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Tylophorine Analogs Allosterically Regulates Heat Shock Cognate Protein 70 And Inhibits Hepatitis C Virus Replication.

ABSTRACT: Tylophorine analogs have been shown to exhibit diverse activities against cancer, inflammation, arthritis, and lupus in vivo. In this study, we demonstrated that two tylophorine analogs, DCB-3503 and rac-cryptopleurine, exhibit potent inhibitory activity against hepatitis C virus (HCV) replication in genotype 1b Con 1 isolate. The inhibition of HCV replication is at least partially mediated through cellular heat shock cognate protein 70 (Hsc70). Hsc70 associates with the HCV replication complex by primarily binding to the poly U/UC motifs in HCV RNA. The interaction of DCB-3503 and rac-cryptopleurine with Hsc70 promotes the ATP hydrolysis activity of Hsc70 in the presence of the 3' poly U/UC motif of HCV RNA. Regulating the ATPase activity of Hsc70 may be one of the mechanisms by which tylophorine analogs inhibit HCV replication. This study demonstrates the novel anti-HCV activity of tylophorine analogs. Our results also highlight the importance of Hsc70 in HCV replication.

SUBMITTER: Wang Y

PROVIDER: S-EPMC5577180 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Tylophorine Analogs Allosterically Regulates Heat Shock Cognate Protein 70 And Inhibits Hepatitis C Virus Replication.

Wang Ying Y Lee Sangwon S Ha Ya Y Lam Wing W Chen Shao-Ru SR Dutschman Ginger E GE Gullen Elizabeth A EA Grill Susan P SP Cheng Yao Y Fürstner Alois A Francis Samson S Baker David C DC Yang Xiaoming X Lee Kuo-Hsiung KH Cheng Yung-Chi YC

Scientific reports 20170830 1

Tylophorine analogs have been shown to exhibit diverse activities against cancer, inflammation, arthritis, and lupus in vivo. In this study, we demonstrated that two tylophorine analogs, DCB-3503 and rac-cryptopleurine, exhibit potent inhibitory activity against hepatitis C virus (HCV) replication in genotype 1b Con 1 isolate. The inhibition of HCV replication is at least partially mediated through cellular heat shock cognate protein 70 (Hsc70). Hsc70 associates with the HCV replication complex ...[more]

PMID: 28855547

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Project description:BACKGROUND: Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Because of the limited effectiveness of existing vaccines and drugs, development of novel antiviral strategies is urgently needed. Heat stress cognate 70 (Hsc70) is an ATP-binding protein of the heat stress protein 70 family. Hsc70 has been found to be required for HBV DNA replication. Here we report, for the first time, that combined siRNAs targeting viral gene and siHsc70 are highly effective in suppressing ongoing HBV expression and replication. METHODS: We constructed two plasmids (S1 and S2) expressing short hairpin RNAs (shRNAs) targeting surface open reading frame of HBV(HBVS) and one plasmid expressing shRNA targeting Hsc70 (siHsc70), and we used the EGFP-specific siRNA plasmid (siEGFP) as we had previously described. First, we evaluated the gene-silencing efficacy of both shRNAs using an enhanced green fluorescent protein (EGFP) reporter system and flow cytometry in HEK293 and T98G cells. Then, the antiviral potencies of HBV-specific siRNA (siHBV) in combination with siHsc70 in HepG2.2.15 cells were investigated. Moreover, type I IFN and TNF-? induction were measured by quantitative real-time PCR and ELISA. RESULTS: Cotransfection of either S1 or S2 with an EGFP plasmid produced an 80%-90% reduction in EGFP signal relative to the control. This combinational RNAi effectively and specifically inhibited HBV protein, mRNA and HBV DNA, resulting in up to a 3.36 log10 reduction in HBV load in the HepG2.2.15 cell culture supernatants. The combined siRNAs were more potent than siHBV or siHsc70 used separately, and this approach can enhance potency in suppressing ongoing viral gene expression and replication in HepG2.2.15 cells while forestalling escape by mutant HBV. The antiviral synergy of siHBV used in combination with siHsc70 produced no cytotoxicity and induced no production of IFN-?, IFN-? and TNF-? in transfected cells. CONCLUSIONS: Our combinational RNAi was sequence-specific, effective against wild-type and mutant drug-resistant HBV strains, without triggering interferon response or producing any side effects. These findings indicate that combinational RNAi has tremendous promise for developing innovative therapy against viral infection.

Project description:BackgroundPhylogenetic and functional analysis was conducted on an Anopheles gambiae gene, ENSANGG00000017398. Based on phylogenetic analysis, this gene belongs to the same lineage as Heat shock protein cognate 70-4 (Hsc70-4) in Drosophila. Accordingly, we propose to name this gene Heat shock protein cognate 70B (HSC70B). We previously reported that expression of HSC70B and other genes including elongation factor-1alpha (EF-1alpha) and the agglutinin attachment subunit (agglutinin) were up-regulated in o'nyong-nyong virus (ONNV)-infected female An. gambiae. Double-stranded RNA interferences have been applied to further investigate HSC70B, EF-1alpha and the agglutinin functions in ONNV replication in An. gambiae.ResultsAmong these three RNAi silenced genes, only dsRNAs of HSC70B (dsHSC70B) promoted ONNV replication in adult An. gambiae compared to the control mosquitoes that were co-injected with ONNV and dsRNA of beta-galactosidase (dsbeta-gal). ONNV titers from mosquitoes co-injected with dsHSC70B were about 9-fold higher at 6 days post-injection (d.p.i.) as compared to the control mosquitoes. By using ONNV tagged with enhanced green fluorescent protein (ONNV-eGFP), co-injection of ONNV-eGFP with dsHSC70B also showed approximately 2 ~ 3-fold higher GFP expression rates than the controls in the head, thorax, and abdomen of the mosquito. Furthermore, co-injection of ONNV with dsHSC70B significantly reduced the lifespan of adult mosquitoes as compared with the control, co-injection of ONNV with dsbeta-gal treated mosquitoes.ConclusionThese results indicate that HSC70B plays important roles in homeostasis and suppression of ONNV replication in the vector, An. gambiae. Biological implications of these findings are that while mosquitoes allow ONNV to replicate in them, they also check viral titers so that ONNV infection will result in no harmful effect on mosquitoes. Therefore, mosquitoes can function as vectors of ONNV transmission to humans while ONNV infection in An. gambiae remains asymptomatic.

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Tylophorine Analogs Allosterically Regulates Heat Shock Cognate Protein 70 And Inhibits Hepatitis C Virus Replication.

Publications

Tylophorine Analogs Allosterically Regulates Heat Shock Cognate Protein 70 And Inhibits Hepatitis C Virus Replication.

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