A549 cells, the EC50 of berbamine against JEV and ZIKV was approximately 1.62 mM and 2.17 mM, respectively. Consistently, when we performed a virus titration assay, we could confirm that berbamine significantly inhibited the infectious progeny viral particle production of both ZIKV (Fig. 2E) and JEV (Fig. 2F). Taken together, these data not only demonstrate that berbamine is an effective anti-flavivirus agent, but also confirm the efficacy of our high-content image screening platform.
In addition to flaviviruses, we also assessed the activity of berbamine against coronavirus (e.g. MERS-CoV and SARS-CoV-2) infection in vitro. We treated primary human lung fibroblasts with berbamine and then infected them with MERS-CoV, after which we performed qRT-PCR to measure the amount of intra- or extracellular viral RNA. We showed that berbamine significantly decreased both the intracellular (Fig. 3A) and extracellular (Fig. 3B) level of MERs-CoV RNA. We also assessed the anti- SARS-CoV-2 activity of berbamine in VeroE6 cells, and found that berbamine significantly inhibited the viral yield, as quantified by qRT-PCR (Fig. 3C). The EC50 of berbamine against SARS-CoV-2 in Vero cells is ~2.3 mM. In summary, these data indicate that in addition to being an effective anti-flavivirus agent, berbamine is also an effective anti-SARS-CoV-2 and MERS-CoV drug.
Analogues of berbamine also inhibit flavivirus and EV-A71 infection in vitro. In our screen of the different compounds, tetrandrine (a berbamine analogue isolated from Stephania tetrandra 36, 37, 38), also significantly inhibited JEV (Fig. S2A and S2B) and ZIKV (Fig. S2C) infection, as shown by our high content image assay. The EC50 of tetrandrine against JEV and ZIKV in A549 cells was ~1.73 mM and ~1.19 mM, respectively. Likewise, tetrandrine markedly inhibited the JEV infection-induced expression of JEV-NS1 in 4T1 cells (Fig. S2D), and significantly decreased the infectious progeny viral particle production of JEV (Fig. S2E) and ZIKV (Fig. S2F) in A549 cells.
In addition to berbamine and tetrandrine, we also assessed the ability of three additional bis-benzylisoquinoline alkaloids (i.e., isotetrandrine, fangchinoline, and E6 berbamine; Fig. S3A), to inhibit JEV or ZIKV infection of host cells. As shown in Figs. S3B-S3D, isotetrandrine, fangchinoline and E6 berbamine all significantly inhibited infection of JEV or ZIKV in host cells. Similarly, tetrandrine, isotetrandrine, and fangchinoline all significantly inhibited DENV2 infection in A549 cells in a concentration-dependent manner (Fig. S4A).
We also performed the high-content image assay to assess the effect of berbamine or tetrandrine on EV-A71 infection. As shown in Figure S4B and S4C, treatment with berbamine or tetrandrine significantly inhibited EV-A71 infection in RD cells, with EC50 values of ~17. 3 mM or ~7.6 mM, respectively. Taken together, these data indicate that these bis-benzylisoquinoline alkaloids act as pan anti-(+)ss RNA viral agents.
Berbamine can protect mice from a lethal challenge of JEV. We subsequently assessed the cytotoxicity of berbamine (Fig. 4A) and tetrandrine (Fig. 4B) in different cell lines, and found that the IC50 for berbamine in different cell lines ranged from ~90 μM to ~126 μM, whereas the IC50 of tetrandrine was from ~20 μM to ~28 μM (Fig. 4C). We also calculated the selectivity index (SI) of berbamine or tetrandrine for JEV and ZIKV infection. The SI helps to determine the window between cytotoxicity and antiviral activity by dividing the EC50 over its IC50 value (i.e., IC50/EC50). As shown in Fig. 4D, the SI values of berbamine were much higher than tetrandrine for these two viruses.
We, thus, assessed the protective effects of berbamine against JEV infection in a mouse model. As shown in Figs. 4E and 4F, berbamine (15 mg/kg, twice per day) indeed protected mice from a lethal challenge of JEV, as demonstrated by the higher survival rate (i.e., 75% in the berbamine-treated group versus 12.5% in the control group) and the better body weight recovery. In summary, these results suggest that in vivo, berbamine is also a potential anti-(+)ss RNA viral drug or at least an anti-flaviviral drug.
Berbamine prevents the entry of (+)ss RNA viruses by decreasing the levels of their receptor(s) at the plasma membrane in host cells. Since chelation of intracellular Ca2+ by BAPTA-AM rendered cells resistant to JEV internalization (Fig. 1B), we examined whether berbamine might block the entry of viruses into cells. Thus, A549 cells were treated with berbamine for 1h, and then then incubated with JEV on ice for 1h before putting back to 37 0C for another 80 min before fixation. In situ RNA hybridization was subsequently performed to detect the JEV vRNA. As expected, JEV vRNA was only detected in untreated control cells, and not in cells pretreated with berbamine or tetrandrine (Fig. 5A). Therefore, these data demonstrate that berbamine inhibits the entry of JEV into host cells.
