No abstract

Enterovirus 71 (EV71) is a causative agent of an array of childhood diseases with severe neurological manifestations implicated. EV71 infection is known to induce caspase-dependent apoptosis in cell cultures and animal models. However, whether an alternative apoptotic pathway independent of caspase activation can be triggered by EV71 infection has not been explored. In this study, we showed that calcium (Ca 2+ )-activated calpains are capable of mediating caspaseindependent pathway activation during EV71-induced apoptosis in HeLa cells. Results from subcellular fractionation analysis and confocal imaging indicated that during EV71 infection, apoptosis-inducing factor (AIF), a primary mediator of the caspase-independent pathway, became truncated and translocated from the mitochondrion to nucleus. This was accompanied by the release of cytochrome c, and sharply decreased mitochondrial membrane potential. AIF knockdown data indicated significant protection against apoptotic cell death, with greater protection provided by the addition of a pan-caspase inhibitor. The Ca 2+ -dependent, calpain isoforms 1 and 2, but not cathepsins, were proven crucial for the altered AIF behaviour as studied by the pharmacological inhibitor and the knockdown approaches. We then analysed Ca 2+ dynamics in the infected cells and found elevated levels of mitochondrial Ca 2+ . Treatment with ruthenium red, a mitochondrial Ca 2+ influx inhibitor, significantly blocked calpain activations and AIF cleavage. Our conclusion was that calpain activation via Ca 2+ flux plays an essential role in eliciting an AIF-mediated, caspase-independent apoptotic pathway in EV71-infected cells. These findings should be useful for understanding the virus-induced cytopathology and the impact of Ca 2+ homeostasis on EV71 infection.

A real-time assay system that allows monitoring of intracellular human enterovirus (HEV) protease activity was established using the principle of fluorescence resonance energy transfer (FRET). It was accomplished by engineering cells to constitutively express a genetically encoded FRET probe. The FRET-based probe was designed to contain an enterovirus 71 3C protease (3C pro ) cleavage motif flanked by the FRET pair composed of green fluorescent protein 2 and red fluorescent protein 2 (DsRed2). Efficient FRET from the stable line was detected in a real-time manner by fluorescence microscopy, and the disruption of FRET was readily monitored upon HEV infection. The level of the repressed FRET was proportional to the input virus titer and the infection duration as measured by the fluorometric method. The FRET biosensor cell line was also responsive to other related HEV serotypes, but not to the phylogenetically distant herpes simplex virus, which was confirmed by Western blot analysis. The FRET biosensor was then utilized to develop a format for the determination of antiviral susceptibility, as the reduced FRET appeared to reflect viral replication. Evaluations of the FRET biosensor system with representative HEV serotypes demonstrated that their susceptibilities to a 3C pro inhibitor, rupintrivir, were all accurately determined. In summary, this novel FRET-based system is a means for rapid detection, quantification, and drug susceptibility testing for HEVs, with potential for the development of a high-throughput screening assay.

No abstract

BACKGROUND Luminescent nanobioprobes with cell-targeting specificity are likely to find important applications in bioanalysis, biomedicine, and clinical diagnosis. Quantum dots (QDs) are unique and promising materials for such a purpose because of their fluorescence and large surface area for attaching cell-targeting molecules. METHODS We produced water-dispersible QDs by coating hydrophobic QDs with small amphiphilic polyethylene glycol (PEG) molecules via hydrophobic interactions. We covalently coupled folate (FA) onto the water-dispersible PEG-coated QDs (PEG-QDs) to produce FA-coupled PEG-QDs (FA-PEG-QDs). RESULTS These FA-PEG-QD nanoparticles functioned as fluorescent nanobioprobes that specifically recognized folate receptors (FRs) overexpressed in human nasopharyngeal cells (KB cells) but not in an FR-deficient lung carcinoma cell line (A549 cells). Using confocal fluorescence microscopy, we demonstrated uptake of FA-PEG-QDs by KB cells but no uptake of folate-free PEG-QDs. The specificity of this receptor-mediated internalization was confirmed by comparing the uptake by KB vs A549 cells. CONCLUSIONS Our results suggest that such cell-targeting fluorescent nanobioprobes are potentially very powerful tools for recognizing target cells and delivering and tracking drugs and other therapeutic materials.