- Email: [email protected]
Treatment with PTEN-Long protein inhibits hepatitis C virus replication
Virology 511 (2017) 1–8
Contents lists available at ScienceDirect
Virology journal homepage: www.elsevier.com/locate/yviro
Treatment with PTEN-Long protein inhibits hepatitis C virus replication Qi Wu
a,b,1
a,c,1
, Zhubing Li
MARK
a,b,c,⁎
, Qiang Liu
a
Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, 120 Veterinary Road, Saskatoon, Saskatchewan, Canada S7N 5E3 b Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada c School of Public Health Vaccinology and Immunotherapeutics, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
A R T I C L E I N F O
A BS T RAC T
Keywords: PTEN-Long Hepatitis C virus Viral replication Core Protein - protein interaction
Hepatitis C virus (HCV) infection is a confirmed risk factor for hepatocellular carcinoma (HCC). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) possesses tumor suppression function that is frequently defective in HCC tumors. PTEN-Long, a translation isoform of PTEN, functions in a cell non-autonomous manner. In this study, we demonstrated that intracellular overexpression of PTEN-Long inhibits HCV replication. More importantly, we showed that treatment with extracellular PTEN-Long protein inhibits HCV replication in a dose-dependent manner. Furthermore, we showed that PTEN-Long interacts with HCV core protein and this interaction is required for HCV replication inhibition by PTEN-Long. In summary, we demonstrated, for the first time, that PTEN-Long protein, an isoform of the canonical PTEN and in the form of extracellular protein treatment, inhibits HCV replication. Our study offers an opportunity for developing additional anti-HCV agents.
1. Introduction Despite the approval of anti-viral drugs, hepatitis C virus (HCV) infection continues to be a significant public health problem with hepatocellular carcinoma (HCC) as the most deadly clinical outcome (Messina et al., 2015). HCV has a single-stranded RNA genome encoding a polyprotein that is processed by cellular and viral proteases to generate structural and non-structural proteins (Scheel and Rice, 2013). Although it is a structural protein, HCV core has been reported to possess many regulatory functions including tumorigenesis (Kao et al., 2016; Moriya et al., 1998). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a dual phosphatase with lipid and protein phosphatase activities (Song et al., 2012; Leslie et al., 2008). These phosphatase activities render PTEN to function as a tumor suppressor (Song et al., 2012). PTEN has been characterized as one of the most frequently mutated or deleted genes in various tumors including HCC (Zhang and Yu, 2010; Song et al., 2012). For instance, three single amino acid mutations in the phosphatase domain have been identified: C124S (lipid and protein phosphatase defective), G129E (lipid phosphatase activity defective), and Y138L (protein phosphatase defective) (Davidson et al., 2010). As protein products translated from alternative
start codons upstream of the ATG initiation sequence of PTEN, four longer isoforms of PTEN, termed PTEN-Long/PTENα, PTEN-M/ PTENβ, PTEN-N, and PTEN-O, respectively, were discovered (Malaney et al., 2017). Whether these translation isoforms have similar or different functions in comparison to canonical PTEN has not been well characterized. PTEN-Long, the focus of the current study, has 173 N-terminal extra amino acids with a poly-arginine region. This region enables PTEN-Long to be exported into extracellular compartments, enter neighboring cells, and induce signaling events in recipient cells (Hopkins et al., 2013; Wang et al., 2015). PTEN-Long possesses comparable or even higher constitutive lipid phosphatase activity than PTEN (Masson et al., 2016; Hopkins et al., 2013; Johnston and Raines, 2015). Similar to the canonical PTEN, PTEN-Long can suppress PI3KAkt activation (Hopkins et al., 2013; Wang et al., 2015; Liang et al., 2017). Interestingly, a G to R point mutation in PTEN-Long (G302R) has the same phenotype as the corresponding G129R mutation in canonical PTEN in regulating Akt activity (Wang et al., 2015; Hopkins et al., 2013), suggesting a conserved functional role for the amino acid residues amongst different PTEN isoforms. Hence, other point mutations of PTEN, such as C124S and Y138L, have also been transferred to the corresponding residues in longer PTEN isoforms in functional studies (Liang et al., 2017).
⁎ Corresponding author at: Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, 120 Veterinary Road, Saskatoon, Saskatchewan, Canada S7N 5E3. E-mail address: [email protected] (Q. Liu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.virol.2017.08.002 Received 23 June 2017; Received in revised form 26 July 2017; Accepted 2 August 2017 0042-6822/ © 2017 Elsevier Inc. All rights reserved.
