Mechanistic Insights into Translation and Replication of Hepatitis C Virus RNA : Exploring Direct-Acting Antivirals
Hepatitis C virus (HCV), a blood-borne pathogen, is a small enveloped RNA virus belonging to the Hepacivirus genus of the Flaviviridae family. HCV infection represents one of the major health concerns affecting approximately 170 million people globally. Patients with chronic HCV infection are at risk of developing hepatic fibrosis, cirrhosis and hepatocellular carcinoma. No protective anti-HCV vaccine is available yet. Until recently, standard therapy based on pegylated interferon plus ribavirin, was inadequate in treating all the patients as it results in a sustained virological response in only 40 to 50 percent of patients infected with the most common genotype (gt 1). Advances in understanding host-HCV interactions have helped developing newer anti-HCV agents such as telaprevir and boceprevir. However, treatment success is still limited due to different factors including genotype specificity, high cost, potential drug-drug interactions, substantial side effects etc. The positive-sense single-stranded RNA genome of HCV is approximately 9.6kb long which is flanked by highly structured and conserved 5’ and 3’ untranslated regions (UTRs) at both ends. Unlike cap-dependent translation of host cell mRNAs, HCV translation is mediated by an internal ribosomal entry site (IRES) present majorly within the 5’UTR. Several reports have demonstrated the interaction of different cellular proteins with HCV-5’UTR and/or 3’UTR, which include human La protein, polypyrimidine tract binding protein (PTB), poly (rC)-binding protein 2 (PCBP2) etc. These interactions of trans-acting factors with the UTRs may be important for HCV translation and/or replication. Earlier study from our laboratory revealed the importance of interaction of human La protein, by its central RNA recognition motif (RRM), with the HCV IRES around a tetranucleotide sequence GCAC near initiator AUG in influencing HCV translation. However, the role of this interaction, if any, in HCV RNA replication was not known. In the first part of the thesis, we characterized the interaction between human La protein and the GCAC to understand its role in HCV replication. We incorporated mutation, which altered the binding of La, in the GCAC motif in HCV monocistronic replicon and checked HCV RNA replication by reverse transcriptase polymerase chain reaction (RT-PCR). The mutation drastically inhibited HCV replication. Interestingly, overexpression of La could reverse the effect of this mutation and significantly enhanced HCV RNA levels. Using a bicistronic replicon, we observed that decrease in replication was independent of translation inhibition. Furthermore, mutation at the GCAC motif reduced the association between La and viral polymerase, NS5B as seen in co-immunoprecipitation assays. Moreover, this mutation affected translation to replication switch regulated by the interplay between HCV-NS3 protease and human La protein. Our analyses of point mutations, based on RT-PCR and luciferase assays, revealed distinct roles of each nucleotide of the GCAC motif in HCV replication and translation. Finally, 5’-3’ crosslink assays revealed that specific interaction of the GCAC motif with human La protein is important for linking 5’ and 3’ends of HCV genome. Results clearly demonstrate the mechanism of regulation of HCV replication by interaction of cis-acting element GCAC within the HCV IRES with human La protein. HCV is highly species-specific. Under natural conditions, HCV infects only humans and chimpanzees. This restricted host-tropism has prevented the development of a small animal model to study HCV infection in vivo. Although several human-specific entry factors have been identified to be responsible for this species selectivity, full multiplication of the HCV in animals (other than humans and chimpanzees) is still not possible. In the second part of the thesis, we showed that a post-entry host factor –‘La protein’ may also contribute in determining HCV host tropism. We aligned La protein sequences from different species and interestingly we found that HCV RNA interacting beta-turn sequence (KYKETDL) in central RRM (residues 112-184) is conserved only in human and chimpanzee. Earlier, it was shown from our laboratory that a heptameric peptide comprising of this sequence (derived from human La) could inhibit HCV translation by competing with La interaction with the IRES element. However, in the current study, another peptide corresponding to the mouse La sequence (KYKDTNL) was unable to inhibit HCV RNA translation. Similarly, wild-type mouse La (mLa) failed to stimulate HCV IRES function, but addition of chimeric mouse La protein bearing human beta-turn sequence (mLahN7) significantly increased HCV IRES mediated translation in vitro. Also, exogenous supplementation of mLahN7 enhanced HCV translation in cell culture system. Moreover, quantitative as well as tagged RT-PCR analyses showed an enhanced HCV replication upon overexpression of mLahN7. The findings obtained in this part raise a possibility of creating HCV mouse model using human specific cellular entry factors and a humanized form of La protein. Hepatitis C has emerged as a major challenge to the medical community. Developing more potent and safe anti-HCV regimens is need of the hour. As described above, a linear hepatapeptide (KYKETDL) was synthesized and shown to reduce HCV translation. However, this linear peptide was stable only for a shorter time scale. Therefore, in the third part of the thesis, effect of a more stable cyclic form of this peptide has been described. NMR spectroscopy suggested that the beta turn conformation is preserved in cyclic peptide as well. Also, using in vitro bicistronic reporter assay, we demonstrated that cyclic peptide inhibits HCV translation in a dose dependent manner. In fact, due to its higher stability, cyclic peptide reduced HCV translation and replication more efficiently than the corresponding linear peptide at longer post-treatment time point. Additionally, we observed that cyclic peptide is non-toxic in cell culture system. Our results suggest that cyclic peptide might emerge as a promising lead compound against hepatitis C. Due to availability of only partially effective liver protective drugs in modem medicine, complementary and alternative medicine approach, based on plant derived compounds, is also being utilised against HCV. Plant derived compounds have advantages of having high chemical diversity, drug-likeliness properties and ability of being metabolized by the body with little or no toxicity than synthetic ones. Different studies have shown that phytochemicals may exert anti-HCV activities by acting as direct-acting antivirals and play a potential therapeutic role in treating HCV infection. Also, from our laboratory, it was shown that methanolic extract of Phyllanthus amarus (P. amarus) plant inhibited HCV replication. The fourth part of the thesis describes the study on the anti-HCV properties of several bioactive components from P. amarus extract. Using a fluorimetric assay, we demonstrated that two principal components of this extract, phyllanthin and corilagin reduced the HCV NS3 protease activity significantly in vitro. We also observed a sharp reduction in HCV negative sense RNA levels in cell culture system. Structural knowledge-based molecular docking studies showed interactions of phyllanthin and corilagin with the amino acid residues of the catalytic triad of NS3 protease. Further, these compounds were found to be non-toxic in cell culture. Also, phyllanthin and corilagin displayed antioxidant properties by blocking HCV induced oxidative stress generated by reactive oxygen species suggesting their hepatoprotective nature. More importantly, our in vivo toxicity analyses and pharmacokinetics studies proved their safety, tolerability, metabolic stability, and systemic oral bioavailability and support their potential as novel anti-HCV therapeutic candidates. Altogether, the study deciphers mechanistic details of translation and replication of HCV RNA and demonstrates novel antiviral agents targeting these important viral processes.
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