Robert Y.L. Wang

Emerging picture of host chaperone and cyclophilin roles in RNA virus replication

Many plus-strand (+)RNA viruses co-opt protein chaperones from the host cell to assist the synthesis, localization and folding of abundant viral proteins, to regulate viral replication via activation of replication proteins and to interfere with host antiviral responses. The most frequently subverted host chaperones are heat shock protein 70 (Hsp70), Hsp90 and the J-domain co-chaperones. The various roles of these host chaperones in RNA virus replication are presented to illustrate the astonishing repertoire of host chaperone functions that are subverted by RNA viruses. This review also discusses the emerging roles of cyclophilins, which are peptidyl-prolyl isomerases with chaperone functions, in replication of selected (+)RNA viruses.

Induction of Apoptosis by Sinulariolide from Soft Coral through Mitochondrial-Related and p38MAPK Pathways on Human Bladder Carcinoma Cells

Sinulariolide, an isolated compound from the soft coral Sinularia flexibilis, possesses the anti-proliferative, anti-migratory and apoptosis-inducing activities against the TSGH bladder carcinoma cell. The anti-tumor effects of sinulariolide were determined by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, cell migration assay and flow cytometry, respectively. Sinulariolide inhibited the growth and migration of bladder carcinoma cells in a dose-dependent manner, as well as induced both early and late apoptosis as determined by the flow cytometer. Also, the sinulariolide-induced apoptosis is related to the mitochondrial-mediated apoptosis via caspase-dependent pathways, elucidated by the loss of mitochondrial membrane potential, release of cytochrome C, activation of caspase-3/-9, Bax and Bad, as well as suppression of Bcl-2/Bcl-xL/Mcl-1. Detection of the PARP-1 cleaved product suggested the partial involvement of caspase-independent pathways. Moreover, inhibition of p38MAPK activity leads to the rescue of the cell cytotoxicity of sinulariolide-treated TSGH cells, indicating that the p38MAPK pathway is also involved in the sinulariolide-induced cell apoptosis. Altogether, these results suggest that sinulariolide induces apoptosis against bladder cancer cells through mitochondrial-related and p38MAPK pathways.

Identification of heat-shock protein 90 beta in Japanese encephalitis virus-induced secretion proteins

Five host cellular proteins were identified in the secretion medium from Japanese encephalitis virus (JEV)-infected baby hamster kidney-21 (BHK-21) cells, including three molecular chaperones: Hsp70, GRP78 and Hsp90. Hsp90 isoforms were characterized further. Hsp90α was observed to be retained inside the nuclei, whereas Hsp90β associated with virus particles during assembly and was released into the secretion medium upon JEV infection. The association of Hsp90β and viral E protein was demonstrated by using sucrose-density fractionation and Western blot analysis. Moreover, JEV viral RNA replication was not affected by treatment with geldanamycin, an Hsp90 inhibitor, but impaired virus infectivity that was determined by a plaque-forming assay. Our results show that Hsp90β, not Hsp90α, is present in the JEV-induced secretion medium and is required for JEV infectivity in BHK-21 cells.

13-Acetoxysarcocrassolide Induces Apoptosis on Human Gastric Carcinoma Cells Through Mitochondria-Related Apoptotic Pathways: p38/JNK Activation and PI3K/AKT Suppression

13-acetoxysarcocrassolide (13-AC), an active compound isolated from cultured Formosa soft coral Sarcophyton crassocaule, was found to possess anti-proliferative and apoptosis-inducing activities against AGS (human gastric adenocarcinoma cells) gastric carcinoma cells. The anti-tumor effects of 13-AC were determined by MTT assay, colony formation assessment, cell wound-healing assay, TUNEL/4,6-Diamidino-2-phenylindole (DAPI) staining, Annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining and flow cytometry. 13-AC inhibited the growth and migration of gastric carcinoma cells in a dose-dependent manner and induced both early and late apoptosis as assessed by flow cytometer analysis. 13-AC-induced apoptosis was confirmed through observation of a change in ΔΨm, up-regulated expression levels of Bax and Bad proteins, down-regulated expression levels of Bcl-2, Bcl-xl and Mcl-1 proteins, and the activation of caspase-3, caspase-9, p38 and JNK. Furthermore, inhibition of p38 and JNK activity by pretreatment with SB03580 (a p38-specific inhibitor) and SP600125 (a JNK-specific inhibitor) led to rescue of the cell cytotoxicity of 13-AC-treated AGS cells, indicating that the p38 and the JNK pathways are also involved in the 13-AC-induced cell apoptosis. Together, these results suggest that 13-AC induces cell apoptosis against gastric cancer cells through triggering of the mitochondrial-dependent apoptotic pathway as well as activation of the p38 and JNK pathways.

