Ezetimibe inhibits Dengue virus infection in Huh-7 cells by blocking the cholesterol transporter Niemann–Pick C1-like 1 receptor

Despite the importance of Dengue virus (DENV) infection in human health, there is not a fully effective vaccine or antiviral treatment against the infection. Since lipids such as cholesterol are required during DENV infection, its uptake and synthesis are increased in infected cells. Ezetimibe is an FDA-approved drug that reduces cholesterol uptake in humans by inhibiting the endocytosis through Niemman-Pick C1-Like 1 (NPC1L1) receptor, expressed on the membrane of enterocytes and hepatocytes. Our results indicate that an increase in the amount of NPC1L1 occurs on the surface of Huh-7 cells during DENV infection, which correlates with an increase in cholesterol levels. Blockage of NPC1L1 with ezetimibe in concentrations up to 50 μM does not reduce cell viability but diminished total cellular cholesterol, the percentage of infected cells, viral yield, viral RNA and protein synthesis without affecting DENV binding and/or entry to Huh-7 cells. Moreover, ezetimibe inhibited DENV replicative complex formation and lipid droplets accumulation. All these results indicate that ezetimibe is an excellent drug to inhibit DENV infection and confirm that cholesterol is a key target to inhibit viral infection.


The amount of NPC1L1 receptor on cell surface increases during DENV infection.
Since the main role of the Niemann-Pick C1-Like 1 (NPC1L1) receptor is the cholesterol uptake, and cholesterol is essential for DENV infection, the first step was to determine if this receptor was present on the cell surface during DENV infection. Thus, non-permeabilizing uninfected and infected Huh-7 cells were incubated with the anti-NPC1L1 receptor antibody and analyzed by confocal microscopy and flow cytometry. As it can be observed, during the first hour postinfection (hpi) an increase in the amount of NPC1L1 receptor on the cell surface compared to the uninfected cells (MOCK-infected) was observed ( Fig. 1 A and B).
However, at 3 and 6 hpi an abrupt reduction in the fluorescence detected on the surface of DENV infected cells was detected, suggesting that the receptor was internalized ( Fig. 1 A and B). However, at 12 hpi the receptor was located again on the surface of the infected cells ( Fig. 1 A and B). These results suggest that DENV infection induces an increase of the NPC1L1 receptor on the cell surface during viral entry (at 1hpi) and during viral replication (at 12 hpi). The decrease in the amount of the receptor at 3 and 6 hpi could be related to the cholesterol uptake by internalization and recycling of the receptor (Ge et al., 2008).

Ezetimibe reduces DENV infection.
Once we determined that the amount of NPC1L1 receptor on the surface of Huh-7 cells increase early after infection, we decided to analyze its importance during DENV infection. To investigate this fact, cells were treated with different concentrations of ezetimibe or vehicle (0 μ M). Although ezetimibe has not a negative effect on cell viability evaluated by IP and MTT methods ( Fig. 2 A and B), it causes an arrest of the receptor on the cell membrane of infected cells at 1, 3, 6 and 12 hours post-infection, as could be observed by confocal microscopy from non-permeabilized cells (Fig. 2 C). Interestingly, ezetimibe was able to inhibit the amount of DENV 2 infected cells, evaluated by flow cytometry at 48 h postinfection with an IC50 value of 13.07 µM (95% Confidence Interval = 9.60 -16.54 µM) (Fig. 3 A).
Remarkably, when 25 and 50 μ M of ezetimibe were used, a significant reduction in viral yield, mainly at the concentration of 50 μ M for DENV 2 (more than one log of UFF/mL, 95% decrease) (Fig. 3 B) and DENV 4 (2 logs of UFF/mL, 99% decrease) (Fig. 3 C) (p = 0.0146 and 0.0141, respectively) was detected. Since the best inhibitory effect of ezetimibe was observed at the concentration of 50 μ M, this concentration was used for subsequent assays. The next step was to evaluate the effect of ezetimibe in the number of infected cells. A significant reduction in the amount of cells labeled with the anti-prM-E antibody was observed in ezetimibe treated DENV 2 and DENV 4 infected cells compared with vehicle-treated cells by confocal microscopy (Fig. 4 A). When these differences were quantified by flow cytometry, the significant reduction in the percentage of infected cells was confirmed in ezetimibe treated cells infected with DENV2 (38.97% (p= 0.0140)) and DENV4 (39.43% (p= 0.0355)) ( Fig. 4 B), compared with vehicle-treated cells. All these results confirm that the NPC1L1 receptor plays an essential role during DENV infection.
To exclude the possibility that the ezetimibe could have an off-target effect the effect of this drug was tested in Vero cells, which are permissive to DENV infection but do not express the NPC1L1 receptor. As it can be observed, ezetimibe did not induce a reduction in the percentage of infected cells supporting the idea that the inhibition of DENV infection induced by ezetimibe in Huh7 cells is due to the blockage of the NPC1L1 receptor (Supplemental material, Fig. 1).
Moreover, the inhibition in DENV infection induced by ezetimibe in the presence of fetal calf serum (FCS), which contains cholesterol as low density (LDL) and high density (HDL) lipoproteins as well as free cholesterol (Forte et al., 1981), was less pronounced than in cells incubated in the absence of FCS, supporting the idea that the cholesterol levels in infected cells are maintained by cholesterol synthesis and also by cholesterol uptake trough at least, two receptors: LDLr and NPC1L1, because this last one was inhibited by ezetimibe (Supplemental material, Fig. 2).

