| Backgrounds and objectivesHepatitis C virus(HCV) infection is a significant global public health problem, which affects approximately 3% of world population, and causes 350,000 death cases every year. HCV infection is very likely to progress into chronic infection. As the disease is insidious and advances gradually, patients are usually diagnosed with irreversible chronic liver diseases such as liver steatosis, fibrosis or even hepatocellular carcinoma. In the early time, the therapy against HCV consisted of pegylated interferon(peg-IFN) and ribavirin, and its efficacy varies in different HCV genotypes with long treatment duration and lots of adverse reactions. In recent years, direct acting antiviral agents(DAA) which directly target specific viral protein have undergone rapid development, and largely increased sustained virological response(SVR) rate in HCV patients. However, the usage of DAA is limited due to a series of problems including emergence of resistant mutants, drug-related side effects and high cost of therapy expenses. Therefore, further research about HCV infection is needed. The development of novel drugs targeting different stages of viral life cycle and deciphering the key factors involved in the interaction between virus and host cells will deepen our understanding about the mechanism of HCV infection and provide research foundation for novel antiviral agents with better efficacy and tolerance.HCV infection involves viral entry, translation, replication, assembly and release. The current DAAs in the market inhibit viral replication mainly by interfering the maturation and enzyme activity of viral proteins. However, the genetic barrier of these drugs tend to be low, and reinfection of the graft in end stage HCV patients cannot be prevented by these drugs. HCV entry is a highly coordinated process which consist of virion non-specific attachment, interaction with host entry factors, viral endocytosis and membrane fusion with host cells. This process provided multiple novel targets for anti-HCV therapy, especially those targeting host components are with higher genetic barrier. Since entry inhibitors block the very beginning stage of viral life cycle and the viral genome is not released in the cells, these agents are with both prophylactic and therapeutic activity. The combination of entry inhibitors and current anti-HCV drugs, which represents a multi-target therapy against different stages of viral life cycle, is likely to become the main orientation of future anti-HCV therapy. Thus, the research and development of novel entry inhibitors covering pan-genotypes is of great necessity.HCV infection causes intracellular environmental changes in host cells, resulting in expression alteration of multiple host factors. Among these, some will affect viral infection in return. It will largely promote the research of antiviral therapy against HCV if we better understand the molecular mechanism of these factors especially those with inhibitory effect on HCV infection. Non-coding RNA(nc RNA) is previously considered as elements with no practical functions. Recently, as the development of biological technology, more and more researchers find that ncRNA play various important functions in physiological or pathological processes. MicroRNAs(miRNA) which are less than 200 bp in length has been studied well, and their modes of action and mechanisms have been clarified clearly. MiR-122 is reported to promote virus replication by interacting with 5’UTR of viral genome, and its antagonist has entered clinical trials for treating HCV infection. Our lab also reported that miR-221 enhances the antiviral activity of IFN by targeting the inhibitory molecule in IFN pathway. However, the study of long non-coding RNAs(lncRNA) which are more than 200 bp in length is still in an early stage, and the amount is even less in virus infection especially in HCV infection. Therefore, the discussion about the role of lncRNA in HCV infection is necessary to illuminate the interaction between virus and host cells, and provide insights and targets for antiviral drugs.Currently, most anti-HCV agents are synthesized compound targeting a specific target with high production cost and some adverse reactions. Cell-based therapy is one of the most popular therapies in recent years, and mesenchymal stem cells(MSC) are the most common source of cell-based therapy. The effect of MSC could be realized by paracrining some biological substances from the cells, and exosomes are the critical transmitter of this process. Studies show that exosomes could transfer both infectious pathogens and protective substances against pathogens to the target cells. It has been reported that exosomes secreted from HCV infected cells could transmit infectious viral particles to other cells. Nevertheless, there is no study