Virology 511 (2017) 1–8
Q. Wu et al.
for normalization (Qiao et al., 2013). Relative changes in RNA levels were analyzed by the 2-ΔΔct method using the iQ5 program (Bio-Rad Laboratories). The results were analyzed for statistical differences by the Student's t-test. A p value of ≤ 0.05 was considered statistically significant.
We recently demonstrated that PTEN can inhibit HCV replication through interacting with HCV core protein (Wu et al., 2017). Whether PTEN-Long affects HCV replication has not been investigated. In this study, we showed that intracellular expression of or extracellular treatment with PTEN-Long protein can inhibit HCV replication. Furthermore, we showed that PTEN-Long - HCV core protein interaction is involved in the inhibitory effect of PTEN-Long on HCV replication.
2.4. Co-immunoprecipitation (Co-IP) and Western blotting For Flag co-IP experiments, cells were harvested by radioimmunoprecipitation assay (RIPA) buffer and incubated with anti-Flag (SigmaAldrich) antibody at 4 ℃ overnight and then incubated with Protein G Sepharose (GE Healthcare) at 4 ℃ for 4 h. The mixtures were centrifuged at 10,000 rpm for 10 min and the supernatants were removed. The pellets were resuspended in SDS lysis buffer and boiled for 10 min to elute the proteins. For Western blotting, proteins were subjected to SDS-PAGE and then blotted onto nitrocellulose membranes. The membranes were blocked in 5% skim milk in PBS and incubated with a primary antibody overnight at 4 ℃. Membranes were washed and incubated with a secondary antibody for 1 h at room temperature. After a wash with PBST (PBS+0.1% Tween 20), membranes were scanned using Li-Cor Odyssey scanner (ODY-CLx) and band intensities were determined by Quantity One software (Bio-Rad Laboratories). The primary antibodies used were HCV core (Anogen), Flag (Sigma-Aldrich), PTEN and β-actin (Cell Signaling Technology), and PTEN-Long (Gly-2) provided by Ramon Parsons (Hopkins et al., 2013). The secondary antibodies used were IRDye 800CW goat antimouse IgG and IRDye 680RD goat anti-rabbit IgG (Li-Cor Biosciences).
2. Materials and methods 2.1. Plasmids and in vitro transcription Plasmids containing HCV-2a J6/JFH-1(p7-RLuc-2A) or HCV-2a J6/JFH-1(p7-RLuc-2A) ΔE1E2 sequences were provided by Charles Rice (Jones et al., 2007). Plasmid expressing Flag-PTEN was provided by Jack Dixon (Maehama and Dixon, 1998). Plasmids expressing PTEN-Long-V5-His6 (Addgene plasmid #49417) and PTEN-V5-His6 (Addgene plasmid #49420) in the bacterial JpExpress404 vector or in the pcDNA3 vector were obtained from Addgene or Ramon Parsons (Hopkins et al., 2013). Bacterial or eukaryotic expression plasmids encoding red fluorescent protein (RFP) fusion proteins with PTEN or PTEN-Long were constructed as described (Hopkins et al., 2013). Plasmids expressing PTEN-Long mutants with single amino acid substitutions C297S, G302E, or Y311L were generated in reference to the corresponding mutations of PTEN (Peyrou et al., 2013). His6-blue fluorescent protein (BFP)-expressing plasmid in the pT7 His6-SUMO vector (Lucigen) was described previously (Hoffman et al., 2015). Plasmid expressing a fusion protein between HCV-2a J6 core and enhanced green fluorescent protein (EGFP) was also constructed. All plasmids were generated using standard methods and confirmed by DNA sequencing when necessary. HCV RNA was generated by in vitro transcription using the MEGAscript T7 In Vitro Transcription reagents (Thermo Fisher Scientific).
2.5. Purification of recombinant proteins The expression of recombinant proteins in E. coli was induced by IPTG (Thermo Fisher Scientific). His6-tagged PTEN, PTEN-Long, BFP, PTEN-RFP, PTEN-Long-RFP, and RFP proteins were purified by NiNTA agarose (Qiagen) as previously described (Hoffman et al., 2015).
2.2. Cell culture, transfection, fluorescent microscopy, and HCV infection
3. Results
Huh-7 and Huh-7.5 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) fetal bovine serum (FBS). Huh-7 cells with replicating HCV-2a J6 core-Flag/JFH-1(p7-RLuc-2A) or HCV-2a J6 core R50A-Flag/JFH-1(p7-RLuc-2A) RNAs were maintained with culture medium plus G418 (Enzo Life Sciences) as described previously (Wu et al., 2017). Cells were transfected with plasmid DNA or HCV RNA using the calcium phosphate precipitation method (Jackel-Cram et al., 2007) or the jet-PEI transfection reagent (Polyplus Transfection) (Shi et al., 2016). Cells transfected with fluorescent protein-expressing plasmids were visualized with appropriate settings under a Leica TCS SP5 confocal microscope (Leica Microsystems). HCV-2a JFH-1 rLuc subgenomic replicon cells and infection with HCV-2a J6 core-Flag/JFH-1(p7-RLuc-2A) virus were described previously (Wu et al., 2017).