Cyclophilin A and Nuclear Factor of Activated T Cells Are Essential in Cyclosporine‐Mediated Suppression of Polyomavirus BK Replication

Immunosuppressants have impacts on the development of polyomavirus‐associated nephropathy. We previously demonstrated that cyclosporin A (CsA) suppressed polyomavirus BK (BKV) replication. The role of cyclophilin A (CypA) and nuclear factor of activated T cells (NFAT) in CsA‐imposed suppression of BKV replication was determined in this study. Results demonstrated that knockdown of CypA but not CypB significantly reduced BKV large T antigen (TAg) expression and BKV titer. Overexpression of CypA reversed CypA siRNA‐induced inhibition in BKV TAg expression. In addition, CypA overexpression attenuated the suppressive effect of CsA on TAg expression, suggesting CypA implicated in CsA‐mediated anti‐BKV effect. Knockdown of NFATc3 abrogated TAg expression, while overexpression of NFATc3 promoted TAg expression and augmented BKV promoter activity. NFATc3 binding to the BKV promoter was verified by chromatin immunoprecipitation assay and electrophoretic mobility shift assay. Renal histology also displayed an increase in NFATc3 expression in tubulointerstitium of BKV‐associated nephropathy. Furthermore, overexpression of NFATc3 rescued CsA‐mediated inhibition of BKV load and TAg expression. A CsA analog, NIM811, which cannot block NFAT functionality, failed to suppress TAg expression. In conclusion, CypA and NFAT are indispensable in BKV replication. CsA inhibits BKV replication through CypA and NFAT, which may be potential targets of anti‐BKV treatment.

Heat shock protein-90-beta facilitates enterovirus 71 viral particles assembly

Molecular chaperones are reported to be crucial for virus propagation, but are not yet addressed in Human Enterovirus 71 (EV71). Here we describe the specific association of heat shock protein-90-beta (Hsp90β), but not alpha form (Hsp90α), with EV71 viral particles by the co-purification with virions using sucrose density gradient ultracentrifugation, and by the colocalization with viral particles, as assessed by immunogold electron microscopy. The reduction of the Hsp90β protein using RNA interference decreased the correct assembly of viral particles, without affecting EV71 replication levels. Tracking ectopically expressed Hsp90β protein associated with EV71 virions revealed that Hsp90β protein was transmitted to new host cells through its direct association with infectious viral particles. Our findings suggest a new antiviral strategy in which extracellular Hsp90β protein is targeted to decrease the infectivity of EV71 and other enteroviruses, without affecting the broader functions of this constitutively expressed molecular chaperone.

A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells

The roles of virus-derived small RNAs (vsRNAs) have been studied in plants and insects. However, the generation and function of small RNAs from cytoplasmic RNA viruses in mammalian cells remain unexplored. This study describes four vsRNAs that were detected in enterovirus 71-infected cells using next-generation sequencing and northern blots. Viral infection produced substantial levels (>105 copy numbers per cell) of vsRNA1, one of the four vsRNAs. We also demonstrated that Dicer is involved in vsRNA1 generation in infected cells. vsRNA1 overexpression inhibited viral translation and internal ribosomal entry site (IRES) activity in infected cells. Conversely, blocking vsRNA1 enhanced viral yield and viral protein synthesis. We also present evidence that vsRNA1 targets stem-loop II of the viral 5′ untranslated region and inhibits the activity of the IRES through this sequence-specific targeting. Our study demonstrates the ability of a cytoplasmic RNA virus to generate functional vsRNA in mammalian cells. In addition, we also demonstrate a potential novel mechanism for a positive-stranded RNA virus to regulate viral translation: generating a vsRNA that targets the IRES.

