Chutima Thepparit

Identification of GRP 78 (BiP) as a liver cell expressed receptor element for dengue virus serotype 2

Summary.This study sought to identify receptor elements for dengue virus serotype 2 on human liver cells (HepG2) using the viral overlay protein binding assay (VOPBA) technique and Mass Spectrometry fingerprinting. A single major and several minor virus binding bands were observed, and mass spectrometry identified a candidate binding protein for the major binding band as GRP 78 (BiP). GRP78 expression on the cell surface was confirmed, and antibodies directed against both the N and C-terminus of GRP 78 (BiP) altered both the binding of the virus to the cell surface as well as the infectivity profile of HepG2 cells in response to dengue serotype 2 infection. GRP 78 (BiP), which has previously been identified as a co-receptor protein for coxsackievirus A9, is the first non-Fc receptor protein identified for the dengue virus, although GRP78 probably functions as part of a receptor complex.

Serotype-Specific Entry of Dengue Virus into Liver Cells: Identification of the 37-Kilodalton/67-Kilodalton High-Affinity Laminin Receptor as a Dengue Virus Serotype 1 Receptor

ABSTRACT Dengue virus, the causative agent of dengue fever, dengue shock syndrome, and dengue hemorrhagic fever, infects susceptible cells by initially binding to a receptor(s) located on the host cell surface. Evidence to date suggests that receptor usage may be cell and serotype specific, and this study sought to identify dengue virus serotype 1 binding proteins on the surface of liver cells, a known target organ. By using a virus overlay protein binding assay (VOPBA), in both nondenaturing and denaturing gel systems, a putative dengue virus serotype 1 binding protein of approximately 37 kDa expressed on the surface of liver (HepG2) cells was identified. Mass spectrometry analysis identified a candidate protein, the 37/67-kDa high-affinity laminin receptor. Entry of the dengue virus serotype 1 was significantly inhibited in a dose-dependent manner by both antibodies directed against the 37/67-kDa high-affinity laminin receptor and soluble laminin. No inhibition of virus entry was seen with dengue virus serotypes 2, 3, or 4, demonstrating that the 37/67-kDa high-affinity laminin receptor is a serotype-specific receptor for dengue virus entry into liver cells.

Isolation of a Rickettsial Pathogen from a Non-Hematophagous Arthropod

Rickettsial diversity is intriguing in that some species are transmissible to vertebrates, while others appear exclusive to invertebrate hosts. Of particular interest is Rickettsia felis, identifiable in both stored product insect pests and hematophagous disease vectors. To understand rickettsial survival tactics in, and probable movement between, both insect systems will explicate the determinants of rickettsial pathogenicity. Towards this objective, a population of Liposcelis bostrychophila, common booklice, was successfully used for rickettsial isolation in ISE6 (tick-derived cells). Rickettsiae were also observed in L. bostrychophila by electron microscopy and in paraffin sections of booklice by immunofluorescence assay using anti-R. felis polyclonal antibody. The isolate, designated R. felis strain LSU-Lb, resembles typical rickettsiae when examined by microscopy. Sequence analysis of portions of the Rickettsia specific 17-kDa antigen gene, citrate synthase (gltA) gene, rickettsial outer membrane protein A (ompA) gene, and the presence of the R. felis plasmid in the cell culture isolate confirmed the isolate as R. felis. Variable nucleotide sequences from the isolate were obtained for R. felis-specific pRF-associated putative tldD/pmbA. Expression of rickettsial outer membrane protein B (OmpB) was verified in R. felis (LSU-Lb) using a monoclonal antibody. Additionally, a quantitative real-time PCR assay was used to identify a significantly greater median rickettsial load in the booklice, compared to cat flea hosts. With the potential to manipulate arthropod host biology and infect vertebrate hosts, the dual nature of R. felis provides an excellent model for the study of rickettsial pathogenesis and transmission. In addition, this study is the first isolation of a rickettsial pathogen from a non-hematophagous arthropod.

