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The roles of Dicer and TRBP in HCV replicationZhang, Chao 24 September 2010
MicroRNAs (miRNAs) are non-coding small RNAs that regulate eukaryotic gene activity at the post-transcriptional level by a process termed miRNA gene suppression. MicroRNA-122 (miR-122) is predominantly expressed in human liver cells and recent studies indicated that miR-122 promotes Hepatitis C Virus (HCV) replication and translation through physical interaction with two tandem binding sites located in the 5 untranslated region (5UTR) of the HCV genome (Jopling, et al., 2006; Jopling, et al., 2008). It has been reported that host genes that are also implicated in the miRNA gene suppression pathway are key regulators of HCV replication (Randall, et al., 2007). Two proteins, Dicer, a key RNaseIII enzyme, and its binding partner TRBP are essential proteins for miRNA activity. They are part of a protein complex called the RNA induced silencing complex (RISC) which also includes Argonaute proteins, and function in miRNA biogenesis loading the miRNA into RISC. As such, they are intriguing targets to study host-viral interplay during HCV replication.<p>
In our study, we designed siRNAs to knock down Dicer and TRBP and then observed the effects of gene knockdown on full length J6/JFH-1-RLuc HCV (genotype 2a chimeric genome) replication and translation. The results showed that knocking down Dicer and TRBP reduced wild type (wt) J6/JFH-1-RLuc replication but had almost no effects on HCV translation in human liver cells. However, since knocking down Dicer and TRBP did not significantly alter miR-122 levels in the cell, it appears that the role of Dicer and TRBP was not solely the biogenesis of miR-122. This was confirmed by an experiment in which we observed that knocking down Dicer and TRBP also attenuated replication of a mutant virus in which replication is dependent on a exogenously supplied miRNA instead of endogenous miR-122.<p>
Taken together, the results supported the hypotheses that Dicer and TRBP facilitate HCV infection mainly through HCV replication but not translation. The effects of Dicer and TRBP on HCV replication are not solely due to miR-122 biogenesis, and may be due to RISC loading functions in steps of miRNA gene suppression.<p>
This study has set some essential groundwork for investigating potential roles of host factors in the RNAi machinery modulating HCV replication/translation and exploring novel antiviral targets.
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The roles of Dicer and TRBP in HCV replicationZhang, Chao 24 September 2010 (has links)
MicroRNAs (miRNAs) are non-coding small RNAs that regulate eukaryotic gene activity at the post-transcriptional level by a process termed miRNA gene suppression. MicroRNA-122 (miR-122) is predominantly expressed in human liver cells and recent studies indicated that miR-122 promotes Hepatitis C Virus (HCV) replication and translation through physical interaction with two tandem binding sites located in the 5 untranslated region (5UTR) of the HCV genome (Jopling, et al., 2006; Jopling, et al., 2008). It has been reported that host genes that are also implicated in the miRNA gene suppression pathway are key regulators of HCV replication (Randall, et al., 2007). Two proteins, Dicer, a key RNaseIII enzyme, and its binding partner TRBP are essential proteins for miRNA activity. They are part of a protein complex called the RNA induced silencing complex (RISC) which also includes Argonaute proteins, and function in miRNA biogenesis loading the miRNA into RISC. As such, they are intriguing targets to study host-viral interplay during HCV replication.<p>
In our study, we designed siRNAs to knock down Dicer and TRBP and then observed the effects of gene knockdown on full length J6/JFH-1-RLuc HCV (genotype 2a chimeric genome) replication and translation. The results showed that knocking down Dicer and TRBP reduced wild type (wt) J6/JFH-1-RLuc replication but had almost no effects on HCV translation in human liver cells. However, since knocking down Dicer and TRBP did not significantly alter miR-122 levels in the cell, it appears that the role of Dicer and TRBP was not solely the biogenesis of miR-122. This was confirmed by an experiment in which we observed that knocking down Dicer and TRBP also attenuated replication of a mutant virus in which replication is dependent on a exogenously supplied miRNA instead of endogenous miR-122.<p>
Taken together, the results supported the hypotheses that Dicer and TRBP facilitate HCV infection mainly through HCV replication but not translation. The effects of Dicer and TRBP on HCV replication are not solely due to miR-122 biogenesis, and may be due to RISC loading functions in steps of miRNA gene suppression.<p>
This study has set some essential groundwork for investigating potential roles of host factors in the RNAi machinery modulating HCV replication/translation and exploring novel antiviral targets.
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Characterization of Host Protein Interactions with HCV RNA : Implications in Viral Translation, Replication and Design of AntiviralsBhat, Prasanna January 2014 (has links) (PDF)
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.
