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Hepatitis C virus non-structural proteins modulate cellular kinases for increased cytoplasmic abundance of host factor HuR and facilitate viral replication [1]

['Harsha Raheja', 'Department Of Microbiology', 'Cell Biology', 'Indian Institute Of Science', 'Bangalore', 'Biju George', 'Sachin Kumar Tripathi', 'Sandhini Saha', 'Regional Centre For Biotechnology', 'Faridabad']

Date: 2023-08

Host protein HuR translocation from nucleus to cytoplasm following infection is crucial for the life cycle of several RNA viruses including hepatitis C virus (HCV), a major causative agent of hepatocellular carcinoma. HuR assists the assembly of replication-complex on the viral-3′UTR, and its depletion hampers viral replication. Although cytoplasmic HuR is crucial for HCV replication, little is known about how the virus orchestrates the mobilization of HuR into the cytoplasm from the nucleus. We show that two viral proteins, NS3 and NS5A, act co-ordinately to alter the equilibrium of the nucleo-cytoplasmic movement of HuR. NS3 activates protein kinase C (PKC)-δ, which in-turn phosphorylates HuR on S318 residue, triggering its export to the cytoplasm. NS5A inactivates AMP-activated kinase (AMPK) resulting in diminished nuclear import of HuR through blockade of AMPK-mediated phosphorylation and acetylation of importin-α1. Cytoplasmic retention or entry of HuR can be reversed by an AMPK activator or a PKC-δ inhibitor. Our findings suggest that efforts should be made to develop inhibitors of PKC-δ and activators of AMPK, either separately or in combination, to inhibit HCV infection.

Hepatitis C virus is a major human pathogen, which exploits cellular machinery for its propagation in liver cells. The cytoplasmic availability of cellular components is crucial for their direct influence on processes involving the viral RNA, which lacks any nuclear history. Our results establish the involvement of viral proteins, NS3 and NS5A in achieving increased cytoplasmic abundance of a host factor HuR, an RNA binding protein (RBP) critical for HCV replication. This is achieved via direct post-translational modification of HuR and indirect regulation of its nuclear carrier by coercing two host kinases, PKC-δ and AMPK-α. RBPs are emerging as novel targetable candidates for gene regulation. Similar studies with other RBPs and targeting protein modifications, in place of whole protein knockdown, could usher in a revolutionary strategy to neutralize emerging RNA virus-based diseases, while preserving their cellular functions.

Funding: SD acknowledges the J.C. Bose Fellowship from the Department of Science and Technology (DST), Govt. of India, for research support. This study was also supported by the DBT-IISc partnership program (to SD), the Research support from Indian Institute of Science (to SD), DST Fund for Improvement of Science and Technology Infrastructure (DST- FIST) level II infrastructure (to SD), and the University Grants Commission Centre of Advanced Studies. We also acknowledge the funding through Indo-Swiss Joint Research Program (ISJRP) from Department of Biotechnology. HR is supported by the research fellowship from the Council of Scientific and Industrial Research (CSIR-SPM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Cells adopt multiple strategies to regulate the subcellular localization of HuR and, consequently, the stability of its cognate transcripts under different conditions. Here, we investigated the viral mechanism driving the nuclear export of HuR, the cytoplasmic partitioning of HuR, and the effect of its altered localization. We report that PKC-δ and AMPK work in a coordinated manner to achieve cytoplasmic localization of HuR via direct modification and indirect regulation of its nuclear carrier. Thus, HCV modulates two separate kinases to promote replication and pathogenesis. Relocalization of HuR and its interaction with cytoplasmic RNAs could reveal the molecular basis of HCV-induced hepatocellular carcinoma and provide an attractive target for preventing its progression.