3.1. Intracellular PTEN-Long inhibits HCV replication We recently showed that ectopic expression of PTEN inhibits HCV replication (Wu et al., 2017). Whether PTEN-Long, a longer isoform of PTEN, can also regulate HCV replication has not been studied. To investigate the effect of PTEN-Long on HCV replication, we transfected Huh-7 cells harboring HCV-2a J6 core-Flag/JFH-1(p7-rLuc-2A) replicating RNA with vector, plasmids expressing PTEN or PTEN-Long and measured HCV replication by luciferase assay. We also used a set of plasmids expressing fusion proteins with RFP, which will enable subsequent experiments. Consistent with our previous results, overexpression of PTEN significantly inhibited HCV replication in comparison to vector (Fig. 1A). Expression of the fusion protein PTEN-RFP also inhibited HCV replication as effectively as PTEN itself, while RFP expression had no effect (Fig. 1A). Overexpression of PTEN-Long or PTEN-Long-RFP could reduce HCV replication to the same extent as PTEN (Fig. 1A). The intracellular expression of PTEN and PTEN-Long proteins was demonstrated by Western blotting using a PTEN-specific antibody (Fig. 1B). It is interesting to point out that while the endogenous PTEN protein could be readily detected in the Huh-7 cells as shown in Fig. 1B, the endogenous PTEN-Long protein was not visible in Western blotting using a PTEN-Long-specific antibody (Gly2) (Hopkins et al., 2013) (data not shown). These results indicated that both PTEN and PTEN-Long can inhibit HCV replication when expressed intracellularly. Because Huh-7 cells harboring HCV-2a J6 /JFH-1(p7-rLuc-2A) or HCV-2a J6 core-Flag/JFH-1(p7-rLuc-2A) replicating RNA have been shown to produce infectious virus particles (Jones et al., 2007; Wu
2.3. Luciferase and RT-qPCR assays Cells were lysed in Passive Lysis Buffer (Promega) and the firefly or renilla luciferase activities were measured by Luciferase Assay reagents (Promega) in a TD 20/20 Luminometer (Turner Designs). Luciferase levels were normalized to the protein concentrations determined by the Bradford assay (Bio-Rad Laboratories). To determine the HCV RNA levels, total RNA was extracted by Trizol (Thermo Fisher Scientific) and reverse transcribed into cDNA by SuperScript II Reverse Transcriptase (Thermo Fisher Scientific). Real time PCR was performed using primers HCV-FD (5`-AGAGCCATAGTGGTCTGCGGAAC-3`) and HCV-rev (5`-CCTTTCGCAACCCAACGCTACTC-3`) (Lim and Hwang, 2011). The transcript levels of GUSB, a house keeping gene, were used 2
Virology 511 (2017) 1–8
Q. Wu et al.
Fig. 1. Intracellular expression of PTEN-Long inhibits HCV replication. (A and B) Huh-7 cells harboring HCV-2a J6/JFH-1(p7-rLuc-2A) replicating RNA were transfected with plasmid vector, or plasmids expressing RFP (red fluorescent protein), PTEN-RFP, PTEN-Long-RFP (PTEN-L-RFP), PTEN, or PTEN-Long (PTEN-L). All proteins have a C-terminal V5-His6 tag. (A) At 48 h after transfection, luciferase assay was performed and the statistical differences between samples were demonstrated as ** if p ≤ 0.01, or NS for not significant. (B) The expression of PTEN and PTEN-Long proteins was demonstrated by Western blotting using an anti-PTEN antibody. The levels of β-actin were also determined as loading controls. (C and D) Huh-7.5 cells were co-transfected with HCV ΔE1E2 or HCV full-length genomic RNA together with plasmid vector or a plasmid expressing PTEN-Long with a C-terminal V5-His6 tag. (C) At 48 h after transfection, luciferase assay was performed and the statistical differences between samples were demonstrated as * if p ≤ 0.05. (D) The expression of PTEN-Long protein was demonstrated by Western blotting using an anti-V5 antibody. The levels of β-actin were also determined as loading controls. (E and F) Huh-7.5 cells were transfected with plasmid vector or plasmid expressing PTEN-Long. At 48 h after transfection, cells were infected with a cell culture derived HCV-2a J6 core-Flag/JFH-1(p7-RLuc-2A) virus. At 72 h after infection, luciferase activity (C) and HCV RNA levels (D) were determined. * = p ≤ 0.05.
