Entry of bovine viral diarrhea virus into ovine cells occurs through clathrin-dependent endocytosis and low pH-dependent fusion

Although mechanisms of bovine viral diarrhea virus (BVDV) entry into bovine cells have been elucidated, little is known concerning pestivirus entry and receptor usage in ovine cells. In this study, we determined the entry mechanisms of BVDV-1 and BVDV-2 in sheep fetal thymus cells. Both BVDV-1 and BVDV-2 infections were inhibited completely by chlorpromazine, β-methyl cyclodextrin, sucrose, bafilomycin A1, chloroquine, and ammonium chloride. Simultaneous presence of reducing agent and low pH resulted in marked loss of BVDV infectivity. Moreover, BVDV was unable to fuse with ovine cell membrane by the presence of reducing agent or low pH alone, while combination of both led to fusion at low efficiency. Furthermore, sheep fetal thymus cells acutely infected with BVDV-1 or BVDV-2 were found protected from heterologous BVDV infection. Taken together, our results showed for the first time that entry of both BVDV-1 and BVDV-2 into ovine cells occurred through clathrin-dependent endocytosis, endosomal acidification, and low pH-dependent fusion following an activation step, besides suggesting the involvement of a common ovine cellular receptor during attachment and entry.     

Vaccinia virus entry into cells via a low-pH-dependent endosomal pathway

Previous studies established that vaccinia virus could enter cells by fusion with the plasma membrane at neutral pH. However, low pH triggers fusion of vaccinia virus-infected cells, a hallmark of viruses that enter by the endosomal route. Here, we demonstrate that entry of mature vaccinia virions is accelerated by brief low-pH treatment and severely reduced by inhibitors of endosomal acidification, providing evidence for a predominant low-pH-dependent endosomal pathway. Entry of vaccinia virus cores into the cytoplasm, measured by expression of firefly luciferase, was increased more than 10-fold by exposure to a pH of 4.0 to 5.5. Furthermore, the inhibitors of endosomal acidification bafilomycin A1, concanamycin A, and monensin each lowered virus entry by more than 70%. This reduction was largely overcome by low-pH-induced entry through the plasma membrane, confirming the specificities of the drugs. Entry of vaccinia virus cores with or without brief low-pH treatment was visualized by electron microscopy of thin sections of immunogold-stained cells. Although some virus particles fused with the plasma membrane at neutral pH, 30 times more fusions and a greater number of cytoplasmic cores were seen within minutes after low-pH treatment. Without low-pH exposure, the number of released cores lagged behind the number of virions in vesicles until 30 min posttreatment, when they became approximately equal, perhaps reflecting the time of endosome acidification and virus fusion. The choice of two distinct pathways may contribute to the ability of vaccinia virus to enter a wide range of cells.     

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.     

Hepatitis B virus entry into HepG2‐NTCP cells requires clathrin‐mediated endocytosis

Hepatitis B virus (HBV) is a leading cause of cirrhosis and hepatocellular carcinoma worldwide, with 250 million individuals chronically infected. Many stages of the HBV infectious cycle have been elucidated, but the mechanisms of HBV entry remain poorly understood. The identification of the sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor and the establishment of NTCP‐overexpressing hepatoma cell lines susceptible to HBV infection opens up new possibilities for investigating these mechanisms. We used HepG2‐NTCP cells, and various chemical inhibitors and RNA interference (RNAi) approaches to investigate the host cell factors involved in HBV entry. We found that HBV uptake into these cells was dependent on the actin cytoskeleton and did not involve macropinocytosis or caveolae‐mediated endocytosis. Instead, entry occurred via the clathrin‐mediated endocytosis pathway. HBV internalisation was inhibited by pitstop‐2 treatment and RNA‐mediated silencing (siRNA) of the clathrin heavy chain, adaptor protein AP‐2 and dynamin‐2. We were able to visualise HBV entry in clathrin‐coated pits and vesicles by electron microscopy (EM) and cryo‐EM with immunogold labelling. These data demonstrating that HBV uses a clathrin‐mediated endocytosis pathway to enter HepG2‐NTCP cells increase our understanding of the complete HBV life cycle.     