HuR contains three RNA recognition motifs (RRMs) and a hinge region, also known as the HuR nuclear shuttling domain (HNS domain) [ 14 – 16 ]. Phosphorylation of HuR residues regulates its structure, RNA-binding affinity, and subcellular localization [ 17 ]. HuR is phosphorylated in a cell-cycle dependent manner by CDKs. CDK1 phosphorylates HuR on S202, which is required for its nuclear retention during the S and G2 phases of the cell cycle [ 18 ]. Phosphorylation on the same site by CDK5 has been shown to reduce its binding to cyclin A mRNA, leading to cell cycle arrest in glioma cells [ 19 ]. Checkpoint kinase 2 (CHK2) phosphorylates HuR on S88, S100, and T118, which modulates HuR binding to RNA by decreasing its affinity for certain mRNAs (e.g., SIRT1) and increasing it for others (e.g., Myc and occludin) [ 20 ]. Phosphorylation on S242 augments the amount of HuR in the cytoplasm, regardless of stress conditions such as exposure to short-wavelength ultraviolet light [ 21 ]. The MAP kinase p38, which is activated during inflammatory response, phosphorylates HuR on T118, increasing its binding to and the stabilization of inflammatory mRNAs, such as p21, COX2, and cPLA 2 α, in addition to promoting HuR accumulation in the cytoplasm [ 22 , 23 ]. Several members of the protein kinase C (PKC) family are also involved in HuR phosphorylation. PKC-α phosphorylates HuR on S158 and S221, which leads to its cytoplasmic relocalization and enhanced binding to targets, such as COX2 and PTGS2 [ 24 ]. PKC-δ-mediated phosphorylation on S318 and S221 has been shown to induce cytoplasmic translocation and increased binding to COX2, CCNDA2, and CCND1 mRNAs [ 25 , 26 ]. Abrogation of phosphorylation on S318 hinders the migration and invasion of colon cancer cells [ 27 ]. Other kinases, such as IKKα, ERK8, and JAK3, also phosphorylate HuR to regulate its localization and function [ 28 – 30 ]. Activated JAK3 phosphorylates HuR on Y200, which prevents its inclusion in stress granules formed upon arsenic treatment [ 28 ]. Phosphorylation of Y5, Y95, Y105, and Y200 residues by SRC and ABL1 kinases is important for centrosomal accumulation of HuR and, therefore, genomic stability [ 31 ]. In addition to phosphorylation, HuR is methylated on R217 by CARM1, which correlates with its cytoplasmic localization in non-small cell lung carcinoma [ 32 ]. Ubiquitination on K313 and K326 leads to the dissociation of HuR from p21, MKP1, and SIRT1 mRNA [ 33 ]; whereas neddylation on K283, K313, and K326 promotes nuclear localization and protein stability [ 34 ].

Multiple host factors are required at various stages of the viral life cycle. Upon viral infection, proteins, such as some IRES trans-acting factors and Nups relocate from the nucleus to the cytoplasm to regulate the viral life cycle [ 6 – 8 ]. Our laboratory has previously shown that human antigen R (HuR), also known as ELAVL1, relocates from nucleus to the cytoplasm upon HCV infection. HuR binds to the viral 3’-UTR, where it displaces the replication inhibitor protein PTB and assists in the binding of the La protein. La further recruits the entire replication complex to initiate viral replication [ 9 ]. HuR is an RNA-binding protein (RBP), that shuttles between nucleus and cytoplasm. Its endogenous function is to regulate the transport, stability, and translation of cellular RNAs [ 10 – 12 ]. Infection-induced relocalization of HuR to the cytoplasm is required for interaction with viral RNA, which guides viral replication. In a genome wide CRISPR screen, HuR was identified as a major host factor required for HCV replication and the specific knockout was found to abolish the HCV RNA replication in cells [ 13 ].

Hepatitis C virus (HCV) is an RNA virus, whose 9.6-kb genome codes for three structural and seven non-structural proteins [ 1 ]. Structural proteins form the viral capsid and envelope, whereas non-structural proteins are involved in the regulation of various viral life processes, including double-membrane vesicle formation, replication, translation, packaging, and release [ 2 – 5 ]. The viral open reading frame is flanked by two regulatory elements, the 5’-untranslated region (UTR) and 3’-UTR, which are involved in initiating viral translation and replication, respectively [ 5 ].