deleted (Jones et al., 2007) in a co-transfection together with plasmid vector or a plasmid expressing PTEN-Long. We also used the wild-type HCV RNA as a control. In both cases, PTEN-Long expression resulted in significantly lower RLuc activities than vector control (Fig. 1C). The
et al., 2017), HCV RNA levels measured at 48 h after transfection with PTEN-Long expressing plasmid may not only reflect the degree of HCV replication. To explicitly determine the effect of PTEN-Long on HCV replication, we used an HCV genomic RLuc RNA with the E1 and E2
3
Virology 511 (2017) 1–8
Q. Wu et al.
and used to infect naïve Huh-7.5 cells. At 72 h after infection, luciferase activity was determined. As shown in Fig. 2F, significantly lower luciferase activity was detected after PTEN-Long-RFP treatment in comparison to RFP. These results indicated that extracellular PTENLong treatment inhibits HCV propagation. Taken together, these results demonstrated that extracellular PTEN-Long protein can enter the cells and inhibit HCV replication.
expression of PTEN-Long protein was demonstrated by Western blotting (Fig. 1D). These results indicated that intracellular PTENLong can inhibit HCV replication. To determine whether PTEN-Long regulates HCV RNA replication after infection, Huh-7.5 cells transfected with plasmid vector or plasmid expressing PTEN-Long were infected with a cell culture derived HCV-2a J6 core-Flag/JFH-1(p7-RLuc-2A) virus. HCV replication was analyzed by luciferase assay (for luciferase activity) and RTqPCR (for HCV RNA level). Results of both assays demonstrated a significantly lower replication after PTEN-Long expression in comparison to control (Figs. 1E and F). These results indicated that intracellular PTEN-Long can inhibit HCV replication after viral infection.
3.3. PTEN-Long interacts and co-localizes with HCV core We recently showed that HCV core protein interacts with PTEN through the arginine residue at position 50 (R50) and the interaction is required for the inhibitory effect of PTEN on HCV replication (Wu et al., 2017). To determine whether HCV core also interacts with PTEN-Long and the role of R50, we performed Flag co-immunoprecipitation experiment after transfecting PTEN-Long expressing plasmid into HCV-2a J6 core-Flag/JFH-1(p7-rLuc-2A) and HCV-2a J6 core R50A-Flag/JFH-1(p7-rLuc-2A) replicon cells. As shown in Fig. 3A, PTEN-Long was detected in the immunoprecipitates using lysates containing wild-type, but not R50A, HCV core. Since PTEN is translated (internally) from the PTEN-Long coding sequence, PTEN protein was also present/absent in the immunoprecipitates (Fig. 3A). These results indicated that HCV core interacts with PTEN-Long and requires R50. To determine whether HCV core and PTEN-Long proteins colocalize in the cell, Huh-7 cells were co-transfected with plasmids expressing fluorescent fusion proteins HCV core-EGFP and PTENLong-RFP. Plasmids expressing EGFP, RFP, or PTEN-RFP were included for control purposes. Consistent with the literature (Liang et al., 2017), confocal microscopic images in Fig. 3B showed mostly cytoplasmic localization of PTEN-Long and both nuclear and cytoplasmic distribution of PTEN. Substantial co-localization of HCV core with both PTEN and PTEN-Long could be observed (Fig. 3B). Protein expression was demonstrated by Western blotting (Fig. 3C).