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Mechanistic Insights into Translation and Replication of Hepatitis C Virus RNA : Exploring Direct-Acting AntiviralsKumar, Anuj January 2014 (has links) (PDF)
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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Study of cell host factors involved in Hepatitis C virus tropism / Etude des facteurs cellulaires de l'hôte impliqués dans le tropisme du virus de l'hépatite CDa Costa, Daniel 18 September 2012 (has links)
Le virus de l’hépatite C (HCV) est un problème majeur de santé publique. Le développement de nouveaux traitements pour lutter contre le HCV a été ralenti par l’absence de modèles d’études in vitro et in vivo convenables. Le but de mon travail de thèse a été, dans un premier temps, de caractériser les facteurs déterminant le tropisme hépatique du HCV. En exprimant des facteurs clés dans une lignée cellulaire humaine non-hépatocytaire, nous avons reconstitué in fine l’ensemble du cycle viral dans ces cellules. L’entrée du virus dans la cellule hôte fait intervenir différents récepteurs d’entrée dont CD81, occludin (OCLN), claudin-1 (CLDN1) et scavenger receptor class B type I (SR-BI). L’expression de ces quatre récepteurs sur cette lignée la rend hautement permissive à l’entrée du virus, mais ne permet pas de rétablir la réplication du virus. L’expression du micro-ARN 122, un micro-RNA important pour l’infection du HCV, dans les cellules exprimant les quatre récepteurs, restaure une forte réplication de l’ARN viral mais ne permet pas de détecter une production de particules infectieuses. L’expression de l’apolipoprotein E (apoE), jouant un rôle primordial dans l’assemblage et la sécrétion, rétablis cette dernière étape du cycle viral du HCV dans la lignée cellulaire humaine non-hépatocytaire. Dans un second temps, j’ai utilisé la stratégie, précédemment établie, pour étudier la spécificité d’espèce de l’infection du HCV dans plusieurs lignées hépatocytaires murines. Nous avons pu rendre ces cellules permissives à l’entrée du HCV et pu détecter une très faible réplication. L’ensemble de mes travaux apportent de nouvelles informations sur la compréhension des facteurs clés nécessaire au cycle viral du HCV dans des cellules murines et humaines. / Hepatitis C virus (HCV) is a global health burden. The development of new therapeutics to treat HCV infection has been hampered by the lack of convenient in vitro and in vivo model systems. The goal of my PhD work was, in a first time, to characterize the factors determining the hepatotropism of HCV. By expressing key factors within a non-hepatic cell line, we reconstituted in fine the full HCV life cycle in those cells. Virus entry into the host cell requires different entry factors which are CD81, occludin (OCLN), claudin-1 (CLDN1) and the scavenger receptor class B type I (SR-BI). The expression of these four factors in this cell line renders it highly permissive to viral entry, but does not allow restoring replication of the virus. The expression of miR-122, a micro-RNA important for HCV infection, into the cell lines expressing the four HCV entry factors restore a strong replication of the HCV RNA but does not allow detecting infectious viral particle production. Further expression of the apolipoprotein E (apoE), which plays a critical role in the assembly and release process, restore the last step of the HCV life cycle in a non-hepatic cell line. In a second part of my PhD, I have used the previously developed strategy to study the species specificity of HCV infection using different mouse hepatoma cell lines. We have been able to render these cell lines permissive to HCV entry and have been able to detect a slight replication. Altogether, my results bring new information on the understanding of key factors important for HCV life cycle in mouse and human cells.