We next investigated the mechanism by which berbamine prevents (+)ss RNA viruses from entering host cells, and focused on JEV. The early stages of the flavivirus life cycle involve the attachment of viral particles to the surface of the cell membrane. These viral particles bind to membrane receptors and then enter into the cell via receptor-mediated endocytosis, after which the viral RNA is released from the endosomes, due to the fusion of flavivirus E proteins with the endosome membrane. To search for potential JEV receptor(s) on host cells, we generated pools of siRNA, and individually knocked down potential virus receptors reported in the literature. In this way, we found that knockdown of the low-density lipoprotein receptor (LDLR) markedly decreased JEV-induced JEV-NS1 protein expression in A549 cells (Fig. 5B and 5C), which suggests that the LDLR might be involved in JEV infection. The LDLR is a calcium binding protein, which has been reported to function as a receptor for the hepatitis C, rhino-, and vesicular stomatitis viruses 39, 40, 41, 42, 43. We then assessed whether LDLR is involved in JEV entry into cells. Thus, control or LDLR knockdown A549 cells were incubated with JEV on ice for 1h and were then incubated with warm medium at 37 0C for another 80 min followed by in situ RNA hybridization to detect the JEV vRNA. As expected, JEV vRNA was only detected in control cells, not in LDLR knockdown cells (Fig. 5D), indicating that LDLR is required for JEC entry. In addition, we incubated live A549 cells on ice with an anti-LDLR antibody and an Alexa Fluor 488-tagged secondary antibody to label the cell surface LDLR, followed by infection with JEV. Thereafter, cells were incubated at 37˚C, fixed at the indicated time points, and subjected to in situ RNA hybridization to detect the vRNA of the JEV (Fig. 5E). We showed that JEV infection induced the internalization of LDLR (green puncta in Fig. 5E), and the internalized LDLR-positive endosomes exhibited strong co- localization with the vRNA particles (red puncta in Fig. 5E) by 30 min after virus infection. The vRNA particles then became dissociated from the LDLR-positive endosomes by 60 min after virus infection (the lower panel in Fig. 5E), and we suspect that this is likely due to the release of viral genome RNA from the endosome. In addition, JEV infection markedly increased the level of LDLR in a time-dependent manner, whereas berbamine treatment abolished this increase (Fig. 5F). These results suggest that LDLR might be one of the receptors used by JEV to gain entry into the host cells, and berbamine decreases the level of LDLR at the plasma membrane thereby rendering cells refractory to JEV infection. We conducted LDLR immunostaining in cells treated with or without berbamine, and confirmed that in the presence of the drug, the level of LDLR at the plasma membrane was significantly decreased, but there was little effect on cell morphology (Fig. 5G).
Since SARS-CoV-2 and MERS-CoV are known to target ACE2 44 and DPP4 45, respectively, for their entry into host cells, we suspect that the anti-SARS-CoV-2 and anti-MERS-CoV activity of berbamine might also be due to its effect on these two receptors at the cell surface. By immunolabeling ACE2 or DPP4 in cells treated with or without berbamine, we showed that the drug indeed significantly decreased the level of both of these receptor proteins at the plasma membrane (Fig. 5H and 5I). In summary, these results suggest that berbamine prevents (+)ss RNA viruses from entering host cells by decreasing their respective receptors levels at the plasma membrane. Berbamine inhibits endolysosomal trafficking and induces the secretion of extracellular vesicles to decrease viral receptor levels at the plasma membrane. Since berbamine decreased the levels of LDLR at the plasma membrane (Fig. 5F), we assessed whether berbamine might affect the endolysosmal trafficking of LDLR by performing LDLR and LAMP1 co-immunostaining in cells treated with or without berbamine. In brief, A549 cells were first incubated with an anti-LDLR antibody on ice for 90 min. The internalization of the LDLR-antibody complex was then initiated when cells were warmed to 37°C 46. In the control cells, after ~30 min to 1 h the internalized LDLR- antibody complex was found in the late endosomes or lysosomes, as shown by the level of co-localization between LDLR and LAMP1, and by ~3 h, most of the internalized LDLR was degraded (top panel in Fig. 6A). However, in berbamine-treated cells, the LDLR-antibody complex was internalized normally but failed to be sent to lysosomes for degradation (bottom panel in Fig. 6A).