3.2. Extracellular PTEN-Long protein treatment inhibits HCV replication In comparison to the canonical PTEN, a unique feature of PTENLong lies in its ability to enter cells as a secreted protein (Hopkins et al., 2013). We therefore were interested in determining whether PTEN-Long as an extracellular protein regulates HCV replication. To this end, we purified His6-tagged PTEN and PTEN-Long proteins with or without fusion with RFP as previously described (Hopkins et al., 2013). Huh-7 cells harboring HCV-2a J6 core-Flag/JFH-1(p7-RLuc2A) replicating RNA were treated with increasing amounts of PTEN or PTEN-Long protein and HCV replication quantified. PBS and BFP protein were used as controls. As shown in Fig. 2A, while HCV replication was not affected by treatment with PBS, BFP, or PTEN proteins in three escalating doses, HCV replication was reduced upon PTEN-Long protein treatment in a dose-dependent manner. These results indicated that extracellular PTEN-Long protein can inhibit HCV replication. We previously showed that the lipid phosphatase deficient G129E mutant of PTEN can inhibit HCV replication as effectively as the wildtype PTEN, whereas the protein phosphatase deficient Y138L and the lipid and protein phosphatase deficient C124S mutants have no effect (Wu et al., 2017). Next, we wanted to determine whether these mutations in PTEN-Long as an extracellular protein will have a similar effect. We therefore transferred these point mutations of PTEN to the corresponding positions in PTEN-Long and purified these proteins for use in the treatment. Fig. 2B showed that while PTEN-Long proteins with C297S and Y311L mutations no longer inhibited HCV replication, the G302E mutant protein had the same inhibitory effect as the wildtype PTEN-Long. These results indicated that the functions of the three amino acids are conserved between PTEN and PTEN-Long in inhibiting HCV replication. To confirm that PTEN-Long but not PTEN protein can enter the cells, we fused PTEN-Long or PTEN with RFP and detected red fluorescence after treatment. As shown in Fig. 2C, red fluorescence was observed after treatment with PTEN-Long-RFP in more than 60% of the cells (50 positive cells out of 80 in total), but not with PTEN-RFP or RFP, indicating that only extracellular PTEN-Long protein can enter the cells whereas PTEN-RFP or RFP cannot. Fusion with RFP did not affect the function of PTEN and PTEN-Long in regulating HCV replication (compare Figs. 2D and 1A). To rule out the possibility that the changes in Renilla luciferase activity observed after PTEN-Long protein treatment was due to an effect of PTEN-Long on the Renilla luciferase itself, we measured HCV RNA levels by RT-qPCR. As shown in Fig. 2E, treatment with PTENLong-RFP protein resulted in a significantly lower HCV RNA level than RFP treatment. These results indicated that PTEN-Long protein indeed regulates HCV RNA replication. To determine whether extracellular treatment with PTEN-Long downregulates HCV propagation, Huh-7 cells harboring HCV-2a J6 core-Flag/JFH-1(p7-RLuc-2A) replicating RNA were once again treated with RFP or PTEN-Long proteins. The supernatants containing infectious HCV reporter viruses were collected at 24 h after treatment
3.4. PTEN-Long inhibits HCV replication through interaction with HCV core Next, we wanted to investigate the role of HCV core - PTEN-Long interaction in regulating HCV replication. HCV-2a J6 core R50A-Flag/ JFH-1(p7-rLuc-2A) replicating Huh-7 cells were treated with 24 nM of BFP, PTEN, PTEN-Long and phosphatase mutant proteins, and PBS before HCV replication measured by luciferase assay for luciferase activity (Fig. 4A) or RT-qPCR assay for HCV RNA levels (Fig. 4B). No effects on HCV replication were observed. These results indicated that PTEN-Long interacts with HCV core, which is required for HCV replication inhibition. To corroborate these findings, we treated HCV rLuc reporter subgenomic replicon cells that do not express HCV structural proteins with PTEN-Long-RFP protein or RFP protein as a control. When HCV replication was measured by both luciferase and RT-qPCR assays, we found that PTEN-Long could no longer inhibit, but instead increased, HCV RNA replication in comparison to RFP treatment (Figs. 4C and D). These results once again indicated that the inhibition of HCV replication by PTEN-Long requires the core protein. 4. Discussion Viruses are the etiologic agents for more than 10% of tumors in human (Mirvish and Shuda, 2016). However, the mutual interactions between PTEN-Long and oncogenic viruses have yet to be characterized. As an extension to our recent research on the effect of the canonical PTEN on HCV replication (Wu et al., 2017), we characterized the role of PTEN-Long in this study. We showed that intracellular expression of PTEN-Long could inhibit HCV replication as effectively as the PTEN protein (Fig. 1). More importantly, we found that HCV 4
Virology 511 (2017) 1–8
Q. Wu et al.
Fig. 2. Extracellular treatment with PTEN-Long protein inhibits HCV replication. Huh-7 cells with HCV-2a J6/JFH-1(p7-rLuc-2A) replicating RNA were treated with BFP, RFP, PTEN, PTEN-Long, PTEN-RFP, or PTEN-Long-RFP proteins or equal volume of PBS (phosphate-buffered saline). All proteins had a His6-tag and were purified from E. coli through affinity chromatography. In (A), 1.5, 6, and 24 nM of PTEN or PTEN-Long proteins and 24 nM of BFP protein were used. In (B, C, D and E), cells were treated with 24 nM of proteins. At 24 h after treatment, luciferase (A, B and D) or RT-qPCR (E) assays were performed to determine HCV replication. The statistical differences between samples were demonstrated as * if p ≤ 0.05, ** if p ≤ 0.01, *** if p ≤ 0.001, or NS for not significant. In (C), cells were stained with DAPI at 6 h after treatment. Images were collected by confocal microscopy and analyzed by the Image J software. In (F), the supernatants containing infectious HCV reporter viruses were collected at 24 h after treatment with RFP or PTEN-Long-RFP proteins and used to infect naïve Huh-7.5 cells. At 72 h after infection, luciferase assay was performed. ** = p ≤ 0.01.