Entry of Rice dwarf virus into cultured cells of its insect vector involves clathrin-mediated endocytosis

Electron microscopy revealed that the entry of Rice dwarf virus (RDV) into insect vector cells involved endocytosis via coated pits. The treatment of cells with drugs that block receptor-mediated or clathrin-mediated endocytosis significantly reduced RDV infectivity. However, the drug that blocks caveola-mediated endocytosis had a negligible effect on such infection. Infection was also inhibited when cells had been pretreated with bafilomycin A1, which interferes with acidification of endosomes. Moreover, immunofluorescence staining indicated that the virus is internalized into early endosomes. Together, our data indicate that RDV enters insect vector cells through receptor-mediated, clathrin-dependent endocytosis and is sequestered in early endosomes.     

FGF21 inhibits apolipoprotein(a) expression in HepG2 cells via the FGFR1-ERK1/2-Elk-1 pathway

Lipoprotein(a) [Lp(a)] is a highly atherogenic lipoprotein, whose metabolism is poorly understood. Efficient and secure drugs that can lower elevated plasma Lp(a) concentrations are currently lacking. Fibroblast growth factor-21 (FGF-21), a member of the FGFS super family, regulates glucose and lipid metabolism in hepatocytes and adipocytes via FGFR-ERK1/2 signaling. In this study, we investigated the molecular mechanisms that influence apolipoprotein(a) [apo(a)] biosynthesis. We also determined the effects of FGF21 on HepG2 cell apo(a) expression and secretion, as well as the mechanism of FGF21 in these effects. Results showed that FGF21 inhibited apo(a) expression at both mRNA and protein levels in a dose- and time--dependent manner and then suppressed the secretion of apo(a). These effects were attenuated by PD98059 (ERK1/2 inhibitor) and Elk-1 siRNA. PD166866 (FGFR1 inhibitor) also attenuated the FGF21-mediated inhibition of apo(a) expression and inhibited ERK1/2 and Elk-1 activation. These results demonstrate that FGF21 suppresses apo(a) expression via the FGFR1-ERK1/2-Elk-1 pathway.     

Herpes simplex virus type 1 enters human epidermal keratinocytes, but not neurons, via a pH-dependent endocytic pathway

Herpes simplex virus (HSV) enters some laboratory cell lines via a pH-dependent, endocytic mechanism. We investigated whether this entry pathway is used in human cell types relevant to pathogenesis. Three different classes of lysosomotropic agents, which raise endosomal pH, blocked HSV entry into primary and transformed human keratinocytes, but not into human neurons or neuroblastoma lines. In keratinocytes, incoming HSV particles colocalized with markers of endocytic uptake. Treatment with the isoflavone genistein, an inhibitor of protein tyrosine kinases, reduced the delivery of incoming viral particles to the nuclear periphery and virus-induced gene expression in keratinocytes but not neurons. Moreover, in keratinocyte monolayer islets, HSV infected both the inner and outer cells in a genistein-sensitive manner, suggesting viral endocytosis from both basolateral and apical plasma membrane surfaces. Together, the results indicate that HSV enters human epidermal keratinocytes, but not neurons, by a low-pH, endocytic pathway that is dependent on host tyrosine phosphorylation. Thus, HSV utilizes fundamentally different cellular entry pathways to infect important target cell populations.     