Results

Phosphorylation of HuR regulates its cellular localization upon HCV infection It has been shown that HuR relocalises from nucleus to cytoplasm after 48h of HCV infection and is important for HCV replication [9,13]. These studies have been done in Huh7.5 and Huh7.5.1 cells. Therefore, we first studied the time kinetics of this relocalisation using nuclear-cytoplasmic fraction of mock and HCV-JFH1 virus infected Huh7.5 cells at 12h and 24h post infection (S1A and S1B Fig). We calculated the ratio of nuclear to cytoplasmic HuR intensities and found that the increase in cytoplasmic HuR begins from 12h post infection and increases till 24h. These time points correlate with the replication phase of the virus wherein translation to replication switch occurs 12-18h post infection and highlight the importance of HuR relocalisation for HCV replication. Viruses are known to modify host proteins [37]. To study the post-translational modifications of host proteins after HCV infection, total protein extracts prepared from mock (control) and HCV-RNA transfected Huh7.5 cells were analyzed by two-dimensional gel electrophoresis and HuR-specific spots were detected using anti-HuR antibody. In control cells, two spots were observed for HuR, one at the acidic end towards pH 3 and the other at the basic end towards pH 10. The spots had the same molecular weight, suggesting the presence of two sub-populations of HuR with separate charged modifications that did not alter overall protein weight. An additional spot, slightly more acidic than the one at pH 10 (marked with an asterisk in Fig 1A), was observed upon HCV RNA transfection (Fig 1A and 1B), indicating a negatively charged modification on HuR. PPT PowerPoint slide

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TIFF original image Download: Fig 1. HuR gets phosphorylated upon HCV infection. (A) Huh7.5 cells were transfected with HCV-JFH1 RNA. Mock and transfected cells were harvested after 48h and cell lysates were precipitated using TCA precipitation. The precipitated proteins were run in 2 dimensions as described in the methods, followed by Western blotting of the gel. Western blotting was done with anti-HuR and anti-Actin antibodies. Appropriate HRP-conjugate secondary antibodies were used. (B) HCV infection was detected by western blotting using Anti-HCV core antibody for same lysates run on 12% SDS-PAGE. (C) Schematic for experimental protocol followed. (D) Huh 7.5 cells were transfected with Myc-tagged overexpression construct of WT HuR, S318A HuR mutant, S242A HuR mutant, Y200F HuR mutant, T118A HuR mutant and S221A HuR mutant and mock transfected. Cells were processed for immunofluorescence staining using Alexa Fluor conjugated secondary antibody against Myc (Green) and viral protein Core (Red). The nucleus was stained with DAPI (Blue). Scale bar represents 10 μm. (E) Nuclear and cytoplasmic ratio for overexpressed HuR (Myc) was quantified for images in (D) using Zen 2.3 lite software. (F) Images of HCV transfected cells following the schematic in (C). (G) Nuclear and cytoplasmic ratio for overexpressed HuR (Myc) was quantified for images in (F) using Zen 2.3 lite software. n = 10. Student t-test was performed for statistical analysis. * = p<0.05, ** = p<0.01, *** = p<0.001. https://doi.org/10.1371/journal.ppat.1011552.g001 Phosphorylation is a post-translational modification that imparts a negative charge on proteins. Therefore, we examined the involvement of phosphorylation on HuR following HCV infection. Some phosphorylation sites known to impact HuR subcellular localization include T118, Y200, S221, S242, and S318. The phospho-dead mutants of these sites were generated using site-directed mutagenesis, wherein the serine or threonine residues were replaced by alanine (T118A, S221A, S242A, S318A), and tyrosine was replaced by phenylalanine (Y200F). The mutants were verified by sequencing and employed in the relocalization assay. Endogenous HuR translocates from the nucleus to the cytoplasm 48h after HCV-RNA transfection [9]. If phosphorylation of a particular residue is crucial for guiding this translocation, the corresponding phospho-dead mutant would prevent the cytoplasmic relocalization of the mutant protein upon viral infection. Wild-type (WT) HuR overexpression was used as a positive control for HuR localization. Using immunostaining, the subcellular localization of WT and mutant HuR proteins was detected in uninfected control and HCV-infected cells. Myc-tagged WT and mutant HuR was overexpressed in Huh7.5 cells, followed by HCV-RNA transfection (Fig 1C). An anti-Myc-tag antibody was used to visualize the overexpressed protein, the HCV Core protein was used to identify HCV-infected cells, and DAPI was used to mark the nuclei. In untransfected control cells, WT and all mutant HuR proteins localized to the nucleus (Fig 1D and 1E). HCV-RNA transfection led to the relocalization of WT HuR to the cytoplasm. The same was observed for HuR mutants S221A and S242A. In contrast, mutants S318A, Y200F, and T118A remained localized to the nucleus even after 48h of HCV-RNA transfection (Fig 1F and 1G). This finding suggests that phosphorylation of S318, Y200, and T118 might be involved in relocalization of HuR to the cytoplasm upon HCV infection. The relocalisation of WT and S318A HuR was also analysed by nuclear- cytoplasmic fractionation, wherein WT/S318A HuR expressing Huh7.5 cells were either Mock transfected or transfected with HCV-RNA and localisation of HuR examined by western blotting using anti-HuR antibody (S1C and S1D Fig). We observed that the ratio of Nuclear to cytoplasmic intensity of overexpressed WT HuR decreased, but that of S318A HuR slightly increased upon HCV-RNA transfection, suggesting and corroborating the imaging results of nuclear retention of S318A HuR upon HCV RNA transfection. The effect on relocalisation of WT and mutant HuR was marginal because the percentage of cells co-transfected with the HuR overexpression construct and HCV-RNA is very low and therefore, immunofluorescence staining serves as a better technique for monitoring the relocalisation. Also, in the HCV infected cells, there could be other signalling in addition to 318 site phosphorylation which might influence HuR relocalisation.