5
Virology 511 (2017) 1–8
Q. Wu et al.
Fig. 3. PTEN-Long interacts and co-localizes with HCV core. (A) Huh-7 cells harboring HCV-2a J6 core-Flag/JFH-1(p7-rLuc-2A) or HCV-2a J6 core R50A-Flag/JFH-1(p7-rLuc-2A) replicating RNAs were transfected with plasmid expressing PTEN-Long. Forty eight hours after transfection, cell lysates were subjected to co-immunoprecipitation (co-IP) using an antiFlag antibody. Input and co-IP products were analyzed by Western blotting (WB) using anti-PTEN and anti-HCV core antibodies, respectively. (B and C) Huh-7 cells were co-transfected with plasmids expressing EGFP, HCV core-EGFP, RFP, PTEN-RFP, or PTEN-Long-RFP as indicated. At 48 h after transfection, cells were stained with DAPI and observed under a confocal microscope with appropriate settings (B). Co-localization sites were indicated by a square. Protein expression was demonstrated in Western blotting (C).
replication was inhibited by incubation with purified PTEN-Long protein in a dose-dependent manner (Fig. 2). This inhibition is due to the permeability feature of the PTEN-Long protein because we
showed that PTEN-Long in the form of an extracellular protein, but not the PTEN protein, can enter HCV replicating cells (Fig. 2). As for the mechanisms, we found that the C297S and Y311L 6
Virology 511 (2017) 1–8
Q. Wu et al.
Fig. 4. PTEN-Long protein inhibits HCV replication by interacting with HCV core. (A and B) Huh-7 cells harboring HCV-2a J6 core R50A-Flag /JFH-1(p7-rLuc-2A) replicating RNA were treated with 24 nM of BFP, PTEN, PTEN-Long proteins or equal volume of PBS. Luciferase (A) or RT-qPCR (B) assays were performed at 24 h after treatment to determine HCV RNA replication. The statistical differences between samples were demonstrated as NS for not significant. (C and D) Huh-7 cells harboring HCV-2a JFH-1 subgenomic replicating RNA were treated with 24 nM of RFP or PTEN-Long-RFP proteins. Luciferase (C) or RT-qPCR (D) assays were performed at 24 h after treatment to determine HCV RNA replication. * = p ≤ 0.05.
consideration, these results suggest that PTEN-Long, once present inside the cell, very likely exerts its inhibitory effects on the replication of HCV full genomic RNA through similar mechanisms as the canonical PTEN. However, given the fact that the canonical PTEN has no effect on the replication of subgenomic HCV RNA (Wu et al., 2017), PTENLong may have additional mechanisms to regulate HCV RNA replication in the absence of the core protein. In conclusion, we demonstrated that PTEN-Long inhibits HCV replication. To the best of our knowledge, this is the first report on characterizing the effect of PTEN-Long on a viral replication. In addition, although direct-acting antivirals (DAAs) are very effective in achieving sustained virological responses in hepatitis C patients, the long-term effects of this therapy on liver diseases, especially hepatocellular carcinoma (HCC), in these patients are controversial and remain to be characterized (Janjua et al., 2017; van der Meer and Berenguer, 2016). The fact that PTEN-Long exerts its inhibitory function on HCV replication (this study) and on tumor growth (Hopkins et al., 2013) in the form of an extracellular protein should provide an additional avenue for developing therapeutics for HCV infection and the resultant HCC.
mutants of PTEN-Long do not inhibit HCV replication (Fig. 2), suggesting the involvement of these two amino acid residues of PTEN-Long in this process. The corresponding amino acid residues in canonical PTEN, namely C124 and Y138, are both involved in regulating the protein phosphatase activity of PTEN (Davidson et al., 2010). If the functions of these amino acids are conserved, our data would suggest that the protein phosphatase activity of PTEN-Long is required for inhibiting HCV replication. This is consistent with a previous study where the protein phosphatase activity was found to be involved in regulating HCV egress (Peyrou et al., 2013). It has been shown that the protein phosphatase mutant of PTEN induced larger lipid droplets and increased cholesterol esters biosynthesis (Peyrou et al., 2013). Furthermore, we showed an interaction and co-localization between PTEN-Long and HCV core proteins (Fig. 3). We also found that the arginine residue at position 50 of the HCV core protein is critical for the interaction and PTEN-Long could no longer inhibit HCV RNA replication in the absence of this R50 residue, suggesting a role for protein interaction (Figs. 4A and B). In an effort to further confirm these findings, we used an HCV subgenomic replicon that does not express the structural proteins, including core. PTEN-Long indeed could not inhibit the replication of the subgenomic HCV RNA. Somewhat to our surprise, we found that PTEN-Long could increase HCV subgenomic RNA replication (Figs. 4C and D). Taking our recent study on how PTEN inhibits HCV replication (Wu et al., 2017) into
