Host-Pathogen Interactions in Hepatitis C Virus Infection : Deciphering the Role of Host Proteins and MicroRNAs
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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