Phosphorylation of S318 influences HCV replication The location of the S318 site on HuR was visualized using Pymol software. The X-ray structure of HuR RRM3 (PDB ID: 6GD2) complexed with RNA indicated that the S318 site was on the surface of the protein and interacted directly with the bound RNA (Fig 3A). Accordingly, phosphorylation of S318 might affect the RNA binding of HuR. To study the impact of phosphorylation on the affinity for HCV-RNA, we used surface plasmon resonance. Biotinylated HCV 3’-UTR RNA was immobilized on a streptavidin-coated SPR chip (SA Chip) and recombinant purified WT, S318A, and S318D HuR proteins were passed over as analytes to calculate the binding affinity (Fig 3B–3D). The Kd values for WT and S318A HuR were 25.7 nM and 46.2 nM, respectively; whereas the phospho-mimic mutant S318D showed approximately five-fold better affinity (Kd 6.36 nM) (Fig 3E). This result indicates that, along with relocalization to the cytoplasm, S318 phosphorylation of HuR also augments the binding affinity for HCV 3’-UTR. To assess the impact of S318 phosphorylation on HCV replication, WT, S318A, and S318D constructs were overexpressed in Huh7.5 cells, followed by HCV-RNA transfection (Fig 3F). The effect on replication was checked by analyzing the levels of HCV negative-strand RNA compared to the vector-overexpression control. Viral replication was increased upon overexpression of WT HuR but not S318A HuR, which did not relocalize to the cytoplasm and was rescued by overexpression of S318D HuR (Fig 3G and 3H). We analysed the S318D distribution in untreated cells and observed increased but not complete cytoplasmic localization as compared to WT HuR (S2 Fig). We further aimed to overexpress the WT and mutant HuR in the background of endogenous HuR silencing. For this, we generated a mutation in the overexpression constructs at the site of siHuR seed sequence binding (S3A Fig). The siRNA resistant overexpression constructs were transfected in the background of siHuR transfection. This was followed by HCV-JFH1 RNA transfection. 48h post transfection, the levels of HCV negative strand RNA were determined to assess HCV replication. The treatment of cells with siHuR reduced the HCV replication (S3B Fig). In this background, the overexpression of WT HuR increased the viral replication, S318A overexpression was unable to induce this increase while S318D overexpression exhibited a rescue in the RNA levels (S3C and S3D Fig). These findings confirm the importance of S318 phosphorylation in HuR localization and hence, HCV replication. PPT PowerPoint slide