Acknowledgements This work was supported by grants from Canadian Institutes of 7
Virology 511 (2017) 1–8
Q. Wu et al.
PTENbeta is an Alternatively Translated Isoform of PTEN That Regulates rDNA TranscriptionNat. Commun. 8, 14771. Lim, Y.S., Hwang, S.B., 2011. Hepatitis C virus NS5A protein interacts with phosphatidylinositol 4-kinase type III alpha and regulates viral propagation. J. Biol. Chem. 286, 11290–11298. Maehama, T., Dixon, J.E., 1998. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5trisphosphate. J. Biol. Chem. 273, 13375–13378. Malaney, P., Uversky, V.N., Dave, V., 2017. PTEN proteoforms in biology and disease. Cell Mol. Life Sci. 74, 2783–2794. Masson, G.R., Perisic, O., Burke, J.E., Williams, R.L., 2016. The intrinsically disordered tails of PTEN and PTEN-L have distinct roles in regulating substrate specificity and membrane activity. Biochem. J. 473, 135–144. Messina, J.P., Humphreys, I., Flaxman, A., Brown, A., Cooke, G.S., Pybus, O.G., Barnes, E., 2015. Global distribution and prevalence of hepatitis C virus genotypes. Hepatology 61, 77–87. Mirvish, E.D., Shuda, M., 2016. Strategies for human tumor virus discoveries: from microscopic observation to digital transcriptome subtraction. Front Microbiol. 7, 676. Moriya, K., Fujie, H., Shintani, Y., Yotsuyanagi, H., Tsutsumi, T., Ishibashi, K., Matsuura, Y., Kimura, S., Miyamura, T., Koike, K., 1998. The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat. Med. 4, 1065–1067. Peyrou, M., Clement, S., Maier, C., Bourgoin, L., Branche, E., Conzelmann, S., Kaddai, V., Foti, M., Negro, F., 2013. PTEN protein phosphatase activity regulates hepatitis C virus secretion through modulation of cholesterol metabolism. J. Hepatol. 59, 420–426. Qiao, L., Wu, Q., Lu, X., Zhou, Y., Fernandez-Alvarez, A., Ye, L., Zhang, X., Han, J., Casado, M., Liu, Q., 2013. SREBP-1a activation by HBx and the effect on hepatitis B virus enhancer II/core promoter. Biochem. Biophys. Res. Commun. 432, 643–649. Scheel, T.K., Rice, C.M., 2013. Understanding the hepatitis C virus life cycle paves the way for highly effective therapies. Nat. Med. 19, 837–849. Shi, Q., Hoffman, B., Liu, Q., 2016. PI3K-Akt signaling pathway upregulates hepatitis C virus RNA translation through the activation of SREBPs. Virology 490, 99–108. Song, M.S., Salmena, L., Pandolfi, P.P., 2012. The functions and regulation of the PTEN tumour suppressor. Nat. Rev. Mol. Cell Biol. 13, 283–296. van der Meer, A.J., Berenguer, M., 2016. Reversion of disease manifestations after HCV eradication. J. Hepatol. 65, S95–S108. Wang, H., Zhang, P., Lin, C., Yu, Q., Wu, J., Wang, L., Cui, Y., Wang, K., Gao, Z., Li, H., 2015. Relevance and therapeutic possibility of PTEN-long in renal cell carcinoma. PLoS One 10, e114250. Wu, Q., Li, Z., Mellor, P., Zhou, Y., Anderson, D.H., Liu, Q., 2017. The role of PTEN – HCV core interaction in hepatitis C virus replication. Sci. Rep. 7, 3695. Zhang, S., Yu, D., 2010. PI(3)king apart PTEN's role in cancer. Clin. Cancer Res. 16, 4325–4330.