Since the endolysosomal degradation of LDLR was compromised in the berbamine-treated cells (Fig. 6A), we reasoned that this should lead to an increase in the total amount of LDLR in these cells. However, we found that berbamine markedly decreased the total amount of LDLR in cells (Fig. 6B). We speculated that the reduced levels of LDLR that occurred in berbamine-treated cells might be due to an increase in the secretion of the endosomes containing these membrane receptors, out of cells. To verify this possibility, we quantified the concentration of extracellular vesicles (EVs) in the cell culture medium of control or berbamine-treated cells using a nanoparticle analyzer. As expected, berbamine significantly promoted the secretion of EVs (Fig. 6C). We then examined whether these EVs contain elevated levels of membrane receptors in the berbamine-treated group when compared with the control group. Thus, EVs in the culture medium from the control and berbamine-treated cells were collected by ultracentrifugation, and the protein levels of several previously reported cell membrane receptors or membrane binding proteins for (+)ss RNA viruses (e.g., ITGB3 47, 48, 49, 50, 51, SCARB1 52, 53, LDLR 54, 55, 56, 57 and ANXA2 58, 59, 60), were analyzed by immunoblot analysis. We showed that the levels of LDLR, ANXA2, ITGB3, and SCARB1, similar to TSG10 (which is an exosome surface protein marker), were all markedly increased in EVs collected from the berbamine-treated cell culture medium, when compared with the control group (Fig. 6D). Similarly, we examined the effect of berbamine on the levels of ACE2 and DPP4 in cells and EVs. We found that when compared with the control cells, berbamine treatment markedly decreased the levels of ACE2 and DPP4 in both A549 cells (Fig. 6E)and Vero cells (Fig. 6F), but markedly increased the level of both in EVs (Fig. 6G). Taken together, these results suggest that berbamine inhibits the endolysosomal trafficking of the plasma membrane receptors of (+)ss RNA viruses. This leads to an increase in the level of secretion of these receptors via EVs and a concomitant decrease in the level of the receptors at the plasma membrane, thereby preventing these viruses from entering host cells.
Berbamine prevents JEV infection by inhibiting lysosomal TRPMLs. It has been previously reported that tetrandrine prevents the entry of Ebola virus into host cells by blocking two-pore channels (TPCs) 25 and TPCs has also been shown to mediate MERS-CoV pseudovirus translation 61. TPCs are Ca2+-permeable non-selective cation channels in the endo-lysosomal system 62, 63. Thus, we first examined whether berbamine might affect the lysosomal Ca2+ levels by assessing the ability of Gly-Phe β-naphthylamide (GPN) to trigger Ca2+ release from lysosomes in cells treated with or without berbamine. We found that berbamine significantly mitigated the GPN-induced cytosolic Ca2+ increase, which suggests that it inhibits lysosomal Ca2+ channels (Fig. 7A). However, Ned-19, an antagonist of nicotinic acid adenine dinucleotide phosphate (NAADP), which can inhibit NAADP-mediated Ca2+ release from lysosomes or endosomes via TPCs 64, failed to inhibit ZIKV infection in A549 cells (Fig. S5A). We also knocked down the expression of TPC1 or TPC2 in 4T1 cells (Fig. S5B), and found that knockdown of either TPC2 or TPC1 had little effect on JEV infection in 4T1 cells (Figs S5C and S5D). In addition, double knockdown of both TPC1 and TPC2 failed to inhibit JEV infection in 4T1 cells (Fig. S5E). These data indicate that berbamine does not target TPCs to inhibit flavivirus infection.
In addition to TPCs, several other ion channels in endosomes and lysosomes have been reported to modulate endolysosomal trafficking 65, 66. Among them, TRPMLs in lysosomes and endosomes play critical roles in membrane trafficking, autophagy, and exocytosis 31, 32, 67. Therefore, we assessed whether berbamine might inhibit virus infection via modulating TRPMLs. As shown in Figure 7B, berbamine significantly decreased the TRPML-mediated Ca2+ release from lysosomes, which was triggered by ML-SA1, a selective and potent TRPMLs agonist 68. Consistently, knockdown of TRPML2, TRPML3, or both markedly inhibited JEV infection (Fig. 7D and 7E), although TRPML1 knockdown failed to affect JEV infection (Fig. 7C). In addition, knockdown of both TRPML2 and TRPML3 decreased the levels of LDLR and ACE2 in cells (Fig. 7F). Interestingly, treatment of cells with ML-SA1 reduced the ability of berbamine to decrease the LDLR levels (Fig. 7G and 7H). In summary, these data suggest that berbamine compromises the endolysosomal trafficking of viral receptors via inhibition of TRPMLs, and this leads to a decrease in the levels of viral receptors for (+)ss RNA viruses, thereby preventing these virus particles from entering the host cells.