Infectious spleen and kidney necrosis virus (a fish iridovirus) enters Mandarin fish fry cells via caveola-dependent endocytosis

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus from the family Iridoviridae. Megalocytiviruses have been implicated in more than 50 fish species infections and currently threaten the aquaculture industry, causing great economic losses in China, Japan, and Southeast Asia. However, the cellular entry mechanisms of megalocytiviruses remain largely uncharacterized. In this study, the main internalization mechanism of ISKNV was investigated by using mandarin fish fry (MFF-1) cells. The progression of ISKNV infection is slow, and infection is not inhibited when the cells are treated with ammonium chloride (NH(4)Cl), chloroquine, sucrose, and chlorpromazine, which are inhibitors of clathrin-dependent endocytosis. The depletion of cellular cholesterol by methyl-β-cyclodextrin results in the significant inhibition of ISKNV infection; however, the infection is resumed with cholesterol replenishment. Inhibitors of caveolin-1-involved signaling events, including phorbol 12-myristate 13-acetate (PMA), genistein, and wortmannin, impair ISKNV entry into MFF-1 cells. Moreover, ISKNV entry is dependent on dynamin and the microtubule cytoskeleton. Cofraction analysis of ISKNV and caveolin-1 showed that ISKNV colocates with caveolin-1 during virus infection. These results indicate that ISKNV entry into MFF-1 cells proceeds via classical caveola-mediated endocytosis and is dependent on the microtubules that serve as tracks along which motile cavicles may move via a caveola-caveosome-endoplasmic reticulum (ER) pathway. As a fish iridovirus, ISKNV entry into MFF-1 cells is different from the clathrin-mediated endocytosis of frog virus 3 entry into mammalian cells (BHK-21) at 28°C, which has been recognized as a model for iridoviruses. Thus, our work may help further the understanding of the initial steps of iridovirus infection.     

Caffeine attenuates lipid accumulation via activation of AMP-activated protein kinase signaling pathway in HepG2 cells

The main purpose of this study is to examine the effect of caffeine on lipid accumulation in human hepatoma HepG2 cells. Significant decreases in the accumulation of hepatic lipids, such as triglyceride (TG), and cholesterol were observed when HepG2 cells were treated with caffeine as indicated. Caffeine decreased the mRNA level of lipogenesis-associated genes (SREBP1c, SREBP2, FAS, SCD1, HMGR and LDLR). In contrast, mRNA level of CD36, which is responsible for lipid uptake and catabolism, was increased. Next, the effect of caffeine on AMP-activated protein kinase (AMPK) signaling pathway was examined. Phosphorylation of AMPK and acetyl-CoA carboxylase were evidently increased when the cells were treated with caffeine as indicated for 24 h. These effects were all reversed in the presence of compound C, an AMPK inhibitor. In summary, these data indicate that caffeine effectively depleted TG and cholesterol levels by inhibition of lipogenesis and stimulation of lipolysis through modulating AMPK-SREBP signaling pathways. [BMB Reports 2013; 46(4): 207-212]     

Anti-proliferative effects of recombinant iron superoxide dismutase on HepG2 cells via a redox-dependent PI3k/Akt pathway

The coding sequence for an iron superoxide dismutase (fe-sod) was amplified from the Nostoc commune genome. Recombinant Fe-SOD was overexpressed in Escherichia coli, accounting for ∼76% of total bacterial protein. Fe-SOD was purified from bacterial lysate by Ni-NTA column chromatography and used to generate an anti-SOD antibody. The purified Fe-SOD was encapsulated in liposomes and delivered to HepG2 liver tumor cells to eliminate cellular superoxide anions. The SOD-loaded cells exhibited lower reactive oxygen species (ROS) levels and higher reduced glutathione (GSH) levels. In Fe-SOD-treated cells, the cell cycle was delayed in the G₁ phase, and HepG2 cell growth slowed in association with dephosphorylation of the serine–threonine kinase Akt. Low-dose H₂O₂ stimulated Akt phosphorylation, implying that Akt activation in HepG2 cells is redox-sensitive. Akt phosphorylation was abrogated by phosphatidylinositol 3-kinase (PI3K) inhibitors, suggesting that PI3K is an upstream mediator of Akt activation in HepG2 cells. This study provides insight into recombinant Fe-SOD-induced signaling mechanisms in liver tumor cells and suggests the feasibility of using Fe-SOD as an antitumor agent. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Anticancer effect of calycopterin via PI3K/Akt and MAPK signaling pathways,ROS-mediated pathway and mitochondrial dysfunction in hepatoblastoma cancer (HepG2) cells

