Mean Inhibits Hepatitis C Virus Replication Through Interfering With Poly Pyrimidine Tract Binding Protein

Background and PurposeHCV is a single-strand, positive-sense RNA virus belonging to the family Flaviviridae. As a blood-borne pathogen, HCV is a hepatotropic virus and can lead to acute/chronic hepatitis, cirrhosis, and liver cancer. It is estimated that approximately185million individuals, i.e.3%of the world population, are chronically infected with HCV. Each year,3million people are newly infected with HCV and approximately476,000patients die from HCV-associated end-stage liver disease and its complications. Current therapy for HCV results in sustained responses in only45%of those treated. But this therapy is long, expensive, and toxic. No preventive vaccine against HCV has been produced to date. HCV is an important infectious disease that will still threatens human health for a long time in the future.As a ubiquitous multi-effect cytokine, polypyrimidine tract binding protein (PTB) is able to shuttle between the nucleus and the cytoplasm, a process that is tightly regulated since the diverse cellular functions of PTB are achieved by a combination of changes in its location and its interaction with other proteins. Nuclear PTB is a negative regulator of pre-mRNA alternative splicing and in some cases regulates mRNA polyadenylation. Shuttling PTB plays other cellular roles, such as the transport of mature RNAs to the cytoplasm, and regulation of mRNA translation and stability. In this study, JC1-Luc chimeric virus infected Huh7.5.1cells are used as HCV-infected cell model to explore the effect of small molecule compound MEAN (6-methoxyethylamino-numonafide) on the cytoplasmic shuttling of PTB and HCV replication.Methods1. Analysis of the effect of MEAN on HCV replication Various concentrations of MEAN (0,0.31,0.63,1.25,2.5,5,10,20μM) was added onto Huh7.5.1cells seeded in96-well plate for72h, and cytotoxicity of MEAN on Huh7.5.1cells was evaluated with real time cellular analysis (RTCA). Huh7.5.1cells were transfected with HCV RNA in vitro transcribed from HCV JCl plasmid, and the supernatant was collected and added to naive Huh7.5.1cells in order to establish a HCV-infected cell model (Huh7.5.1-JCl). After24h, medium was aspirated and100μL of serially diluted compound solutions (0,0.63,1.25,2.5,5,10μM) was inoculated onto Huh7.5.1cells in96-well plate. After coincubation for48h, luciferase assay was conducted to determine the anti-HCV effect of MEAN, which was also validated via detecting the level of HCV RNA and Core protein using Q-PCR and western blot, respectively. Moreover, effects of MEAN on the replication of G451R virus and N415D virus and combinational usage of MEAN and IFN-a, ITX5061or RBV were evaluated by detecting luciferase activity.2. Mechanisms underlying the inhibition of MEAN on HCV replication Huh7.5.1cells transfected with HCV IRES dual luciferase reporter plasmid were treated with MEAN. And the influence of MEAN on HCV IRES-mediated translation was explored using dual luciferase assay48h after treatment. The role of PTB in HCV replication was confirmed via siRNA interference experiment. In addition, Q-PCR, Western blot, immunofluorescence assay and heterokaryon assay were used to study the effect of MEAN on the expression level and nucleocytoplasmic shuttling of PTB. mRNA stability experiment was conducted to explore the influence of MEAN on PTB mRNA stability.Results1. MEAN inhibits HCV repliication1) Luciferase assay shows that MEAN significantly inhibits HCV replication (p<0.05), with a50%effective concentration (EC50) of2.36±0.29μM. 2) The results of Q-PCR and western blot reveal that MEAN dramatically reduces the expression level of HCV RNA and Core protein (p<0.05).3) Immunofluorescence assay demonstrates that MEAN treatment dramatically reduces the number of Core+cells found in JCl-luc virus infected cells (p<0.01).4) MEAN is effective in inhibiting N415D mutant virus and G451R mutant virus infections. The EC50s were3.56±0.08μM and3.71±0.14μM, respectively.5) Drug combinational usage analyses demonstrate that MEAN was synergistic with interferon α (IFNa), ITX5061, and ribavirin (RBV).2. MEAN down-regulates the expression and cytoplasmic translocation of PTB1) Dual luciferase assay demonstrates that MEAN fails to down-regulate HCV IRES-directed translation (p>0.05).2) Knockdown of PTB with siRNAs inhibits HCV infection using JCl-luc virus (p <0.01).3) Both PTB RNA and protein expression levels induced by JCl virus infection are markedly reduced in cells treated with MEAN (p<0.01).4) MEAN treatment accelerates the degradation of PTB mRNA.5) Result of western blot shows that strong cytoplasmic accumulation of PTB is observed upon HCV stimulation. Treatment by MEAN significantly blocked the cytoplasmic redistribution of PTB upon infection (p<0.05). The result of immunofluorescence assay also shows that the ratio of nuclear to cytoplasm for PTB fluorescence intensity in MEAN-treated cells is significantly lower than that in HCV-infected cells (p<0.05).6) Heterokaryon assay indicates that EGFP-PTB is exported from the human nuclei and reimport into the mouse nuclei, but the redistribution of EGFP-PTB can be blocked by MEAN treatmentConclusions1. MEAN inhibits HCV replication significantly.2. MEAN exerts its anti-HCV effect via inhibiting the cytoplasmic accumulation of PTB induced by HCV infection.3. MEAN blocks the cytoplasmic redistribution of PTB upon infection through destabilizing PTB mRNA.