Characterization of Host Protein Interactions with HCV RNA : Implications in Viral Translation, Replication and Design of Antivirals

Abstract

HCV genome is a positive sense single-stranded RNA containing a single open reading frame (ORF) flanked by untranslated regions (UTRs), 5’UTR and 3’UTR.Initiation of HCV RNA translation is mediated by internal ribosome entry site (IRES) present in 5’ UTR and this process is independent of cap-structure and requires only a small subset of canonical initiation factors. Hence, HCV IRES-mediated translation initiation mechanism is quite different from canonical cellular mRNA translation initiation. The IRES is organized into highly structured domains, namely domain II, III and IV. High affinity interactions between structured RNA elements present in the IRES and 40S ribosomal proteins mediate 40S recruitment to HCV IRES. However, details of the RNA elements and region of ribosomal proteins involved in these interactions are poorly understood. In recent days, RNA-based molecules like siRNAs, antisense RNAs and RNA decoys have become promising candidates for antiviral molecules. So designing short RNA molecules that target unique HCV translation initiation mechanism might help in developing novel anti-HCV molecules. HCV 3’UTR and antisense-5’ UTRs serve as sites for replication initiation to synthesize negative and positive strand and this process is catalyzed by NS5B protein (RNA-dependent RNA polymerase). Hence, host proteins binding to both 3’UTR and antisense-5’UTR might play important role in HCV replication. This puts the study of HCV RNA–host protein interactions and its role in viral translation and replication in perspective. Studying the HCV IRES-ribosomal protein S5 interactions and its role in HCV IRES function Previous studies from our laboratory have demonstrated that binding of La protein to GCAC close to initiator AUG enhances ribosomal protein S5 (RPS5) binding with HCV IRES and stimulates HCV translation. However in-detail study on HCV IRES–RPS5 interactions and its implication on HCV translation initiation were lacking. In present study computational modelling suggested that domain II and IV interact majorly with the beta hairpin structure and C-terminal helix of RPS5. Filter-binding and UV cross-linking studies with peptides derived from predicated RNA-binding region of RPS5 and mutational studies with RPS5 demonstrated that beta hairpin structure present in RPS5 is critical for IRES–RPS5 interaction. In parallel, we have studied RNA elements involved in the IRES–RPS5 interactions using deletions and substitution mutations, which we had generated on the basis of the computational model. Direct and competition UV cross-linking experiments performed with these IRES mutants and 40S subunits as a source of RPS5 suggested that structure and sequence of both domain II and IV play crucial role in IRES–RPS5 interactions. We further investigated the effect of these mutations on IRES activity by in vitro translation assay and found that all the mutants that were compromised in binding to RPS5 showed reduced IRES activity. Moreover, ribosome assembly experiments on HCV IRES demonstrated that mutations affecting IRES–RPS5 interactions result in reduction of 80S peak and slight increase of 48S peak. Since the 40S subunit had been previously reported to bind with HCV 3’UTR, we explored the possible interaction of RPS5 with HCV 3’UTR. From direct and competition UV cross-linking assays, we found that RPS5 does not bind to 3’UTR and the interaction is unique to IRES (5’UTR). Interestingly, partial silencing of RPS5 preferentially inhibited HCV translation with marginal effect on cap-dependent translation. Recently, reduction in 40S subunit abundance was reported to preferentially inhibit HCV translation. So, we investigated the abundance of free 40S subunit upon silencing RPS5 and results showed reduction in free 40S subunit level. So, we hypothesize that silencing of RPS5 reduces free 40S abundance to inhibit HCV translation. Taken together, results identified specific RNA elements present in HCV IRES that are critical for IRES–RPS5 interactions and demonstrated the role of these interactions in HCV translation initiation. Targeting ribosome assembly on HCV IRES using short RNAs Stem-loops (SL) IIIe and IIIf of HCV IRES are known to play an important role in stable IRES–40S complex formation. However interaction of these