Characterization of dengue virus entry into HepG2 cells.

BACKGROUND: Despite infections by the dengue virus being a significant problem in tropical and sub-tropical countries, the mechanism by which the dengue virus enters into mammalian cells remains poorly described. METHODS: A combination of biochemical inhibition, dominant negative transfection of Eps15 and siRNA mediated gene silencing was used to explore the entry mechanism of dengue into HepG2 cells. RESULTS: Results were consistent with entry via multiple pathways, specifically via clathrin coated pit mediated endocytosis and macropinocytosis, with clathrin mediated endocytosis being the predominant pathway. CONCLUSION: We propose that entry of the dengue virus to mammalian cells can occur by multiple pathways, and this opens the possibility of the virus being directed to multiple cellular compartments. This would have significant implications in understanding the interaction of the dengue virus with the host cell machinery.

siRNA-mediated silencing of the 37/67-kDa high affinity laminin receptor in Hep3B cells induces apoptosis.

The laminin-binding protein, variously called the 37/67-kDa high affinity laminin receptor or p40, mediates the attachment of normal cells to the laminin network, and also has a role as a ribosomal protein. Over-expression of this protein has been strongly correlated with the metastatic phenotype. However, few studies have investigated the cellular consequence of the ablation of this gene's expression. To address this issue, the expression of the 37/67-kDa high affinity laminin receptor was knocked out with several siRNA constructs via RNA interference in transformed liver (Hep3B) cells. In each case where the message was specifically ablated, apoptosis was induced, as determined by annexin V/propidium iodide staining, and by double staining with annexin V and an antibody directed against the 37/67-kDa high affinity laminin receptor. These results suggest that this protein plays a critical role in maintaining cell viability.

cDNA-AFLP analysis of differential gene expression in human hepatoma cells (HepG2) upon dengue virus infection.

In infectious diseases, the disease pathogenesis is the outcome of the interaction between the genome of the host and the genome of the pathogen. Despite the wide distribution of dengue infections in the world, and the large number of annual infections, few studies have investigated how the dengue genome alters the global transcriptional profile of the host cell. To investigate alterations in the liver cell transcriptome in response to dengue virus infection, liver cells (HepG2) were infected with dengue serotype 2 at MOI 5 and at 3 days post-infection RNA extracted and analyzed by cDNA-AFLP in parallel with mock-infected cells. From 73 primer combinations over 5,000 transcription-derived fragments (TDFs) were observed, of which approximately 10% were regulated differentially in response to infection. Sixty-five TDFs were subsequently cloned and sequenced and 27 unique gene transcripts identified. Semi-quantitative reverse transcription (RT)-PCR was used to validate the expression of 12 of these genes and 10 transcripts (CK2, KIAA509, HSP70, AK3L, NIPA, PHIP, RiboS4, JEM-1, MALT1, and HSI12044) were confirmed to be differentially regulated, with four transcripts (HSP70, NIPA, RiboS4, and JEM-1) showing a greater than twofold regulation. These results suggest that the expression of a large number of genes is altered in response to dengue virus infection of liver cells, and that cDNA-AFLP is a useful tool for obtaining information on both characterized and as yet uncharacterized transcripts whose expression is altered during the infection process.