Glucocorticoids Prevent Enterovirus 71 Capsid Protein VP1 Induced Calreticulin Surface Exposure by Alleviating Neuronal ER Stress.

Severe hand-foot-and-mouth disease (HFMD) caused by Enterovirus 71 (EV71) always accompanies with inflammation and neuronal damage in the central nervous system (CNS). During neuronal injuries, cell surface-exposed calreticulin (Ecto-CRT) is an important mediator for primary phagocytosis of viable neurons by microglia. Our data confirmed that brainstem neurons underwent neuronophagia by glia in EV71-induced death cases of HFMD. EV71 capsid proteins VP1, VP2, VP3, or VP4 did not induce apoptosis of brainstem neurons. Interestingly, we found VP1-activated endoplasmic reticulum (ER) stress and autophagy could promote Ecto-CRT upregulation, but ER stress or autophagy alone was not sufficient to induce CRT exposure. Furthermore, we demonstrated that VP1-induced autophagy activation was mediated by ER stress. Meaningfully, we found dexamethasone treatment could attenuate Ecto-CRT upregulation by alleviating VP1-induced ER stress. Altogether, these findings identify VP1-promoted Ecto-CRT upregulation as a novel mechanism of EV71-induced neuronal cell damage and highlight the potential of the use of glucocorticoids to treat severe HFMD patients with CNS complications.

[Construction and toxicity the recombinant SpltMNPV expressing the scorpion toxin gene].

OBJECTIVE: To develop a high toxic recombinant Spodoptera litura multicapsid nucleopolyhedroviruse (SpltMNPV) insecticide. METHODS: We constructed a recombinant transfer vector that was characterized by disrupting of ecdysterioid UDP-glucosyltransferase (egt) gene and expressing the mature peptide of the Chinese scorpion, B. martensi Karsch (BmK ITal) gene at the control of ie-1 promoter. The transfer vector and the SpltMNPV II DNA cotransfected the SpLi cells. Recombinant viruses were purified by the end point dilution and fluorescent spot purification. RESULTS: We successfully screened the recombinant SpltMNPV-deltaegt-Pph-egfp-ie-1-BmK ITal of which the egt gene was knocked out and expressed the mature peptide of the BmK ITal gene at the control of ie-1 promoter. Bioassays showed that, compared to the wide-type SpltMNPV, the speed of the recombinant virus killing the S. litura (LT50) increased by 0.7-0.8 days. CONCLUSION: The insecticidal effect of SpltNPV could be increased by inserting the foreign gene, which provided a further opportunity to develop the SpltNPV into commercially viable products to control the S. litura.

Bacteriophage T4 gp2 interferes with cell viability and with bacteriophage lambda Red recombination.

The T4 head protein, gp2, promotes head-tail joining during phage morphogenesis and is also incorporated into the phage head. It protects the injected DNA from degradation by exonuclease V during the subsequent infection. In this study, we show that recombinant gp2, a very basic protein, rapidly kills the cells in which it is expressed. To further illustrate the protectiveness of gp2 for DNA termini, we compare the effect of gp2 expression on Red-mediated and Int-mediated recombination. Red-mediated recombination is nonspecific and requires the transient formation of double-stranded DNA termini. Int-mediated recombination, on the other hand, is site specific and does not require chromosomal termini. Red-mediated recombination is inhibited to a much greater extent than is Int-mediated recombination. We conclude from the results of these physiological and genetic experiments that T4 gp2 expression, like Mu Gam expression, kills bacteria by binding to double-stranded DNA termini, the most likely mode for its protection of entering phage DNA from exonuclease V.

Generation of mammalian cells expressing stably measles virus proteins via bicistronic RNA.

The proteins of measles virus are believed to be cytotoxic, and have never been expressed stably from the cloned genes in cultured cells. We found that measles viral proteins can be expressed via a bicistronic RNA. The dominantly selectable DHFR* protein-coding region encoding a mutant dihydrofolate reductase was inserted into the 3'-untranslated regions of the measles viral genes encoding nucleoprotein (N), matrix (M) protein, and hemagglutinin (H). The tandemly arranged cistrons were placed under control by the inducible promoter of human metallothionein IIA gene, or the noninducible early promoter of simian virus 40. Upon transfecting into mammalian cells, these gene constructs synthesized bicistronic RNAs. The downstream DHFR* gene conferred resistance to methotrexate (MTX). Cells that survived MTX selection expressed stably the N, M, or H protein of measles virus. Expression of N protein was further inducible by cadmium chloride treatment. This system will be useful for studying the protein functions of measles virus, and could be applied to express other potentially toxic gene products.