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TIFF original image Download: Fig 3. S318 site phosphorylation influences HCV-RNA replication. (A) S318 site in HuR RRM3 in association with AU-rich RNA. The red balls indicate S318 site, directly involved in interaction with associated RNA. (B-D) Sensorgrams for interaction of (B) WT, (C) S318D HuR and (D) S318A HuR with HCV 3’UTR at indicated concentrations. The y-axis represents the change in Response Units in Association and dissociation phases and x-axis represents time. (E) Table for the average Kd value obtained for HuR binding to HCV 3’UTR. Kd values were calculated as Kd = koff/kon where kon represent rate constant of association phase of 100s and koff represents rate constant of dissociation phase of 200s using all the 4 concentrations graphs in each protein (B-D) and the average was calculated. (F) Schematic for the workflow. (G) Following the workflow in (F), total cellular RNA was isolated, and HCV negative strand RNA detected by real time PCR (n = 3). (H) Western blotting for HuR was performed following the workflow in (F). o/e denotes the overexpression of described WT/mutant HuR. Student t-test was performed for statistical analysis. * = p<0.05, ** = p<0.01, *** = p<0.001. https://doi.org/10.1371/journal.ppat.1011552.g003

PKC-δ activity is required for HCV-mediated cytoplasmic export of HuR Once the involvement of S318 phosphorylation in guiding HuR localization was ascertained, we determined the role of the kinase responsible for this modification. An earlier study suggested that PKC-δ participated in the phosphorylation of S318 on HuR and its nucleocytoplasmic shuttling [17,26]. Therefore, the involvement of PKC-δ in regulating HuR localization upon HCV infection was investigated. The levels of total and phosphorylated PKC-δ at different time points after HCV RNA transfection were analyzed. An increase in p-PKC-δ/total PKC-δ after 48h of HCV-RNA transfection was observed, suggesting the activation of PKC-δ upon HCV RNA transfection (Fig 4A). This effect was not observed upon the transfection of pSGR-JFH1/Luc-GND RNA which is a replication defective sub-genomic HCV-JFH1 RNA (S4A Fig), suggesting the effect of replicative viral RNA in inducing PKC-δ phosphorylation. We also observed an increase in cleavage of PKC-δ upon HCV RNA transfection (S4B Fig). This cleavage product retains the catalytic subunit of PKC-δ, while freeing it from the regulatory subunit and is known to be transported to the nucleus [40]. This cleaved subunit might initiate the cascade to HuR phosphorylation for its cytoplasmic export. To visualize the interaction between HuR and PKC-δ in cells, immunoprecipitation of HuR was performed in mock (control) and HCV-virus infected cells, and the presence of PKC-δ in the pull-down fraction confirmed their physical interaction (Fig 4B). The increase in HuR associated with PKC-δ indicated a stronger interaction in cells upon HCV infection. The association was further strengthened by reverse pull-down, wherein immunoprecipitation of PKC-δ was performed and increased HuR association was observed upon HCV infection (Fig 4C). To correlate this finding with HuR localization, an inhibitor of PKC-δ (rottlerin) was used. The effect of PKC-δ inhibition on HCV-induced HuR relocalization was assessed. Huh7.5 cells were transfected with HCV-JFH1 RNA and 3 μM rottlerin was added to the medium after 6h of transfection (Fig 4D). The cells were incubated with the inhibitor for 48h, after which HuR localization was visualized by confocal microscopy. In the absence of the inhibitor, HuR relocated from the nucleus to the cytoplasm upon HCV RNA transfection (Fig 4E and 4F). The presence of the inhibitor did not alter the localization of HuR in control cells, but it prevented its relocalization to the cytoplasm upon HCV RNA transfection. These results confirmed the involvement of PKC-δ in altering the localization of HuR upon HCV infection. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Involvement of PKC-δ in HuR localisation. (A) Huh7.5 cells were transfected with HCV-JFH1 RNA and cells harvested at indicated time-points. Western blotting was done with anti-PKCδ, anti-p-PKCδ, anti-Core and anti-actin antibodies. Appropriate HRP-conjugate secondary antibodies were used. (B) Immuno-pulldown of HuR was performed from Huh7.5 cells, post 48h of HCV-JFH1 virus infection and associated PKC-δ was checked by western blotting with specific antibody. The values at bottom represent ratio of densitometry values of PKC-δ in the IP fraction to that of HuR in IP fraction. (C) Immuno-pulldown of PKC-δ was performed from Huh7.5 cells, post 48h of HCV-JFH1 virus infection and associated HuR was checked by western blot using HuR-specific antibody. The values at bottom represent ratio of densitometry values of HuR in the IP fraction to that of PKC-δ in IP fraction. (D, E) Huh7.5 cells were treated with infectious HCV-JFH1 virus in the presence of 3 μM of Rottlerin and immunofluorescence staining was carried out at 24h post infection using Alexa Fluor conjugated secondary antibodies against HCV-NS3 (Red) or HuR (Green). The nucleus was counterstained with DAPI. Scale bar represents 10 μm. (F) Nuclear and cytoplasmic ratio of HuR (green) was quantified for images in (F) using Zen 2.3 lite software. n = 30. Student t-test was performed for statistical analysis. * = p<0.05, ** = p<0.01, *** = p<0.001. https://doi.org/10.1371/journal.ppat.1011552.g004

