The present study investigates the effects of siRNA on suppression the dengue infection in HepG2 cells by silencing the GRP78, CLTC, and DNM2 genes separately or in combination. The silenced HepG2 cells with specific siRNA duplexes (GRP78, CLTC, and DNM2) revealed a reduction of the percentage of infected cells as shown by flow cytometry analysis, reduction of the intracellular and extracellular viral RNA load when monitored by RT-qPCR, and reduction of generation of infectious virions as observed by plaque assay. The findings have shown that the suppression was specific and efficient. This results are consistent with our previous findings of inhibition of dengue infection in monocytes by silencing the CD-14 associated molecule and clathrin-mediated endocytosis 
. Our previous study has showed a significant reduction of infected cells (85.2%), intracellular viral RNA load (73.0%), and extracellular viral RNA load (63.0%) in silenced monocytes as compared to non-silenced monocytes. As seen in the current study, silenced HepG2 cells showed a more efficient reduction of infected cells (89.7%), intracellular viral RNA load (92.4%), and extracellular viral RNA load (70.7%) compared to monocytes. This variation could be attributed to the more effectiveness of the gene silencing in HepG2 cells than in monocytes as well as the monocytes are primary cells, whereas the HepG2 cells are a cell line.
In dengue infection, evidences have shown that the liver is a major target organ for DENV infection in humans as many pathological findings and liver dysfunction have been detected in the livers of DHF/DSS patients and is a characteristic of severe dengue infection 
. It has been demonstrated that there is a direct correlation between the development of the severe and life-threatening form of the disease and high viral load 
. Hepatocytes were considered as a target in this study to reduce the dengue viral load during the course of infection based on evidences of its crucial role in dengue infection, and the previous findings of the inhibition of dengue infection in monocytes. Prevention the multiplication of DENV in the main target cells could result in reduction of the total viral load during the course of infection. This would potentially prevent the progression of dengue fever to the severe life-threatening form of dengue infection. Furthermore, a decreased of viremia in humans can result in the drop in number of infected vectors and, therefore, break of the transmission chain.
There are various strategies to develop antiviral agents against DENV. Recently, the virus entry step has become an attractive therapeutic strategy 
. DENV-host cell interaction is initiated with the virus binding on attachment receptors on the cell surface followed by stimulation of signals that result in the endocytic internalization of the virus particles. This is critical for successful entry into the host cell and the establishment of infection. This study was designed to target the GRP78 that has been identified as receptor on HepG2 cells for DENV-2 entry 
, and clathrin-mediated endocytosis that known as the main pathway for virus internalization 
. This is a possible way to provide a new therapeutic strategy by targeting the host factors known to be involved in viral infection. Thus, it is expected to control viral infection by making the cellular receptors for viruses on human cells less accessible. Recent studies showed that design and synthesis of agents that prevent DENV binding and entry to the cellular receptor sites could prove to be novel antiviral agents of preventing the disease. RNAi pathway shows a role in modulating DENV replication in previous studies 
In the present study, the designed siRNA showed an efficient knockdown of the target genes when evaluated at the mRNA level 24 hours after transfection. To evaluate the viral entry into HepG2 cells, the silenced cells were infected with a live DNEV 72 hours post transfection to confirm that the target proteins level was reduced as the half-life time of these target proteins is less than 72 hours as shown by previous studies. These proteins possess a half-life of 24–48 hours for GRP78 
, 18–36 hours for CLTC 
, and 24–34 hours for DNM2 
Silencing the GRP78 and clathrin-mediated endocytosis resulted in significant inhibition of dengue infection up to 81.3% and 81.4% for GRP78 and the clathrin-mediated endocytosis respectively. In addition, a more potent inhibition (up to 89.7%) is observed when a combined silencing of both GRP78 and clathrin-mediated endocytosis was done simultaneously. This reduction in viral yield was not due to cell death as could be identified by cytotoxicity and viability tests (>90% live cells) in both silenced and non-silenced HepG2 cells. GRP78 could be up regulated in dengue infected cells as a direct response to productive infection in dengue infected cells and as a secondary consequence, in both dengue infected and bystander cells, of the release of cytokines and factors from dengue infected cells that can induce GRP78 expression. Previous studies have suggested two roles for GRP78 in dengue infection. First, GRP78 serves as part of a receptor complex for DENV entry into hepatocytes 
. Additionally, heat shock treatment of cells in the tissue culture system prior to DENV challenge, which would be expected to up regulate both GRP78 and HSP70, thus enhancing DENV entry and replication 