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Host-Pathogen Interactions in Hepatitis C Virus Infection : Deciphering the Role of Host Proteins and MicroRNAsShwetha, S January 2015 (has links) (PDF)
Host-pathogen interactions in Hepatitis C Virus infection: Deciphering the role of host proteins and microRNAs
Hepatitis C virus (HCV) is a positive sense single stranded RNA virus belonging to the Hepacivirus genus of the Flaviviridae family. HCV genome consists of a single open reading frame flanked by highly structured 5‟ and 3‟ untranslated regions (UTRs) at both ends. Unlike cellular mRNAs, HCV RNA translation is independent of the cap structure and is mediated by an internal ribosomal entry site (IRES) present in the 5‟UTR. HCV replication begins with the synthesis of a complementary negative-strand RNA using the positive strand RNA genome as a template catalyzed by the NS5B RNA dependent RNA polymerase (RdRp). The de novo priming of HCV RNA synthesis by NS5B occurs at the very end of the 3‟UTR. The 3‟UTR is organized into highly structured regions namely the variable region, poly U/UC region and the 3‟X region. These regions contain cis-acting elements that determine the efficiency of viral replication. In addition, the interaction of trans-acting factors with the 3‟ UTR is also important for regulation of HCV replication. HCV 3‟UTR interacts with several cellular proteins such as the human La protein, polypyrimdine tract binding protein (PTB), poly (rC)-binding protein 2 (PCBP2) and Human antigen R (HuR). However, the molecular basis of regulation of viral replication by these proteins is not well understood. Many proteins that are hijacked by HCV as well as other cytoplasmic RNA viruses, such as La, PCBP2, HuR and PTB are RNA binding proteins (RBPs). They are involved in post transcriptional regulation of cellular gene expression. Thus the subversion of these proteins by the virus can affect their normal physiological functions. In addition to proteins, recent reports also describe the involvement of non-coding RNAs including microRNAs (miRNA) and long non coding RNAs (lncRNA) in HCV infection. miRNAs can either directly bind to the HCV genome and regulate its life cycle or indirectly modulate the expression of host proteins required by the virus. miRNAs that are differentially regulated in virus infected tissues or body fluids of infected patients can also serve as biomarkers for diagnosis of various stages of the disease. Hence, it was planned to study the role of host proteins and miRNAs in the HCV life cycle and pathogenesis to have novel insights into the biology of HCV infection. Riboproteomic studies have identified several host proteins that directly interact with the 5‟ and/or 3‟UTRs of the HCV RNA. One of the RNA binding proteins that predominantly interact with the 3‟UTR of HCV RNA was found to be HuR. In the present study, we have extensively characterized the interaction between HuR and HCV 3‟UTR and studied its functional implications in HCV life cycle along with other host factors.
Characterizing the HCV 3’UTR–HuR interaction and its role in HCV replication
HuR is a ubiquitously expressed member of the Hu family which shuttles between the nucleus and cytoplasm in response to stress. Whole genome siRNA knockdown and other studies have suggested that HuR is essential for HCV replication. However, the molecular mechanism of its involvement in this process was not clear. We observed that siRNA mediated knockdown of HuR reduces the HCV RNA and protein levels. Immunofluorescence studies indicated that HuR relocalizes from the nucleus to the cytoplasm in HCV infected cells. Through confocal microscopy and GST pulldown assays, we have demonstrated that HuR co localizes with the viral polymerase, NS5B and directly interacts with the NS5B protein. Membrane flotation assays showed that HuR is present in the detergent resistant membrane fractions which are the active sites of HCV replication. In addition to the interaction of HuR with the viral protein NS5B, we also characterized its interaction with the viral RNA. Direct UV cross linking assays and UV cross linking immunoprecipitation assays were performed to demonstrate the interaction of HuR with the HCV 3‟UTR. The RRM3, hinge region and RRM1 of HuR were found to be important for binding. Further, we observed that HuR competes with PTB for binding to the 3‟UTR when cytoplasmic S10 extracts or recombinant proteins were used in UV cross linking assays. In contrast, the addition of HuR facilitated the binding of La protein to the HCV 3‟UTR in the above assays. Competition UV cross linking assays indicated that both HuR and PTB bind to the poly U/UC region of the 3‟UTR while La binds to the variable region. HuR and La showed higher affinities for binding to the 3‟UTR as compared to PTB in filter binding assays. Since HuR and PTB interact with the same region on the 3‟UTR and HuR showed ~4 fold higher affinity for binding, it could displace PTB from the 3‟UTR. Next, we investigated the roles of HuR, PTB and La in HCV translation and replication in cell culture using three different assay systems, HCV sub genomic replicon, HCV bicistronic SGR-JFH1/Luc replicon as well as the infectious HCV full length RNA (JFH1). Results clearly indicated that HuR and La are positive modulators of HCV replication. Interestingly, PTB facilitated HCV IRES mediated translation but appeared to have a negative effect on HCV replication. The positive effectors, HuR and La showed significant co localization with one another in the cytoplasm in immunofluorescence studies. GST pulldown and coimmunoprecipitation experiments indicated protein-protein interactions between HuR and La but not between HuR and PTB. Through quantitative IP-RT assays, we demonstrated that the overexpression of HuR in HCV RNA transfected cells increases the association of La with the HCV RNA while HuR knockdown reduces the association of La with the HCV RNA. Previous studies in our laboratory have shown that La helps in HCV genome circularization. The addition of HuR significantly increased La mediated interactions between the 5‟UTR and the 3‟UTR of HCV RNA as monitored by 5‟-3‟ co precipitation assays, suggesting a possible mechanism by which cooperative binding of HuR and La could positively regulate HCV replication. Taken together, our results suggest a possible interplay between HuR, PTB and La in the regulation of HCV replication.