Ezetimibe inhibits post-entry steps during DENV replicative cycle.
In order to investigate the specific step in DENV replicative cycle in which the NPC1L1 receptor is playing a role, the drug was added at different times of infection: 1) 6 hrs before infection (pretreatment), 2) 0, 3) 12, 4) 24 or 5) 36 hpi, according to the following scheme ( Fig. 5 A). While no differences in the percentage of infected cells were observed when cells were pretreated with ezetimibe, a significant reduction in the number of infected cells was observed in cells treated with ezetimibe after infection (Fig. 5 B), supporting the idea that NPC1L1 is involved in steps after viral attachment. To analyze in further detail the specific step in which ezetimibe is inhibiting DENV infection, first, a viral binding assay in the presence of ezetimibe was performed. As shown in Figure 6 A and 6 B, the amount of virus bound to the surface of cells infected with DENV2 or DENV4 was the same in the absence or the presence of ezetimibe, suggesting that the NPC1L1 receptor not is involved in viral attachment. To confirm this result and knowing that the binding site of ezetimibe to NPC1L1 is distinct from the anti- To explore the possibility that ezetimibe could be disturbing the internalization of DENV, viral entry was analyzed by confocal microscopy using antibodies against C protein at a 1 hpi. The amount of C protein in the cytoplasm of infected cells was similar in ezetimibe treated and untreated cells, suggesting that the drug not alter DENV entry (Fig. 6 C).
Finally, to analyze if viral translation and/or replication could be altered by ezetimibe, the amount of viral protein and viral genome was quantified by Western blot and qRT-PCR respectively in cells untreated or treated with ezetimibe. In agreement with the results of viral yield, a significant reduction in the RNA copy number ( Fig. 7 A and B) for DENV 2 and DENV 4 (p = 0.0033 and 0.0347, respectively) was detected. In the same way, a significant reduction in the amount NS3 protein (Fig. 7 C-E) from DENV 2 and DENV 4 infected cells (p = <0.0001) at both concentrations of the drug was observed, supporting the idea that ezetimibe blocks DENV infection inhibiting viral translation and replication.

Ezetimibe treatment affects the replicative complexes integrity.
Since the main function of the NPC1L1 receptor is the uptake of cholesterol which is required for replication complexes (RC) formation, and translation and replication take place in these structures, the integrity of the RC in Huh-7 cells infected with DENV 2 and treated with ezetimibe was evaluated. The colocalization of E and NS3 proteins (Anwar et al., 2011) was analyzed by confocal microscopy (Fig. 8 A).
In the absence of ezetimibe, almost 100% of the cells were infected, and a compact and perinuclear distribution of both viral proteins was observed ( All these results indicate that NPC1L1 receptor is involved in cholesterol accumulation required for RC formation during DENV infection. Thus, ezetimibe represents an effective antiviral drug for DENV infection.