about exosomes with anti-HCV potency. Human stem cell derived exosomes are cellular components under physiological culture with low production cost and less side effects. It has become a vital part of regenerative medicine. Therefore, the study of it in HCV infection is very meaningful.Part I: Screening and mechanistic study of natural compounds with anti-HCV activityMethods: The two compounds which have antiviral activities against HCV are screened out through HCV in vitro infection models from hundreds of natural pharmaceutical plants. They are schizandronic acid SZA extracted from schisandra and lignanolide TGN from Caulis Trachelospermi. Single cycle HCV pseudoparticles of different genotypes are applied to check the effect of the compounds on viral entry step and in primary human hepatocytes to evaluate the effect of the compounds on viral entry. The experiment is followed by virus kinetic tests which are designed to pinpoint the probable target of SZA and TGN on HCV infection. Meanwhile, the density gradient ultracentrifugation is carried out to determine whether the two compounds impair lipid density and infectivity of HCV particles. We also perform flow cytometry, western blotting and binding activity test to identify the compounds’ influence on relevant receptors on the target cells. Membrane fusion assay mediated by hydrophobic fluorescent dye DiD is taken to dynamically monitor the effect of SZA on the fusion part of virus entry. Fluorescent dye Prodan is used to test the effect of SZA on fluidity of lipid memebrane. Flow cytometry as well as immunoprecipitation assay are implemented to determine the effect of TGN on the interaction between HCV E2 and CD81. Besides, a prediction analysis is made to predict the probable target of TGN on CD81 LEL. SZA and TGN is also tested for their acitivity on virus cell-to-cell transmission, and they are also used with clinically applied anti-HCV drugs to test their antiviral effect in a combination therapy. The results are integrated and analyzed to illustrate the target and probable mechanism of the compounds in viral life cycle.Results: SZA and TGN have low cell toxicity and strong antiviral effect in both HCVcc and HCVpp models. The antiviral activity of SZA and TGN on HCVpp of different genotypes are consistent. SZA and TGN could also inhibit HCVpp entry into primary human hepatocytes. The result of kinetic tests suggests that the post-binding step of early viral entry might be the action target. SZA and TGN do not break the lipid density or affect viral infectivity of HCV particle, while flow cytometry results, western blotting and binding activity test indicate that neither do the compounds affect the expression of entry relevant receptors on target cells, nor do they impair the receptors’ viral binding activity. DiD mediated membrane fusion assay indicates that SZA blocks virion-host membrane fusion process of post-binding step in the early stage of HCV infection with elevated membrane fluidity under treatment of SZA. TGN inhibits HCV infection by interfering the interaction between HCV E2 and CD81, and the prediction analysis shows that the hydrogen bond formed between Thr 166, Asn 184, Lys187 or Glu188 on CD81 LEL and TGN might contribute to the antiviral effect. Moreover, SZA and TGN block HCV cell-to-cell transmission and have significant synergistic effect on HCV inhibition when combined with IFN or VX-950.Conclusion: Natural plant-derived SZA and TGN inhibit HCV infection significantly. The mode of action of the compounds is interfering the key process of viral entry during early phase of HCV infection. The therapeutic targets are quite different from the ones of traditional DAA. These drugs could be served as phrophylactic interventions and the supplements of current therapy, and have promising application prospect in the future.Part II: High-throughput screening and mechanistic study of long non-coding RNA with anti-HCV activityMethods: By high-throughput sequencing analysis, a series of lncRNA have selected out for their differential expression after HCV infection. Among them, almost all the transcripts of lncRNA GAS5 are found to be upregulated after HCV infection. Overexpression or downregulation of this lncRNA could inhibit or promote viral infection. Single cycle HCV pseudoparticles are utilized to evaluate the effect of GAS5 on virus entry. Transfection of HCV RNA by electroporation or usage of HCV replicon cells is carried out to test the role of GAS5 in viral replication. Truncations of GAS5 are constructed to analyze their function in HCV infection for the identification of its functional sequences. Prediction analysis is used to analyze the probable interacted proteins with GAS5, and HCV replication-related protein is screened out from them. RNA immunoprecipitation(RIP) assay is utilized to confirm