Loss of nuclear PTEN in HCV-infected human hepatocytes
© Bao et al.; licensee BioMed Central Ltd. 2014
Received: 3 October 2013
Accepted: 26 June 2014
Published: 18 July 2014
Hepatitis C virus (HCV) infection is a major risk factor for chronic hepatitis and hepatocellular carcinoma (HCC); however, the mechanism of HCV-mediated hepatocarcinogenesis is not well understood. Insufficiency of PTEN tumor suppressor is associated with more aggressive cancers, including HCC. We asked whether viral non-coding RNA could initiate oncogenesis in HCV infected human hepatocytes. The results presented herein suggest that loss of nuclear PTEN in HCV-infected human hepatocytes results from depletion of Transportin-2, which is a direct target of viral non-coding RNA, vmr11.
The intracellular distribution of PTEN in HCV-infected cells was monitored by immunostaining and Western blots of nuclear and cytoplasmic proteins. Effects of PTEN depletion were examined by comparing expression arrays of uninfected cells with either HCV-infected or vmr11-transfected cells. Target genes suggested by array analyses were validated by Western blot. The influence of nuclear PTEN deficiency on virus production was determined by quantitative analysis of HCV genomic RNA in culture media of infected hepatocytes.
Import of PTEN to the nucleus relies on the interaction of Transportin-2 and PTEN proteins; we show that depletion of Transportin-2 by HCV infection or by the introduction of vmr11 in uninfected cells results in reduced nuclear PTEN. In turn, nuclear PTEN insufficiency correlates with increased virus production and the induction of γ-H2AX, a marker of DNA double-strand breaks and genomic instability.
An HCV-derived small non-coding RNA inhibits Transportin-2 and PTEN translocation to the nucleus, suggesting a direct viral role in hepatic oncogenesis.
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is one of the most commonly targeted tumor suppressor genes in human cancers, encoding a 403-residues dual specificity phosphatase with both lipid and protein phosphatase activities [1–5]. The lipid phosphatase activity of PTEN is a central negative regulator of the Phosphatidylinositol-3-kinase (PI3K) signal cascade for cell growth and proliferation. Deregulation of PTEN has been associated with a spectrum of metabolic disorders related to hepatocarcinogenesis . A recent report  points to post-transcriptional silencing of PTEN by HCV core 3a protein as a possible mechanism of PTEN deregulation.
Nuclear insufficiency of PTEN has been shown to contribute to centromere destabilization and genomic instability [8, 9], a hallmark of cancer, and nuclear PTEN depletion has been associated with more aggressive cancers [10–12]. In this study, we ask whether HCV infection initiates nuclear PTEN insufficiency and, in particular, whether viral non-coding RNA plays a part in regulating the intracellular redistribution of PTEN protein. We present evidence of inhibition of Transportin-2 by a viral non-coding RNA (vmr11). This inhibition restricts nuclear translocation of PTEN in HCV-infected human hepatocytes. We further show that restoring intracellular Transportin-2 levels rescues wild-type levels of nuclear PTEN. Based on the interaction between PTEN and Transportin-2, our results support a novel mechanism of regulation of nuclear PTEN translocation where down-regulation of Transportin-2 by vmr11 correlates with the nuclear exclusion of PTEN protein in HCV-infected human hepatocytes.
Restriction of nuclear PTEN in HCV-infected cells
HCV-derived small non-coding RNAs
Abundance of vmr11 varies with time post HCV infection
Expression arrays of human hepatocytes transfected with vmr11 “mimic” oligonucleotides as compared to HCV-infected cells
To identify vmr11 target genes that may be involved in limiting nuclear PTEN translocation, we contrasted changes in gene expression in HCV-infected hepatocytes (shown as H77-1, -2 and -3 in three independent infections), and separately in cells transfected with vmr11 “mimic” oligonucleotides (mimic-1, -2 and -3), against uninfected (control) cells, using the Agilent microarray platform (Additional file 2). For each comparison, differential expression analyses revealed several hundred up- and down-regulated genes that could serve as potential markers for HCV infection-associated liver disease. Notably, the qualitative and quantitative changes in gene expression of cells transfected with vmr11 “mimic” oligonucleotides (vmr11 ‘mimic’ is defined as a synthetic 22nt oligomer identical in sequence with the ‘wild type’ vmr11) were similar to those in HCV-infected cells (Additional file 1: Figure S2).
