Vaccinia virus induces EMT-like transformation and RhoA-mediated mesenchymal migration.

The emerging outbreak of monkeypox is closely associated with the viral infection and spreading, threatening global public health. Virus-induced cell migration facilitates viral transmission. However, the mechanism underlying this type of cell migration remains unclear. Here we investigate the motility of cells infected by vaccinia virus (VACV), a close relative of monkeypox, through combining multi-omics analyses and high-resolution live-cell imaging. We find that, upon VACV infection, the epithelial cells undergo epithelial-mesenchymal transition-like transformation, during which they lose intercellular junctions and acquire the migratory capacity to promote viral spreading. After transformation, VACV-hijacked RhoA signaling significantly alters cellular morphology and rearranges the actin cytoskeleton involving the depolymerization of robust actin stress fibers, leading-edge protrusion formation, and the rear-edge recontraction, which coordinates VACV-induced cell migration. Our study reveals how poxviruses alter the epithelial phenotype and regulate RhoA signaling to induce fast migration, providing a unique perspective to understand the pathogenesis of poxviruses.

Heparin-Promoted Cellular Uptake of the Cell-Penetrating Glycosaminoglycan Binding Peptide, GBPECP, Depends on a Single Tryptophan.

A 10-residue, glycosaminoglycan-binding peptide, GBPECP, derived from human eosinophil cationic protein has been recently designated as a potent cell-penetrating peptide. A model system containing peptide, glycan, and lipid was monitored by nuclear magnetic resonance (NMR) spectroscopy to determine the cell-penetrating mechanism. Heparin octasaccharide with dodecylphosphocholine (DPC) lipid micelle was titrated into the GBPECP solution. Our data revealed substantial roles for the charged residues Arg5 and Lys7 in recognizing heparin, whereas Arg3 had less effect. The aromatic residue Trp4 acted as an irreplaceable moiety for membrane insertion, as the replacement of Trp4 with Arg4 abolished cell penetration, although it significantly improved the heparin-binding ability. GBPECP bound either heparin or lipid in the presence or absence of the other ligand indicating that the peptide has two alternative binding sites: Trp4 is responsible for lipid insertion, and Arg5 and Lys7 are for GAG binding. We developed a molecular model showing that the two effects synergistically promote the penetration. The loss of either effect would abolish the penetration. GBPECP has been proven to enter cells through macropinocytosis. The GBPECP treatment inhibited A549 lung cancer cell migration and invasion, implying that the cellular microenvironment would be modulated by GBPECP internalization. The intracellular penetration of GBPECP leading to inhibition of epithelial cell migration and invasion depends on the presence of the tryptophan residue in its sequence compared with similar derivative peptides. Therefore, GBPECP shows substantial potential as a novel delivery therapeutic through rapid and effective internalization and interference with cell mobility.

[Therapeutic effects of small interfering RNA targeting MDM2 on human osteosarcoma cells].

OBJECTIVE: To investigate the inhibitory effect of the small interfering RNA targeting mdm2 gene on the growth of osteosarcoma cells. METHODS: PGCsilencerTM-mdm2 siRNA was constructed and transfected into the osteosarcoma cell line U2OS cells. The inhibitory effects on mdm2 were determined by RT-PCR and Western blot analysis. The cell growth activity was determined by MTT assay, and the cell apoptosis was examined by flow cytometry. The therapeutic effects of simdm2 was assessed on the nude mouse model of transplanted tumor. RESULTS: The simdm2 plasmid was successfully constructed. After simdm2 being transfected into the U2OS cells, the expressions of mdm2 gene and protein were significantly inhibited. The ability of cell growth activity decreased greatly and cell apoptosis occurred apparently. There was no significant difference between the negative control group and non-transfected group. The growth of xenograft tumor in simdm2 transfected nude mice was inhibited and the expressions of mdm2 gene and protein were down-regulated remarkably. CONCLUSION: siRNA targeting mdm2 gene inhibits the mdm2 expression in osteosarcoma U2OS cells and the growth of osteosarcoma in nude mice.