Calycopterin is a flavonoid compound isolated from Dracocephalum kotschyi that has multiple medical uses, as an antispasmodic, analgesic, anti-hyperlipidemic, and immunomodulatory agents. However, its biological activity and the mechanism of action are poorly investigated. Herein, we investigated the apoptotic effect of calycopterin against the human hepatoblastoma cancer cell (HepG2) line. We discovered that calycopterin-treated HepG2 cells were killed off by apoptosis in a dose-dependent manner within 24 h, and was characterized by the appearance of nuclear shrinkage, cleavage of poly (ADP-ribose) polymerase and DNA fragmentation. Calycopterin treatment also affected HepG2 cell viability: (a) by inhibiting cell cycle progression at the G2/M transition leading to growth arrest and apoptosis; (b) by decreasing the expression of mitotic kinase cdc2, mitotic phosphatase cdc25c, mitotic cyclin B1, and apoptotic factors pro-caspases-3 and -9; and (c) increasing the levels of mitochondrial apoptotic-related proteins, intracellular levels of reactive oxygen species, and nitric oxide. We further examined the phosphorylation of extracellular signal-related kinase (ERK 1/2), c-Jun N-terminal kinase, and p-38 mitogen-activated protein kinases (MAPKs) and found they all were significantly increased in HepG2 cells treated with calycopterin. Interestingly, we discovered that treated cells had significantly lower Akt phosphorylation. This mode of action for calycopterin in our study provides strong support that inhibition of PI3K/Akt and activation of MAPKs are pivotal in G2/M cell cycle arrest and apoptosis of human hepatocarcinoma cells mediated by calycopterin.     

Nonselective utilization of the endomannosidase pathway for processing glycoproteins by human hepatoma (HepG2) cells.

Endo-alpha-D-mannosidase, a Golgi-situated processing enzyme, provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins (Moore, S. E. H., and Spiro, R. G. (1990) J. Biol. Chem. 265, 13104-13112). The present report demonstrates that at least five distinct glycoproteins secreted by HepG2 cells (alpha 1-antitrypsin, transferrin, alpha 1-acid glycoprotein, alpha 1-antichymotrypsin, and alpha-fetoprotein) as well as cell surface components can effectively utilize this alternate processing route. During a castanospermine (CST)-imposed glucosidase blockade, these glycoproteins apparently were produced with their usual complement of complex carbohydrate units, and upon addition of the mannosidase I inhibitor, 1-deoxymannojirimycin (DMJ), to prevent further processing of deglucosylated N-linked oligosaccharides, Man6-8GlcNAc, but not Man9GlcNAc, were identified; the Man8GlcNAc component occurred as the characteristic isomer generated by endomannosidase cleavage. Although the endomannosidase-mediated deglucosylation pathway appeared to be nonselective, a differential inhibitory effect on the secretion of the various glycoproteins was noted in the presence of CST which was directly related to the number of their N-linked oligosaccharides, ranging from minimal in alpha-fetoprotein to substantial (approximately 65%) in alpha 1-acid glycoprotein. Addition of DMJ to CST-incubated cells did not further decrease secretion of the glycoproteins, although processing was now arrested at the polymannose stage, and a portion of the oligosaccharides were still in the glucosylated form. These latter findings indicate that complex carbohydrate units are not required for secretion of these glycoproteins and that any effect which glucose residues exert on their intracellular transit would be related to movement from the endoplasmic reticulum to the Golgi compartment.     

