Characterization of a Temperature-Sensitive Vertebrate Clathrin Heavy Chain Mutant as a Tool to Study Clathrin-Dependent Events In Vivo

Clathrin and clathrin-dependent events are evolutionary conserved although it is believed that there are differences in the requirement for clathrin in yeast and higher vertebrates. Clathrin is a long-lived protein and thus, with clathrin knockdowns only long-term consequences of clathrin depletion can be studied. Here, we characterize the first vertebrate temperature-sensitive clathrin heavy chain mutant as a tool to investigate responses to rapid clathrin inactivation in higher eukaryotes. Although we created this mutant using a clathrin cryo-electron microscopy model and a yeast temperature-sensitive mutant as a guide, the resulting temperature-sensitive clathrin showed an altered phenotype compared to the corresponding yeast temperature-sensitive clathrin. First, it seemed to form stable triskelions at the non-permissive temperature although endocytosis was impaired under these conditions. Secondly, as a likely consequence of the stable triskelions at the non-permissive temperature, clathrin also localized correctly to its target membranes. Thirdly, we did not observe missorting of the lysosomal enzyme beta-glucuronidase which could indicate that the temperature-sensitive clathrin is still operating at the non-permissive temperature at the Golgi or, that, like in yeast, more than one TGN trafficking pathway exists. Fourthly, in contrast to yeast, actin does not appear to actively compensate in general endocytosis. Thus, there seem to be differences between vertebrates and yeast which can be studied in further detail with this newly created tool

RNA Interference Mediated Inhibition of Dengue Virus Multiplication and Entry in HepG2 Cells

Background: Dengue virus-host cell interaction initiates when the virus binds to the attachment receptors followed by endocytic internalization of the virus particle. Successful entry into the cell is necessary for infection initiation. Currently, there is no protective vaccine or antiviral treatment for dengue infection. Targeting the viral entry pathway has become an attractive therapeutic strategy to block infection. This study aimed to investigate the effect of silencing the GRP78 and clathrin-mediated endocytosis on dengue virus entry and multiplication into HepG2 cells. Methodology/Principal Findings: HepG2 cells were transfected using specific siRNAs to silence the cellular surface receptor (GRP78) and clathrin-mediated endocytosis pathway. Gene expression analysis showed a marked down-regulation of the targeted genes (87.2%, 90.3%, and 87.8 % for GRP78, CLTC, and DNM2 respectively) in transfected HepG2 cells when measured by RT-qPCR. Intracellular and extracellular viral RNA loads were quantified by RT-qPCR to investigate the effect of silencing the attachment receptor and clathrin-mediated endocytosis on dengue virus entry. Silenced cells showed a significant reduction of intracellular (92.4%) and extracellular viral RNA load (71.4%) compared to non-silenced cells. Flow cytometry analysis showed a marked reduction of infected cells (89.7%) in silenced HepG2 cells compared to non-silenced cells. Furthermore, the ability to generate infectious virions using the plaque assay was reduced 1.07 log in silenced HepG2 cells