Human foreskin fibroblast‐like stromal cells can differentiate into functional hepatocytic cells

Foreskin fibroblast‐like stromal cells (FDSCs) are progenitors isolated from human tissue that can differentiate into diverse cell types. Many types of stem cells can differentiate into hepatocyte‐like cells, which could be used for drug testing or in liver regeneration therapy, but whether FDSCs can be converted into functional hepatocytes is unknown. FDSCs show divergent properties when cultured in distinct media, forming spheres in Dulbeccos modified Eagles medium (DMEM) containing F12, epidermal growth factor (EGF), and basic fibroblast growth factor (b‐FGF), but have fibroblast‐like morphology when cultured in DMEM‐based growth medium. Both cell populations express the typical mesenchymal stem cell markers CD90, CD105, and CD73, but the p75 neurotrophin receptor (p75NTR) was detected only in FDSC spheres. Both types of FDSCs can differentiate into hepatocyte‐like cells, which express typical liver markers, including albumin and hepatocyte paraffin 1 (Hep Par1), along with liver‐specific biological activities. When plasmids containing the human hepatitis B virus (HBV) genome were transfected transiently into FDSCs, differentiated hepatocyte‐like cells secrete large amounts of HBe and HBs antigens. FDSCs could be used for clinical hepatic therapy and/or serve as a model of HBV.

Rapid detection and quantification of Enterovirus 71 by a portable surface plasmon resonance biosensor

This study presents the first report on a label-free detection and rapid quantification method for human enterovirus 71 (EV71) using a portable surface plasmon resonance (SPR) system. The SPR sensor instrument was configured to run on low power in a miniaturized platform to improve the device portability for a wider application both in laboratories and in the field. A color tunable organic light emitting diode in red spectrum was attached on a trapezoidal prism for the disposable light source module. The SPR signal processing using integration area under the reflectivity curve is applied for optimum signal to noise ratio (SNR) enhancement. The major capsid protein VP1 of EV71 was selected as the biomarker target in the detection study. The experimental time required for the EV71 quantification was reduced from 6 days using the conventional viral plaque assay to several minutes using the proposed method. The study results establish a detection limit of approximately 67 virus particles per milliliter (vp/ml) of EV71 in a Dulbeccos modified Eagles medium. The VP1 detection in the portable SPR biosensor had a detection limit of approximately 4.8pg/ml in the PBS buffer. Therefore, the proposed direct EV71 viral particle quantification method can be rapidly performed in real time, with high sensitivity and less labor and without assays or fluorescence.

Proteome demonstration of alpha-1-acid glycoprotein and alpha-1-antichymotrypsin candidate biomarkers for diagnosis of enterovirus 71 infection.

Background: Human enterovirus 71 (EV71) is the major causative agents of hand-foot-and-mouth disease and frequently associated with severe complications such as encephalitis and death. Understanding the host response following enteroviral infection may facilitate the development of biomarkers for EV71 infections. Methods: We implemented two-dimensional gel electrophoresis technology on proteins prepared from serum obtained from 4 mild and 4 severe cases of EV71 infections and 4 healthy control children, to investigate the differentially expressed proteins. The differential expressed proteins were further identified with liquid chromatography–mass spectrometry/mass spectrometry analysis and western blotting validation. Results: A total of 27 differentially expressed proteins were picked and identified with liquid chromatography–mass spectrometry/mass spectrometry. Of the 27 identified proteins, 6 proteins were up-regulated in the mild-infected and severe EV71-infected patients in comparison to the healthy control group. Two proteins, alpha-1-acid-glycoprotein (AGP1) and alpha-antichymotrypsin (AACT), were not detected in the EV71-infected patients, but appeared in the control patient. Western blotting analysis demonstrated that AGP1 and AACT proteins were negatively associated with the clinical severity of EV71 infection. Similarly, both of the proteins were not detected in the secretion medium from the EV71-infected neuroblastoma cells, but detected in the mock-infected cells, suggesting that differentially expressed AGP1/AACT protein levels are in response to EV71 infections. Conclusions: Two candidate proteins AGP1 and AACT, whose expression levels were reduced under the EV71 infection pathological condition, provide useful source of information for potential diagnostic biomarkers of EV71 infection in children.