PKC-δ regulates the HCV life cycle through HuR Given that HuR is involved in viral RNA replication, we investigated the effect of rottlerin on HCV-RNA levels. Huh7.5 cells were transfected with HCV-JFH1 RNA and 3 μM rottlerin was added to the medium for 24h (Fig 5A). After 24h, the abundance of HCV-negative strand RNA in the cells was quantified by real-time PCR, which showed that addition of rottlerin decreased HCV-RNA levels by > 90% (Fig 5B). Similarly, confocal microscopy indicated that the percentage of HCV positive cells dropped from ~20% in untreated cells to <2% in rottlerin-treated cells (Fig 5C and 5D). In all assays, the PKA inhibitor KT5720 was used as a negative control, which did not show any effect on HuR localization upon HCV infection. However, we observed an increase in replication upon PKA inhibitor treatment which could be because of the regulation of localisation of another negative regulator of HCV replication, PTB [41]. The effect on viral replication was also assayed using siRNA mediated knockdown of PKC-δ. Huh7.5 cells were transfected with HCV-JFH1 RNA after 24h of siRNA transfection. 48h post HCV RNA transfection, HCV-negative strand RNA was quantified. We observed a dose-dependent decrease in HCV-negative strand RNA with increasing concentration of siRNA targeting PKC-δ (Fig 5E and 5F). PPT PowerPoint slide

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TIFF original image Download: Fig 5. Role of PKC-δ. In HCV life cycle. (A) Workflow of the experiment. (B) According to the workflow, cells were harvested after 24h of addition of inhibitors. Total RNA was isolated from the cells and HCV negative strand RNA was detected using RT-qPCR. Fold change in RNA levels was calculated as compared to RNA levels in cells with no inhibitor treatment. (n = 3). (C) Huh7.5 cells were transfected with HCV-JFH1 RNA in the presence of 3 μM of Rottlerin or 10 μM KT5720 and immunofluorescence staining was carried out at 24h post infection using Alexa Fluor conjugated secondary antibodies against HCV-NS3 (Red). The nucleus was counterstained with DAPI. Scale bar represents 100 μm. (D) Quantification of percentage of HCV positive cells in the confocal images in panel C. (n = 3). (E) Huh7.5 cells were transfected with either non-specific siRNA (Nsp si) or indicated concentrations of siRNA targeting PKC-δ (si PKC-δ). 16h post transfection, cells were transfected with HCV-JFH1 RNA, and 48h post JFH1-RNA transfection cells were harvested, and HCV negative strand RNA levels determined by qRT-PCR using HCV specific primers. (F) The extent of PKC-δ silencing in (E) was determined by western blotting by using anti-PKC-δ antibody. HCV-Core protein served as the marker for HCV-RNA transfection. β-actin was used as loading control. (G) Expression level of PKC-δ (PRKCD gene) in healthy tissue and Virus induced primary HCC tissue samples from TCGA database. (H) Kaplan Meier plot for survival probability for Virus induced primary HCC patients with varying level of expression of PKC-δ. Data taken from TCGA database. Student t-test was performed for statistical analysis. * = p<0.05, ** = p<0.01, *** = p<0.001. https://doi.org/10.1371/journal.ppat.1011552.g005 The expression of PKC-δ in healthy and hepatocellular carcinoma patients (Virus induced HCC patient positive for either HCV, HBV or both) in The Cancer Genome Atlas (TCGA) database was analyzed. Increased PKC-δ expression in virus-induced hepatocellular carcinoma patients suggested its involvement in disease progression (Fig 5G), and survival probability was inversely proportional to PKC-δ expression levels (Fig 5H). This result suggested that patients with elevated PKC-δ would have more and early relocalisation of HuR upon HCV infection, which would lead to enhanced viral replication and, hence, increased likelihood of severe disease progression.