Functional analyses using the KEGG database  revealed several pathways enriched among differentially expressed genes and in common between the two sets of comparisons (H77 and ‘mimic’). In particular, genes involved in lipid biosynthesis, intracellular signaling, and control of cell growth and patterning were enriched among the up-regulated genes; whereas genes involved in the regulation of cell motility, and members of the peroxisome proliferator activated receptors (PPAR) signaling pathway involved in lipid metabolism, were over-represented among the down-regulated genes (Additional file 3).
Import of nuclear PTEN is sensitive to intracellular Transportin-2 levels
Functional domains of the HCV-derived vmr11
Replication of HCV is positively correlated with restriction of nuclear PTEN
Experiments described thus far utilized vmr11 or vmr11 mutant oligonucleotides to artificially increase intracellular concentrations of vmr11. Next, we asked whether competition of vmr11 RNA produced in vivo in HCV-replicating cells would influence TRN-2 protein levels and limit nuclear PTEN. Significantly, such assays allowed us to address whether virus production (HCV genomic RNA recovered from the culture medium of infected cells) increased in response to limiting nuclear PTEN protein.
To compete vmr11 RNA in vivo, we transfected the various vmr11 mutants (shown in Figure 5A) into HCV-infected human hepatocytes. As shown (Figure 5B), base substitutions at nucleotides 7-10 (vmr11 mutants 3 and 4) that lost the capacity to recognize the TRN-2 target site, and hence showed no inhibition of TRN-2 protein or nuclear PTEN, minimally influenced HCV production. By contrast, vmr11 mutants (1, 2, 5, 6 and 7) that apparently do not interfere with TRN-2-target recognition, and hence allow the inhibition of TRN-2 and nuclear PTEN, favored virus production. These results suggest that depletion of nuclear PTEN favors virus production. Western blots of nuclear PTEN and TRN-2 proteins from the same cells that were scored for virus production are shown in Figure 5C. Overall, the results suggest that reduced levels of nuclear PTEN protein in HCV infected cells favors virus production; and that PTEN can be restored in vivo by the introduction of competing vmr11 mutant oligonucleotides.
Depletion of nuclear PTEN correlates with increased γ − H2AX
In an attempt to correlate the effects of HCV infection or transfection with vmr11 RNA to γ-H2AX induction in vivo, we compared immunostaining of γ-H2AX in human hepatocytes either infected with HCV or transfected with vmr11 ‘mimic’ oligonucleotides. Immunostaining for NS5A viral antigen identified the HCV-infected cells (Figure 6C). Visual inspection of the HCV-infected cells and control cells that were transfected with vmr11 ‘mimic’ oligonucleotides identified relatively enhanced γ-H2AX staining nuclei. Overall, these results are consistent with the interpretation that depletion of nuclear PTEN in human hepatocytes, either due to HCV infection or by artificially increasing intracellular vmr11 RNA in uninfected cells, results in the induction of γ-H2AX.To conclude, inhibition of Transportin-2 by the viral non-coding RNA, vmr11 limits PTEN translocation to the nucleus and is sufficient to induce γ-H2AX an indicator of DSB and genomic instability in HCV-infected human hepatocytes (an overview of the results, is depicted in Figure 6D).
One of the critical steps in virus life cycle is their ability to modulate host defenses that favor virus propagation. For oncogenic virus such as HCV, a challenge is to define direct viral role in the depletion of PTEN tumor suppressor that correlates with more aggressive cancer, including HCC. The results described here suggest a novel mechanism of regulation of intracellular distribution of PTEN protein by viral non-coding RNA directed suppression of Transposrtin-2. Viral non-coding RNA mediated inhibition of Transportin-2 and the loss of nuclear PTEN in HCV-infected human hepatocytes implicate a direct viral role in oncogenic transformation. Consistent with that interpretation is our observation that HCV mediated nuclear insufficiency of PTEN correlates with DSB and genomic instability and efficient virus production.
