Hsp90βis critical for the infection of severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome(SFTS)caused by the SFTS virus(SFTSV)is an emerging disease in East Asia with a fatality rate of up to 30%.However,the viral-host interaction of SFTSV remains largely unknown.The heat-shock protein 90(Hsp90)family consists of highly conserved chaperones that fold and remodel proteins and has a broad impact on the infection of many viruses.Here,we showed that Hsp90 is an important host factor involved in SFTSV infection.Hsp90 inhibitors significantly reduced SFTSV replication,viral protein expression,and the formation of inclusion bodies consisting of nonstructural proteins(NSs).Among viral proteins,NSs appeared to be the most reduced when Hsp90 inhibitors were used,and further analysis showed that their translation was affected.Co-immunoprecipitation of NSs with four isomers of Hsp90 showed that Hsp90βspecifically interacted with them.Knockdown of Hsp90βexpression also inhibited replication of SFTSV.These results suggest that Hsp90βplays a critical role during SFTSV infection and could be a potential target for the development of drugs against SFTS.

Construction of Hsp90β gene specific silencing plasmid and its transfection efficiency

The purpose of this work was to construct the plasmid that could direct the synthesis of siRNA-like transcripts and thus mediate strong and specific repression of human heat shock protein 90β(Hsp90β)gene expression and to compare the transfection efficiency of the plasmids in varying conditions of transfection.Three 64 nt oligos corresponding to different regions of the target gene were chemically synthesized and annealed and were then ligated with pSUPER EGFP1 plasmid and double-digested with HindIII and BglII.Recombinant plasmids were transformed into Escherichia coli,DH5a,and the colonies were picked and grown in the Amp-agarose.The presence of positive clones was checked by the means of endodigestion and sequencing.Three cell strains,HepG2,Human umbilicus vein endothelium cells(HUVEC)and HeK293,were cultured.Then the plasmids were transfected into the cells at different ratios of plasmid to Lipofectamine.The transfection efficiency was measured by detection of enhanced green fluorescence protein(EGFP).The presence of positive recombinant clones were verified by double-digestion and sequencing.The bases inserted into the plasmids were correct and the positive colonies were named pSuper-Hsp90β1,pSuper-Hsp90β2 and pSuper-Hsp90β3.After optimizing the ratio of plasmid to Lipofectamine,we achieved high transfection efficiency in HeK293 cells.Transfection efficiency was still low in the HepG2 cells.In conclusion,the si-RNA-synthesizing plasmids targeting Hsp90βwere constructed and transfected into cells with different transfection efficiency.

Homeopathic Thuja 30C ameliorates benzo(a)pyreneinduced DNA damage,stress and viability of perfused lung cells of mice in vitro

OBJECTIVE： To examine if the ultra-highly diluted homeopathic remedy Thuja 30C can ameliorate benzo（a）pyrene （BaP）-induced DNA damage, stress and viability of perfused lung cells of Swiss albino mice in vitro. METHODS： Perfused normal lung cells from mice were cultured in 5% Roswell Park Memorial Institute medium and exposed to BaP, a potent carcinogen, at the half maximal inhibitory concentration dose （2.2 μmol/L） for 24 h. Thereafter, the intoxicated cells were either treated with Thuja 30C （used against tumor or cancer） or its vehicle media, succussed alcohol 30C. Relevant parameters of study involving reactive oxygen species （ROS） accumulation, total glutathione （GSH） content, and generations of heat shock protein （hsp）-90 were measured; the cell viability and other test parameters were measured after treatment with either Thuja 30C or its vehicle media. Circular dichroism spectroscopy was performed to examine if Thuja 30C directly interacted with calf thymus DNA as target. For ascertaining if DNA damaged by BaP could be partially repaired and restituted by the remedy, 4＇,6-diamidino-2-phenylindole staining was performed. RESULTS： Thuja 30C increased cell viability of BaP-intoxicated cells significantly, as compared to drug-untreated or drug-vehicle control. A minimal dose of Thuja 30C significantly inhibited BaP-induced stress level, by down-regulating ROS and hsp-90, and increasing GSH content. Thuja 30C itself had no DNA-damaging effect, and no direct drug-DNA interaction. However, it showed quite striking ability to repair DNA damage caused by BaP. CONCLUSION： Thuja 30C ameliorates BaP-induced toxicity, stress and DNA damage in perfused lung cells of mice and it apparently has no effect on normal lung cells.