Origin of thymidine kinase deficient (TK-) haploid frog cells via an intermediate thymidine transport deficient (TT-) phenotype

Wild-type cultured cells of the frog cell line ICR 2A give rise to 5-bromodeoxyridine (BUdR)-resistant colonies only when the selecting concentration of the drug is 5 × 10^?5 M or lower. The progeny of these colonies multiply in 10^?4 M BUdR; resistance is correlated with the absence of a thymidine (TdR)-specific transport reaction with a K^m in the range of 2–7 × 10^?4 M. All of the TdR transport-deficient (TT-) isolates examined (25) had TdR kinase activity (4% to 100% of wild-type). Variants deficient in TdR kinase activity (5% of wild-type) were obtained by exposing TT-cultures to 10^?3 M BUdR. The TK - variants multply continuously in 10^?3 M BUdR and retain the phenotype after prolonged culture in the absence of the drug. The frequency with which they occur is increased 20 to 50 fold by prior treatment of the culture with ICR 191, an acridine mustard mutagen. In haploid cells, it would be expected that TK- variants would arise in equal numbers from wild-type and TT- cultures if loss TdR kinase occurred independently of loss of the transport reaction. However, wild-type cells give no colonies resistant to 10^?3 M BUdR under conditions the give 1 to 50 colonies per million TT- cells. The TT- phenotype seems to be a required intermediate state in the origin of the TK- phenotype. Therefore, the TK- clones described above are unlikely to be products of mutation at a single genetic locus.     

Depletion of cytosolic or mitochondrial thioredoxin increases CYP2E1-induced oxidative stress via an ASK-1-JNK1 pathway in HepG2 cells

Thioredoxin is an important reducing molecule in biological systems. Increasing CYP2E1 activity induces oxidative stress and cell toxicity. However, whether thioredoxin protects cells against CYP2E1-induced oxidative stress and toxicity is unknown. SiRNA were used to knockdown either cytosolic (TRX-1) or mitochondrial thioredoxin (TRX-2) in HepG2 cells expressing CYP2E1 (E47 cells) or without expressing CYP2E1 (C34 cells). Cell viability decreased 40-60% in E47 but not C34 cells with 80-90% knockdown of either TRX-1 or TRX-2. Depletion of either thioredoxin also potentiated the toxicity produced either by a glutathione synthesis inhibitor or by TNFα in E47 cells. Generation of reactive oxygen species and 4-HNE protein adducts increased in E47 but not C34 cells with either thioredoxin knockdown. GSH was decreased and adding GSH completely blocked E47 cell death induced by either thioredoxin knockdown. Lowering TRX-1 or TRX-2 in E47 cells caused an early activation of ASK-1, followed by phosphorylation of JNK1 after 48 h of siRNA treatment. A JNK inhibitor caused a partial recovery of E47 cell viability after thioredoxin knockdown. In conclusion, knockdown of TRX-1 or TRX-2 sensitizes cells to CYP2E1-induced oxidant stress partially via ASK-1 and JNK1 signaling pathways. Both TRX-1 and TRX-2 are important for defense against CYP2E1-induced oxidative stress.     

Rafts promote assembly and atypical targeting of a nonenveloped virus, rotavirus, in Caco-2 cells

Rotavirus follows an atypical pathway to the apical membrane of intestinal cells that bypasses the Golgi. The involvement of rafts in this process was explored here. VP4 is the most peripheral protein of the triple-layered structure of this nonenveloped virus. High proportions of VP4 associated with rafts within the cell as early as 3 h postinfection. In the meantime a significant part of VP4 was targeted to the Triton X-100-resistant microdomains of the apical membrane, suggesting that this protein possesses an autonomous signal for its targeting. At a later stage the other structural rotavirus proteins were also found in rafts within the cells together with NSP4, a nonstructural protein required for the final stage of virus assembly. Rafts purified from infected cells were shown to contain infectious particles. Finally purified VP4 and mature virus were shown to interact with cholesterol- and sphingolipid-enriched model lipid membranes that changed their phase preference from inverted hexagonal to lamellar structures. Together these results indicate that a direct interaction of VP4 with rafts promotes assembly and atypical targeting of rotavirus in intestinal cells.