Small RNA-based mechanisms, such as RNA interference (RNAi), play important roles in regulating the course of viral infection and viral pathogenesis in plants and animals [24–29]. Examples of virus-derived microRNAs have been reported in the human cytomegalovirus, Epstein Barr virus, Kaposi Sarcoma-associated virus, peach latent mosaic viroid [30, 31], as well as in two viruses with RNA genomes, HIV-1 and BLV [27, 32]. An earlier study  employing high-throughput sequencing predicted, albeit did not functionally validate, the presence of small viral RNAs derived from both positive and negative sense genomic RNA of several viruses, including HCV. By analyzing the conserved hairpin-loop structures of the HCV minus strand genomic RNA as a source of viral microRNA, we identified novel viral small non-coding RNA, vmr11, and have functionally validated the vmr11-dependent restriction of nuclear PTEN.
PTEN is a haploinsufficient tumor suppressor; partial depletion of PTEN is expected to profoundly influence cell cycle deregulation and genomic instability, hallmarks of oncogenic susceptibility. A number of mechanisms have been proposed to explain nucleocytoplasmic partitioning of PTEN [34–36]; however, there are no examples of PTEN restriction mediated by a human oncogenic virus. PTEN lacks both the conventional nuclear import (nuclear localization signal, NLS) and the nuclear export (NES) motifs [1, 35]. No mutation has been reported among all the cases of nuclear PTEN ‘mis-localization’ examined , leaving open the possibility that nuclear translocation of PTEN may be regulated by ‘non-genetic’ mechanisms such as protein-protein interaction involving TRN-2.
PTEN deregulation plays a significant role in hepatopathogenesis [37, 38]. The proposed role of HCV core 3a in modulating the formation of PTEN-dependent large lipid droplets in hepatocytes  implicates a direct viral role in liver disease. Adding to these studies, the results described herein support a novel mechanism for regulation of PTEN translocation to the nucleus in HCV-infected cells. Specifically, nuclear insufficiency of PTEN in HCV-infected cells results from the depletion of Transportin-2, which is mediated by a novel viral non-coding RNA, vmr11. Reduced levels of Transportin-2 in cells that are either transfected with vmr11 ‘mimic’ oligonucleotides, or suffer siRNA knock-down of endogenous TRN-2, restrict the Transportin-2 and PTEN complex at the nuclear membrane. Collectively, these findings point to a novel mechanism of regulation of intracellular distribution of PTEN that is mediated by Transportin-2, a direct target of vmr11. The observation that the TRN-2-PTEN protein complex is restricted at the nuclear membrane leaves open the possibility that factor(s) other than protein-protein interaction may be required for the PTEN translocation to the nucleus.
Perhaps the most intriguing consequence of nuclear PTEN insufficiency in HCV-infected cells is the induction of γH2AX, a major player in the recognition and repair of DNA double-strand breaks (DSB) and a hallmark of genomic instability and cancer susceptibility [8, 39, 40]. We observed enhanced staining foci of γH2AX-positive cells in HCV-infected or vmr11-transfected human hepatocytes. Whether DSB related to the nuclear PTEN insufficiency in HCV infection serve as an early indicator of HCV infection-associated hepatocellular carcinoma can be pursued in future studies in an animal model of HCV-infection associated HCC.
Post-attachment primary hepatocytes (PPH)
A sustained primary human hepatocytes culture system was recently described . Briefly, a co-culture of Hepatic Stellate cell line (CFSC-8B) was used as a ‘feeder layer’, to provide the extracellular matrix for efficient attachment of human primary hepatocytes suspension. The co-culture was maintained in a serum-free Hepatocyte-Defined Medium (HDM) for 30 days, during which the stellate cells (that require serum for their replication) are largely depleted. The primary human hepatocytes form “spheroids” that can be dispersed (with 0.05% trypsin treatment) and propagated as monolayers (independent of stellate cells), as ‘Post-attachment Primary Hepatocytes’ (PPH) in HDM supplemented with 1% FBS. The PPH cultures are dispersed with 0.05% trypsin and reseeded at 1:3 dilutions at weekly intervals.