Internalization and Propagation of the Dengue Virus in Human Hepatoma (HepG2) Cells

Objectives: This study sought to undertake a comparative analysis of the internalization and propagation of all four dengue serotypes in a single cell line of human liver origin, HepG2. Methods: Virus production after infection was determined by the plaque assay technique. Internalization profiles were determined by incubating virus and cells on ice and then raising the temperature for various times. The contribution of extracellular matrix components to internalization was determined by pretreatment of cells with either trypsin or heparinase III. Results: HepG2 cells were able to support the propagation of all four serotypes with mature viruses being produced by 12 h for dengue serotype 4 and by 17–18 h for the remaining serotypes. Virus internalization showed a plateau for serotypes 1, 2 and 4 entry while serotype 3 showed a constant increase in internalization for up to 5 h. Pretreatment of HepG2 cells with heparinase III or trypsin both resulted in a reduction in viral production, with the smallest effect being noted for dengue serotype 3. Conclusion: These results suggest that the interaction between the dengue virus and liver cells is a complex one that requires both protein and nonprotein elements, and has a significant serotype/strain element.

Horizontal transmission of Rickettsia felis between cat fleas, Ctenocephalides felis

Rickettsia felis is a rickettsial pathogen primarily associated with the cat flea, Ctenocephalides felis. Although laboratory studies have confirmed that R. felis is maintained by transstadial and transovarial transmission in C. felis, distinct mechanisms of horizontal transmission of R. felis among cat fleas are undefined. Based on the inefficient vertical transmission of R. felis by cat fleas and the detection of R. felis in a variety of haematophagous arthropods, we hypothesize that R. felis is horizontally transmitted between cat fleas. Towards testing this hypothesis, flea transmission of R. felis via a bloodmeal was assessed weekly for 4 weeks. Rhodamine B was used to distinguish uninfected recipient and R. felis‐infected donor fleas in a rickettsial horizontal transmission bioassay, and quantitative real‐time PCR assay was used to measure transmission frequency; immunofluorescence assay also confirmed transmission. Female fleas acquired R. felis infection more readily than male fleas after feeding on a R. felis‐infected bloodmeal for 24 h (69.3% and 43.3%, respectively) and both Rickettsia‐uninfected recipient male and female fleas became infected with R. felis after cofeeding with R. felis‐infected donor fleas (3.3–40.0%). Distinct bioassays were developed to further determine that R. felis was transmitted from R. felis‐infected to uninfected fleas during cofeeding and copulation. Vertical transmission of R. felis by infected fleas was not demonstrated in this study. The demonstration of horizontal transmission of R. felis between cat fleas has broad implications for the ecology of R. felis rickettsiosis.

Dengue 2 infection of HepG2 liver cells results in endoplasmic reticulum stress and induction of multiple pathways of cell death

BackgroundA number of studies have implicated the direct involvement of the liver in dengue virus (DENV) infection, and it has been widely shown that liver cells subsequently undergo apoptosis. The mechanism by which liver cells undergo apoptosis in response to DENV infection remains unclear. To provide further information on the mechanism of apoptosis in DENV infected liver cells, HepG2 cells were infected with DENV 2 and analyzed for the induction of ER stress, apoptosis and autophagy.ResultsIn response to DENV infection, HepG2 cells showed the induction of both the ER resident unfolded protein response as well as the Noxa/PUMA stress response pathways. Proteolytic activation of caspases 4, 7, 8 and 9 was observed as well as changes in mitochondrial transmembrane potential. Increased monodansylcadaverine staining was observed in DENV infected cells, consistent with the previously reported induction of autophagy.ConclusionsThese results are consistent with a model in which the induction of multiple ER stress pathways is coupled with the induction of multiple cell death pathways as a mechanism to ensure the removal of infected liver cells from the system.

The Involvement of Microglial Cells in Japanese Encephalitis Infections

Despite the availability of effective vaccines, Japanese encephalitis virus (JEV) infections remain a leading cause of encephalitis in many Asian countries. The virus is transmitted to humans by Culex mosquitoes, and, while the majority of human infections are asymptomatic, up to 30% of JE cases admitted to hospital die and 50% of the survivors suffer from neurological sequelae. Microglia are brain-resident macrophages that play key roles in both the innate and adaptive immune responses in the CNS and are thus of importance in determining the pathology of encephalitis as a result of JEV infection.