the interaction between the protein and GAS5. Deletant of GAS5 is constructed to further clarify the functional sequences of this lncRNA. The relevant target and involved mechanism of GAS5 on HCV infection are overall evaluated.Results: GAS5 is upregulated in the cytoplasm of infected cells, and the upregulation is along with the elevated infective doses and progression of the infection. Overexpression of GAS5 in Huh7 cells significantly inhibits HCV infection, while downregulation of GAS5 promotes it. GAS5 does not have inhibitory effect on HCVpp of different genotypes, indicating that it does not block virus entry. Overexpression of GAS5 in HCV replicon cells suppresses intracellular viral RNA levels as well as viral protein expression, downregulation of GAS5 promotes virus infection. Truncations of GAS5 including GAS5-251 can suppress viral infection. Prediction analysis indicates that the front 200 bp of GAS5 can interact with viral NS3 protein. The interacted proteins with GAS5 are pulled down by RNA immunoprecipitation, and analyzed by western-blot. The result shows that overexpression of GAS5 could pull down HCV NS3 protein, providing the proof for potential interaction between GAS5 and viral NS3 protein. Deletant of the front 200 bp of GAS5 loses the original antiviral activity, further confirming that the front 200 bp is the functional sequences that interact with viral protein to inhibit viral infection.Conclusion: LncRNA GAS5 is capable to inhibit HCV infection. The antiviral mechanism involves GAS5’s interaction with viral NS3 protein, interfering the enzyme activity of NS3, thus suppressing viral replication. The upregulation of GAS5 in infected host cells might be due to the defense reactions of host cells to virus infection. This research could provide insights for elucidating molecular mechanism of virus infection and developing future anti-HCV therapy.Part III: Antiviral activity and mechanistic study of mesenchymal stem cell-derived exosomes on HCVMethods: Conditioned medium from umbilical mesenchymal stem cells(uMSC) is used to test its effect on HCV infection. Exosomes are further extracted and purified from the supernatants of uMSC(uMSC-Exo), and its anti-HCV activity is evaluated. Single cycle HCV pseudoparticles are applied to determine the effect of uMSC-Exo on HCV entry. Host cells are transfected with viral RNA or HCV replicon cells are utilized to detect the effect of uMSC-Exo on viral replication. The intracellular and extracellular infectivity are also evaluated to test the effect of uMSC-Exo on viral assembly and release. Proteinase K treatment assay is used to determine which components in uMSC-Exo are the functional substances. Small RNA sequencing is made with uMSC-Exo, and miRNAs with antiviral potency are identified. Function analysis is carried out by overexpression or knock down of relevant miRNAs, and their roles in inhibiting HCV infection are evaluated.Results: uMSC inhibit HCV infection by paracrine, and uMSC-Exo are the main active constituents in this process. uMSC-Exo can enter Huh7 cells and reduce intracellular HCV RNA level as well as viral protein expression in infected cells. uMSC-Exo have no effect on viral entry, but suppress viral replication. Proteinase K treatment assay confirms that the RNA components are the active anti-HCV constituents in uMSC-Exo. Small RNA sequencing of uMSC-Exo indicates their miRNA expression profile. Among them, nine miRNAs are upregulated in the host cells after uMSC-Exo treatment. The functional analysis suggests four miRNAs(let-7f, miR-145, miR-199 a and miR-221) play important roles in HCV infection. The inhibitory effect of uMSC-Exo is lost when the uMSC are transfected with the inhibitors of the four miRNAs. uMSC-Exo exhibit synergistic effect when combined with IFN or VX-950.Conclusion: uMSC-Exo inhibits HCV infection by exosomal mi RNAs(let-7f, miR-145, miR-199 a and miR-221) with antiviral activity on viral replication. This work provides novel insights and possibility for developing anti-HCV therapy.SummaryIn summary, the work uses high-throughput screening of natural compound library, and identifies SZA and TGN with antiviral activity. Further study suggests SZA or TGN inhibits viral entry by targeting either virion membrane fusion or cell entry factor CD81. By high-throughput sequencing, GAS5 is found to be upregulated after HCV infection. The antiviral mechanism concerns interaction with viral NS3 protein, thus interfering normal activity of NS3, and suppressing viral replication. uMSC-Exo are first identified to have antiviral potency against HCV. The mode of action involves specific exosomal miRNA of uMSC to infected target cells. The above research findings provide insights and prospects for deciphering molecular mechanism and developing antiviral agents against HCV infection. |