. Second, GRP78 is known to function as a major ER chaperone and master regulator of unfolded protein responses 
. Dengue infection and viral protein production results in ER stress 
. An overwhelming load of misfolded proteins up regulate the GRP78 
. Increasing the requirement of GRP78 triggers a signaling of unfolded protein response (UPR) pathway 
. Thus, the UPR pathway restores the normal function of the cell by enhancing transcription of ER chaperones, decreasing protein translation to mitigate the ER overload, increasing protein degradation, or activates the apoptotic cell death 
. In this study, silencing GRP78 showed inhibition of dengue infection in HepG2 cells. Based on above explanation, we propose that GRP78 inhibits dengue infection either by interaction with cell-surface attachment and internalization and then multiplication. The other possible way is that GRP78 may function in its traditional role in binding and chaperoning many unfolded proteins, include DENV proteins 
. Our result is consistent with a previous observation showing that cleaving the GRP78 by SubAB toxin can dramatically reduce the releasing of infectious DENV and intracellular virion particles 
. Another study also shows the siRNA knockdown of GRP78 in HepG2 cells decreased infectious-virus production 
. Furthermore, elimination of GRP78 leads to reduction of cytosolic ubiquitination and inhibits ubiquitination-proteosome pathway which is known to regulate the endocytosis of cell-surface receptors 
. Therefore, inhibiting the cellular endocytosis pathway possibly leads to inhibit the internalization of DENV 
and other flaviviruses 
Incomplete inhibition of dengue infection can be interpreted as the result of using an alternative secondary receptor or endocytosis by the DENV to establish the infection or incomplete inhibition by the RNAi machinery. For RNAi based therapy designing, it is necessary to consider that this technology knockdown gene expression, but in general does not eliminate it. Therefore, in some conditions, incomplete down regulation of a pathogenic gene seems to be adequate to produce a clinically appropriate improvement 
The approach of gene silencing is a widely accepted technique and, recently, RNAi technique had shown a potential to achieve the gene therapy goal 
. The effectiveness of siRNA as a therapy against dengue infection will depend on the efficiency of siRNA delivery to the target mammalian cells 
. However, for application of RNAi as a dengue therapeutic, an effective and cell-specific in vivo
delivery system is required. Recently, several studies have described a success in siRNAs delivery, in vivo
, by coupling to antibodies or peptides that recognize cell surface receptors 
. This can provide a further supporting and future potential for the practical utility of this approach. The aim of any therapeutic is to maximize the ratio of desired effects to undesired effects. In some cases, such as chemotherapy, interferon treatment, and highly active anti-retroviral treatment, the ratio is not ideal and a significant degree of toxicity is associated with treatment. While RNAi has the capacity to provide better gene targeting specificity, the exposure of cells to any exogenous molecule (siRNA or transfecting reagent) has the potential to disturb normal cellular functions and needs to be carefully controlled. In this study, the transfection experiment was optimized to produce maximum silencing effect at low-cell toxicity as revealed by measurement the released LDH from cells with compromised membrane and Trypan Blue exclusion assays in cell viability test.
One of the potential weaknesses of the RNAi based therapeutic is the problem of resistance and RNAi escape mutations 
. This problem will probably require the use of RNAi in combination therapy approaches, including multiple RNAi target sequences and/or other synergistic antiviral drugs as well as by targeting the host factors known to be involved in viral infection, such as entry receptor or binding molecules on the susceptible cells. Here, we used three different siRNA pools targeting GRP78, CLTC, and DNM2. Each pool achieved its own silencing level and similar knockdown efficiency of these genes in HepG2 cells was achieved by co-transfection of the three pools simultaneously. The desire to pool three siRNAs was raised primarily from the finding that the silencing efficiency of a single siRNAs was lesser than combined pool of triple siRNAs. These pools have shown a greater potency in the reduction of the target gene expression and elimination of off-targets effect.
We have successfully inhibited the DENV entry and multiplication into HepG2 cells by silencing the GRP78 and clathrin mediated endocytosis. Reduction of the viral load would potentially prevent dengue fever and the severe life-threatening form of dengue infection. Decreasing viremia in humans can result in a decline in the infected vectors numbers, and thus interruption of the transmission chain. This might not only save lives but also curb potential epidemics. This tool might serve as a novel promising therapeutic agent for the attenuation of dengue infection and reduction the progression to severe form of the disease DHF.