Investigation Of The Genes Associated With Antiviral Effect Of Geldanamycin

Objective:Geldanamycin, an ansamycin antibiotic specifically binding heat shock protein 90(Hsp90), inhibits the function of this chaperone, resulting in rapid degradation and inappropriately intracellular trafficking of Hsp90-associated client proteins. GA exhibits not only antitumor activity but also road-spectrum antiviral effect. However, the mechanism of the anti-viral infection has not been understood. To explore the genes associated with the anti-viral infection, we screened and identified some candidate genes which may contribute the anti-viral effect of Geldanamycin in cultured cells.Methods:HeLa cells were infected with herpes simplex virus type 1 or coxsackievirus type B and plaque assay was used to analysis dose-dependent inhibition of GA on virus yields. The stable cell line (293-N) with expressing nucleocapsid protein of SARS-cornavirus was established by transfecting pcDNA3-SARS-N to HEK293 cells and selecting with geneticin. We used flow cytometry to investigate the impact of N protein and GA on cell cycle distribution. The 7267-spot human long oligonucleotide microarrays were applied to investigated the gene expression profiles of untreated HeLa cells, GA-treated(1μM, 24h) HeLa cells, HSV-l-infected(24h) HeLa cells, HSV-1-infected HeLa cells treated with GA(1μM, 24h). The results were confirmed by semi-quantitate RT-PCR.Results: GA significantly inhibited HSV-1 and CBV replication. The titer of HSV-1 was reduced more than 10, 000-fold, and CBV's was reduced more than 1000-fold in the presence of 0. 625μM GA. In the stable cell line expressing SARS-Cov N protein, the cell cycle arrested at the S-G2 transition, and the disturbed cell cycles could be rescued to a normal proliferative phase by GA treatment. The results of genechip demonstrated global impact of GA on host cell gene transcription. It was found that there were fourteen genes up-regulated in the HeLa cells by GA treatment and down-regulated in HeLa cells infected by HSV-1, and ten genes down-regulated in HeLa cells by GA treatment, and up-regulated in HeLa cells infected by HSV-1. The results of semi-quantitate RT-PCR were good agreement with the data of genegchips.Conclusion:GA can inhibit proliferation or replication of many viruses, and it also has a inhibitory effect on the cytopathy causing by some viral components. We utilized micrarray technique to determine the genes involved the antiviral activity of GA at transcription level. GA could up-regulate the expression of ACTG1,BAG3,RAN,S0D1, and down-regulate the expression of HYAL1 which may be related with GA' s antiviral effect.