Akt induces apoptosis in neuroblastoma cells expressing a C98X vasopressin mutant following autophagy suppression

Mutations in the arginine vasopressin (AVP)‐neurophysin II (NP‐II) gene that affect the folding and transport of the prohormone result in loss of secretion of the anti‐diuretic hormone AVP from pituitary nerve terminals and cause autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI). One such mutation consists of the replacement of a Cys residue at position 98 with a stop codon (C98X) in the AVP precursor (corresponding to C67X in NP domain). In neuroblastoma cells over‐expressing this truncated AVP precursor autophagy, a macromolecular degradation process, was shown to be essential for assuring cell survival. In the present study, we investigated the role of the Akt pro‐survival signalling in the regulation of autophagy and of apoptosis linked with the handling of C98X AVP. Impairing autophagy‐lysosomal sequestration or cathepsin D (CD)‐mediated proteolysis triggered the activation of the intrinsic death pathway of apoptosis in C98X‐expressing cells, but not in the wild‐type ‐AVP‐expressing cells. This was shown by the expression of a Vps34 dominant negative, which down‐regulates the PI3k class III‐dependent signalling needed for autophagosome (APH) formation, by genetic silencing as a result of RNA interference (RNAi) of Lamp2, a protein indispensable for the fusion of APHs with lysosomes, and by RNAi silencing of the lysosomal protease CD. Ectopic expression of either the wild‐type or the mutated C98X AVP altered neither the expression nor the phosphorylation of the pro‐survival signalling molecule Akt. Strikingly, the ectopic adenoviral‐directed expression of a constitutively active Akt, instead of preserving cell survival, resulted in the suppression of autophagy, and precipitated Bax‐mediated cell death. The present data demonstrate the need for autophagy‐mediated degradation of mutated C98X peptides, which otherwise become toxic to the cell, and suggest that, in the presence of mis‐folded proteins, the stimulation of the Akt signalling counteracts the beneficial effects of autophagy and precipitates cell death. It follows that growth factors impinging on the Akt pathway may have deleterious effect in neurones expressing mutant neuropeptides. This can provide an explanation for the late onset and progressive neuronal cell loss observed in hypothalamic magnocellular neurones of adFNDI patients.

Involvement of ATP synthase β subunit in chikungunya virus entry into insect cells

Chikungunya virus (CHIKV), the virus responsible for the disease chikungunya fever in humans, is transmitted by Aedes mosquitoes. While significant progress has been made in understanding the process by which CHIKV enters into mammalian cells, far less progress has been made in understanding the CHIKV entry process in insect cells. This study sought to identify mosquito-cell-expressed CHIKV-binding proteins through a combination of virus overlay protein binding assays (VOPBA) and mass spectroscopy. A 50-kDa CHIKV-binding protein was identified as the ATP synthase β subunit (ATPSβ). Co-immunoprecipitation studies confirmed the interaction, and colocalization analysis showed cell-surface and intracellular co-localization between CHIKV and ATPSβ. Both antibody inhibition and siRNA-mediated downregulation experiments targeted to ATPSβ showed a significant reduction in viral entry and virus production. These results suggest that ATPSβ is a CHIKV-binding protein capable of mediating the entry of CHIKV into insect cells.

Interaction of Rickettsia felis with histone H2B facilitates the infection of a tick cell line

Haematophagous arthropods are the primary vectors in the transmission of Rickettsia, yet the molecular mechanisms mediating the rickettsial infection of arthropods remain elusive. This study utilized a biotinylated protein pull-down assay together with LC-MS/MS to identify interaction between Ixodes scapularis histone H2B and Rickettsia felis. Co-immunoprecipitation of histone with rickettsial cell lysate demonstrated the association of H2B with R. felis proteins, including outer-membrane protein B (OmpB), a major rickettsial adhesin molecule. The rickettsial infection of tick ISE6 cells was reduced by approximately 25 % via RNA-mediated H2B-depletion or enzymic treatment of histones. The interaction of H2B with the rickettsial adhesin OmpB suggests a role for H2B in mediating R. felis internalization into ISE6 cells.