Bioinformatics prediction of HCV small non-coding RNAs
The 9,646 bp negative sense HCV type 1 genome (GenBank accession: NC_004102) was divided, starting every 20 bp, into fixed size segments of length L = 70 (80, 90 and 100). RNAfold  was employed to test each short sequence to determine stable hairpin structures that can form viral small RNA precursors. Hairpins that scored in the top 5% of the score distributions (determined by comparison to simulated random samples) were analyzed for conserved secondary structure by comparing 37 HCV type 1 complete genome sequences. Lastly, putative viral microRNA sequences were determined starting from conserved sequences 7 bp or longer (‘seeds’) that could be extended to mature 20-25 viral small RNAs (see Additional file 1 for details).
Transfection of PPH using H77 run-off transcript
pCV-H77c plasmid DNA was linearized for run-off transcription of full length genomic RNA using T7 Ribomax Express (Promega). 5×105 PPH cells were transfected with 1 μg of the H77 transcript using the Fugene 6 (Promega) procedure. Virus replication was monitored by quantitation of genomic equivalents (GE) of HCV RNA or by immune blotting for NS5A or HCV core antigens.
Real time Quantitative PCR of viral small RNAs
1 μg of total RNA was reverse-transcribed using QuantiMir RT Kit (System Biosciences). The cDNA was diluted (100-fold) and amplified using PCR Supermix (Invitrogen) or iTaq SYBR Green Supermix with Rox (Bio-Rad) on GeneAmp PCR System 9700 or on ABI 7300 Real Time PCR system.
Subcellular protein fractionation and Western blot
PPH cells were lysed with either RIPA buffer supplemented with 1X protease inhibitor cocktail (Roche), or with NE-PER nuclear and cytoplasmic extraction reagents (Thermo Pierce) for fractionations of nuclear or cytoplasmic proteins. Normally, 10 μg of protein was analyzed on 10% precast Mini-PROTEIN gel (BIO-RAD). The gels were transferred to PVDF membrane, blocked with 5% non-fat milk and probed with antibodies against PTEN (ab#79156 1:1000), GAPDH (ab8245, 1:5000), Lamin-B1 (ab#16048, 1:500), HCV core (ab50288, 1:1000) or γ-H2AX (ab11174, 1:1000).
5×104 of HCV-infected or uninfected PPH cells were seeded into six-well plates with sterilized cover glasses one day before staining. The cells were washed with PBS and fixed with 4% paraformaldehyde, washed and permeabilized with methanol at -20°C, blocked with 1% BSA and stained with primary antibody against PTEN (ab32199, 1:100) and NS5A (ab20342, 1:100); followed by FITC or Texas Red conjugated secondary antibodies.
Quantitation of virus from the culture medium of infected cells
QIAamp Viral RNA Mini Kit (QIAGEN) was used to extract RNAs from 140 μl of the culture medium of HCV–infected (or control) cells. The cDNA was prepared using QuantiTect Rev Transcription Kit (QIAGEN) and was analyzed with primers that target the 5’ end of HCV (positive sense) genomic RNA. Primer sequences are H77-QRT-Forward (TGTGGAGCTGAGATCACTGG) and H77-QRT-Reverse (CCGCCTTATCTCCACGTATT).
All Quantitative Real time PCR and densitometry data were analyzed using Microsoft Excel 2010.
RNA labeling and array hybridization
Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology).
The Agilent Feature Extraction software (version 184.108.40.206) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed with the GeneSpring GX v12.0 software package (Agilent Technologies). Differentially expressed mRNAs were identified through Fold Change filtering (cutoff 2.0). Pathway analyses were performed using the standard enrichment computation method. Lastly, 3′UTRs of GenBank RefSeq genes for vmr11 putative target analysis with PITA were extracted from the UCSC Genome Browser tables. (Details are provided in Additional file 1).
The authors thank Dr. Charles M. Rice (The Rockefeller University) for Huh-7.5 cell line, Drs. Jens Buch and Robert H Purcell (NIH) for the H77 clone. Supported by Katzen Cancer Research Funds from the George Washington University, and the McCormick Genomics Center.
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