HCV non-structural proteins increase the cytoplasmic abundance of HuR Activation of PKC-δ and relocalization of HuR to cytoplasm seemed to be a viral strategy for its efficient replication. Therefore, candidate viral protein(s) involved in PKC-δ activation were investigated. Over-expression of either the structural protein, Core or all the non-structural proteins together through pSGR-Luc construct was performed in Huh7.5 cells and localization of HuR was assessed after 48h of overexpression. Viral non-structural proteins were found to be sufficient to cause the relocalization of HuR (Fig 6A and 6B). Among non-structural proteins, NS3 is a major pathogenic protein that interacts with multiple host proteins and kinases [42–45]. To assess the physical interaction between NS3 and PKC-δ, interaction studies for NS3 and PKC-δ were performed in Huh7.5 cells. Myc-tagged NS3 overexpression construct was transfected in Huh7.5 cells and 48h post transfection, PKC-δ was pulled down from the cell lysate to assess co-immunoprecipitation of myc-tagged NS3. PKC-δ pull-down could immunoprecipitate NS3 from the cell lysate, establishing their physical association (Fig 6C). This interaction was further confirmed by the colocalization of NS3 and PKC-δ in HCV-JFH1 RNA transfected cells with a colocalization coefficient of 0.898 (Fig 6D). The colocalization assay was performed using HCV infection as well. The staining for HCV-NS3 and HCV-Core was performed to examine their interaction with PKC-δ (Fig 6E). This yielded a colocalization coefficient of 0.184 for Core and PKC-δ colocalization, and a colocalization coefficient of 0.85 for NS3 and PKC-δ colocalization, suggesting the interaction between PKC-δ and NS3 and not Core in HCV infected cells. These assays provide the evidence of direct interaction of HCV-NS3 protein with PKC-δ, which could activate it for HuR phosphorylation at S318 and hence its cytoplasmic localisation upon HCV infection. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Viral protein involved in relocalisation of HuR. (A) Huh7.5 cells were transfected with plasmid expressing HCV protein as indicated. Immunofluorescence staining was carried out at 48h post transfection using Alexa Fluor conjugated secondary antibodies against HuR (Green) and indicated viral proteins (Red). The nucleus was counterstained with DAPI. Scale bar represents 20 μm. (B) Nuclear and cytoplasmic ratio for HuR was quantified for images in (A) using Zen 2.3 lite software. n = 30. Student t-test was performed for statistical analysis. * = p<0.05, ** = p<0.01, *** = p<0.001. (C) Myc-tagged NS3 was overexpressed in Huh7.5 cells and 48h post transfection, cells were harvested for coimmunoprecipitation. Anti-PKC-δ antibody was used to pull down PKC-δ from vector control (pCDNA3.1) or Myc-NS3 transfected cell lysates. Western blotting was performed using anti-Myc antibody to detect the presence of NS3 in pull-down fraction. (D) Huh7.5 cells were transfected with HCV-JFH1 RNA. Immunofluorescence staining was carried out at 48h post transfection using Alexa Fluor conjugated secondary antibodies against NS3 (Green) and PKC-δ (Red). The nucleus was counterstained with DAPI. Scale bar represents 10 μm. An enlarged image of infected cell and the line profile for PKC-δ and NS3 intensity over the indicated arrow in the enlarged image is depicted in the inset. Colocalization coefficient was calculated using Zeiss software. (E) Huh7.5 cells were infected with HCV-JFH1 virus. Immunofluorescence staining was carried out at 48h post transfection using Alexa Fluor conjugated secondary antibodies against NS3, Core (Green) and PKC-δ (Red). The nucleus was counterstained with DAPI. Scale bar represents 10 μm. Colocalization coefficient was calculated using Zeiss software. https://doi.org/10.1371/journal.ppat.1011552.g006

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