Discussion
The opportunity offered by host-directed antiviral therapy (HDA), targeting host factors that are usurped by DENV for replication, is promising. Given the genetic austerity of the viruses, it depends on cellular factors and organelles to complete its viral cycle. One of the cellular components required during DENV  (Soto-Acosta et al., 2017, 2013. Therefore, treatment with statins, which inhibit the activity of HMG-CoA reductase, or metformin which activates APMK in cellular and/or animal models appears promising against DENV infection (Martinez-Gutierrez et al., 2014;Rothwell et al., 2009b;Soto-Acosta et al., 2017, 2013. However, while lovastatin treatment was not able to inhibit DENV infection in a clinical trial in humans (Whitehorn et al., 2016) Fig. 1 A and B). However, unlike what was reported with LDLr in DENV infection, at 12 hpi we observed recovery of the NPC1L1 receptor on the cell membrane. At this time, there is a significant viral replication activity with important cholesterol requirements. It has been described that when cholesterol is removed from the medium and later added to liver cells, 85% of the NPC1L1 receptor is located in the plasma membrane to capture it (Ge et al., 2008). This was observed in the DENV-infected cells when the cells require a higher amount of cholesterol, and it is uptake from the media at early times. This finding suggests that binding or entry of DENV triggers cholesterol accumulation in the cell via LDLr and NPC1L1 receptor, but during viral replication, the cholesterol requirement increases and it is obtained through the NPC1L1 receptor uptake and by the increase in cholesterol synthesis by the HMG-CoA reductase (Soto-Acosta et al., 2017). It is known that the NPC1L1 receptor is involved in the uptake of cholesterol in liver cells, which can be blocked by ezetimibe, a drug approved by the FDA as cholesterol-lowering therapy (Garcia-Calvo et al., 2005). Our results suggest that ezetimibe can reduce the production of new viral particles (Fig. 3 B and C), inhibits the DENV genome synthesis (Fig. 7 A and B) and reduce viral protein synthesis (Fig. 7 C-E) of DENV 2 and DENV 4 by inhibiting the uptake of extracellular cholesterol and blocking the NPC1L1 receptor by arresting it in the cell membrane during the first 12 hours of infection ( Fig. 2 C). In addition, we were able to exclude that ezetimibe could be having an off-target effect since it was unable to inhibit DENV infection in Vero cells which lack the NPC1L1 receptor.
Since treatment with ezetimibe was not able to inhibit DENV binding or entry, we can suggest that the NPC1L1 receptor is not an entry factor for DENV.
This was also the case for hepatitis B virus (HBV), in which ezetimibe blocked post-entry events but not binding or entry (Lucifora et al., 2013). In contrast, ezetimibe inhibited attachment and entry of Hepatitis C virus (HCV) considering to the NPC1L1 receptor as an entry factor (Sainz et al., 2012). Interestingly, ezetimibe had a higher potential to inhibit DENV than HCV and HBV infections (Lucifora et al., 2013;Sainz et al., 2012), because the in vitro IC50 for DENV (13.07 μ M) was lower than the one reported for HBV and HCV, supporting the idea that ezetimibe is a good candidate as an anti-DENV drug. It is important to notice that the reduction in viral yield, the inhibition in the amount of viral genome and proteins after treatment with ezetimibe, can be considered as a consequence of the loss of the integrity of RC (Fig. 8 and 9), because a reduction in cholesterol levels, causes an inhibition in RC formation in DENV-infected cells (Anwar et al., 2011;Miller et al., 2007). The confocal microscopy and TEM analysis demonstrated that ezetimibe affects the integrity of the membrane vesicles reported as RC in DENV infected cells (Junjhon et al., 2014;Reyes-Ruiz et al., 2018;Welsch et al., 2009). Additionally, a large number of LDs at the periphery of the viral RC observed in infected cells (Samsa et al., 2009)         Western blot assays (C-E), respectively. The log of the number of copies of RNA ± SD and relative band intensity from three independent assays in duplicate is presented. *p= <0.05, **p= <0.001.          Supplemental material. Figure 2