Studying the role of HuR- associated cellular RNAs in HCV infection
HuR belongs to the category of mRNA turnover and translation regulatory proteins (TTR-RBPs), which are capable of triggering rapid and robust changes in cellular gene expression. HuR plays a role in several post transcriptional events such as mRNA splicing, export, stability and translation. In the present study, we have investigated the possible consequences of relocalization of HuR on cellular processes in the context of HCV infection. We observed that 72h post transfection of infectious HCV-JFH1 RNA, there is an increase in the mRNA levels of some of the validated targets of HuR including the vascular endothelial growth factor A (VEGFA), dual specificity phosphatise 1 (MKP1) and metastasis - associated lung adenocarcinoma transcript (MALAT1). IP-RT assays demonstrated that the association of HuR with VEGFA and MKP1 was higher in HCV-JFH1 RNA transfected cells as compared to the mock transfected cells indicating that increase in HuR association could probably help in stabilization of these mRNAs. Interestingly, we observed that the association of HuR with the lncRNA MALAT1 decreases in the presence of HCV RNA, while its RNA levels increased. Earlier it has been reported that MALAT1 interacts with HuR and was predicted to interact with La. We confirmed the interaction of both HuR and La proteins with MALAT1 RNA in vitro and in the cell culture system. Results from our time course experiments suggest that relocalization of HuR and La upon HCV infection might decrease their association with the nuclear retained MALAT1 RNA leading to significant reduction in MALAT1 RNA levels at the initial time points. However at later time points, MALAT1 was found to be unregulated through activation of the Wnt/beta-catenin pathway as demonstrated using a chemical inhibitor against β-catenin. Since MALAT1 is a known regulator of epithelial mesenchymal transition (EMT) and metastasis, we further studied the physiological consequence of the observed increase in MALAT1 levels upon HCV infection. Cell migration and cell invasion studies suggested that the knockdown of MALAT1 led to the inhibition of HCV- triggered wound healing and matrigel invasion and also rescued the down regulation of E-Cadherin protein levels, an EMT marker. Our study highlights the importance of the lncRNA, MALAT1 in HCV infection and suggests its possible involvement in HCV induced HCC.
Investigating the role of miRNAs in HCV pathogenesis and replication miRNAs can also regulate HCV infection and pathogenesis in multiple ways. It is known that under disease conditions, there is aberrant expression of intracellular as well as circulating miRNAs. We have investigated the expression profile of 940 human miRNAs in HCV infected patient serum samples to identify the differentially regulated miRNAs. miR-320c, miR-483-5p and the previously reported miR-125b were found to be upregulated in the serum of cirrhotic and non-cirrhotic HCV infected patient serum samples. All three miRNAs were also unregulated in the cell culture supernatant of HCV infected cells as well as within the HCV infected cells. miR-483-5p was specifically enriched in the exosomes isolated from patient serum samples. Knockdown of miR-320c and miR-483-5p did not have significant effect on HCV replication while knockdown of miR-125b affected HCV replication through regulation of one of its target genes, HuR. We observed that with time, miR-125b levels in HCV-JFH1 RNA transfected cells increase while the HuR protein levels decrease. Using luciferase reporter constructs, we demonstrated that the decrease in HuR protein levels is indeed mediated by miR-125b. Mutations in the target site of miR-125b in the HuR 3‟UTR prevented the down regulation of luciferase activity. Next we tested the effect of silencing miR-125b on HCV replication. Knockdown of miR-125b prevented the reduction in HuR protein levels but with no significant effect on HCV replication. It appeared that the HuR protein already present in the cytoplasm could be sufficient to support HCV replication. Hence similar experiments were carried out in cells depleted of HuR using either siRNA against HuR or a chemical inhibitor of nucleocytoplasmic transport of HuR, Leptomycin B. We observed that when the intracellular levels of HuR are reduced using either of the two approaches, there is a decrease in HCV replication. This is in accordance with the results obtained in the first part of the thesis. However when miR-125b was silenced in HuR depleted cells, we noticed an upregulation in the HuR protein levels by western blot analysis and a consequent increase in HCV RNA levels as quantified by qRT-PCR. From our findings, we
can conclude that miR-125b mediated regulation of HuR plays an important role in HCV replication. We hypothesize that this could be a cellular response to HCV infection to which the virus responds by inducing protein relocalization.
Altogether, these studies outline the importance of host factors including cellular proteins and non-coding RNAs in the regulation of HCV life cycle and pathogenesis. Results reveal the mechanistic insights into how HCV infection triggers host defense pathways, which are evaded by the virus by counter strategies.
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