stem-loops with 40S subunit in isolation, independent of other regions of HCV IRES, was not studied. In this study, using electrophoretic mobility shift assay (EMSA) and sucrose gradient centrifugation experiments, we demonstrate that short RNA containing both SLIIIe and SLIIIf together (SLRef RNA) binds to 40S subunit, while short RNAs containing either of the stem-loops (SLRe RNA and SLRf RNA) lose their ability to interact with 40S subunit. Further, SLRef RNA inhibited ribosome assembly on the IRES, whereas SLRe and SLRf RNA failed to inhibit the same. Since SLRef RNA is derived from IRES, we investigated the interaction SLRef RNA with IRES–trans-acting factors (ITAFs). UV cross-linking of radio-labelled HCV IRES with cytoplasmic extract (S10) in presence of unlabelled short RNAs suggested possible interactions of La and RPS5 proteins with SLRef RNA. Studies with recombinant La protein and RPS5 further confirmed their interaction with SLRef RNA. Ex vivo experiments with HCV bicistronic RNA suggested that SLRef RNA specifically inhibits HCV translation. In addition to that SLRef RNA inhibited the HCV RNA synthesis in JFH1 HCV cell culture system. Moreover, specific delivery of pSUPER construct expressing SLRef RNA (pSUPERSLRef) to mice liver along with HCV bicistronic construct using Sendai virosomes demonstrated specific inhibition of HCV IRES activity by SLRef RNA in mice hepotocytes. In summary, short RNA derived from HCV IRES was shown to bind with La protein and RPS5 to inhibit ribosome assembly on HCV IRES. Further, targeted delivery of SLRef RNA into mice liver using Sendai virosome resulted in inhibition of HCV RNA translation in mice hepatocytes. Characterizing the interaction of host proteins with antisense-5’UTR and 3’UTR and its significance in HCV replication Antisense-5’UTR and 3’UTR of HCV RNA are the sites of replication initiation. Hence, host proteins binding to both of these RNA sequences are potential candidates for regulation of HCV replication. In this study, we have investigated host proteins binding with antisense-5’UTR and 3’UTRof HCV RNA by performing UV cross-linking experiments with cytoplasmic extract of Huh7 cells, and found that a protein of ~42kDa protein interacts with both antisense-5’UTR and 3’UTR. Based on earlier report, we predicted that the ~42kDa protein could be hnRNPC1/C2. Results of UV cross-linking followed by immuno pull-down (UV-IP assay) and UV cross-linking experiments with recombinant hnRNPC1 protein confirmed that hnRNPC1 indeed binds to antisense-5’UTR and 3’UTR. Further, filter-binding experiments demonstrated that hnRNPC1 protein binds to 3’UTR with higher affinity compared to antisense-5’UTR. Subsequently, we investigated the regions within 3’UTR and antisense-5’UTR that interact with hnRNPC1protein. Results demonstrated that poly-(U/UC) region of 3’UTR and region containing stem-loops SL-IIIa’, SL-IIIb’, SL-IIIcdef’ and SL-IV’ in antisense-5’UTR were mostly involved in the interaction. Interestingly, studies with confocal microscopy suggested that hnRNPC1/C2 re-localizes from nucleus to cytoplasm upon JFH1 infection, which might in turn influence HCV replication. To investigate the role of hnRNPC1/C2 in HCV replication, partial silencing of hnRNPC1/C2 was performed in HCV cell culture system (JFH1) and results demonstrated that hnRNPC1/C2 is critical for HCV RNA synthesis. However experiments with HCV bicistronic RNA suggested that hnRNPC1/C2 does not play significant role in HCV translation. Taken together, results suggested that hnRNPC1/C2 re-localizes from nucleus to cytoplasm upon JFH1 infection and binds to HCV 3’UTR and antisense- 5’UTR to regulate HCV replication. In summary, this thesis provides novel insights into the interaction of host proteins with HCV RNA and its significance in HCV translation and replication. Inhibition of the ribosome assembly and consequent reduction in HCV translation with mutations interfering with IRES–RPS5 interaction, reported in the present study, unfolds the novel role of this interaction in HCV translation. Further, results obtained in the present study with a small RNA SLRef, derived from HCV IRES, provide proof of concept for using short RNAs to specifically inhibit HCV translation. In addition, studies of interaction of hnRNPC1/C2 with HCV RNA and its re-localization upon HCV infection sheds light on the significance of host–virus interaction in viral RNA replication.