Health Research (CIHR) (FRN# 109427), Saskatchewan Health Research Foundation, and Natural Sciences and Engineering Research Council of Canada to QL. The research leading to these results has received funding from CIHR (FRN# NHC-142832) and the Public Health Agency of Canada (PHAC) in the form of a Ph.D. scholarship to QW. ZL is a recipient of a University of Saskatchewan Vaccinology and Immunotherapeutics Graduate Student scholarship. This article is published with the permission of the Director of VIDOInterVac, journal series no. 790. References Davidson, L., Maccario, H., Perera, N.M., Yang, X., Spinelli, L., Tibarewal, P., Glancy, B., Gray, A., Weijer, C.J., Downes, C.P., Leslie, N.R., 2010. Suppression of cellular proliferation and invasion by the concerted lipid and protein phosphatase activities of PTEN. Oncogene 29, 687–697. Hoffman, B., Shi, Q., Liu, Q., 2015. Arginine 112 is involved in HCV translation modulation by NS5A domain I. Biochem. Biophys. Res. Commun. 465, 95–100. Hopkins, B.D., Fine, B., Steinbach, N., Dendy, M., Rapp, Z., Shaw, J., Pappas, K., Yu, J.S., Hodakoski, C., Mense, S., Klein, J., Pegno, S., Sulis, M.L., Goldstein, H., Amendolara, B., Lei, L., Maurer, M., Bruce, J., Canoll, P., Hibshoosh, H., Parsons, R., 2013. A secreted PTEN phosphatase that enters cells to alter signaling and survival. Science 341, 399–402. Jackel-Cram, C., Babiuk, L.A., Liu, Q., 2007. Up-regulation of fatty acid synthase promoter by hepatitis C virus core protein: Genotype-3a core has a stronger effect than genotype-1b core. J. Hepatol. 46, 999–1008. Janjua, N.Z., Chong, M., Kuo, M., Woods, R., Wong, J., Yoshida, E.M., Sherman, M., Butt, Z.A., Samji, H., Cook, D., Yu, A., Alvarez, M., Tyndall, M., Krajden, M., 2017. Long-term effect of sustained virological response on hepatocellular carcinoma in patients with hepatitis C in Canada. J. Hepatol. 66, 504–513. Johnston, S.B., Raines, R.T., 2015. Catalysis by the tumor-suppressor enzymes PTEN and PTEN-L. PLoS One 10, e0116898. Jones, C.T., Murray, C.L., Eastman, D.K., Tassello, J., Rice, C.M., 2007. Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus. J. Virol. 81, 8374–8383. Kao, C.C., Yi, G., Huang, H.C., 2016. The core of hepatitis C virus pathogenesis. Curr. Opin. Virol. 17, 66–73. Leslie, N.R., Batty, I.H., Maccario, H., Davidson, L., Downes, C.P., 2008. Understanding PTEN regulation: PIP2, polarity and protein stability. Oncogene 27, 5464–5476. Liang, H., Chen, X., Yin, Q., Ruan, D., Zhao, X., Zhang, C., McNutt, M.A., Yin, Y., 2017.
8
Contents lists available at ScienceDirect
Virology journal homepage: www.elsevier.com/locate/yviro
Treatment with PTEN-Long protein inhibits hepatitis C virus replication Qi Wu
a,b,1
a,c,1
, Zhubing Li
MARK
a,b,c,⁎
, Qiang Liu
a
Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, 120 Veterinary Road, Saskatoon, Saskatchewan, Canada S7N 5E3 b Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada c School of Public Health Vaccinology and Immunotherapeutics, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
A R T I C L E I N F O
A BS T RAC T
Keywords: PTEN-Long Hepatitis C virus Viral replication Core Protein - protein interaction
Hepatitis C virus (HCV) infection is a confirmed risk factor for hepatocellular carcinoma (HCC). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) possesses tumor suppression function that is frequently defective in HCC tumors. PTEN-Long, a translation isoform of PTEN, functions in a cell non-autonomous manner. In this study, we demonstrated that intracellular overexpression of PTEN-Long inhibits HCV replication. More importantly, we showed that treatment with extracellular PTEN-Long protein inhibits HCV replication in a dose-dependent manner. Furthermore, we showed that PTEN-Long interacts with HCV core protein and this interaction is required for HCV replication inhibition by PTEN-Long. In summary, we demonstrated, for the first time, that PTEN-Long protein, an isoform of the canonical PTEN and in the form of extracellular protein treatment, inhibits HCV replication. Our study offers an opportunity for developing additional anti-HCV agents.
1. Introduction Despite the approval of anti-viral drugs, hepatitis C virus (HCV) infection continues to be a significant public health problem with hepatocellular carcinoma (HCC) as the most deadly clinical outcome (Messina et al., 2015). HCV has a single-stranded RNA genome encoding a polyprotein that is processed by cellular and viral proteases to generate structural and non-structural proteins (Scheel and Rice, 2013). Although it is a structural protein, HCV core has been reported to possess many regulatory functions including tumorigenesis (Kao et al., 2016; Moriya et al., 1998). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a dual phosphatase with lipid and protein phosphatase activities (Song et al., 2012; Leslie et al., 2008). These phosphatase activities render PTEN to function as a tumor suppressor (Song et al., 2012). PTEN has been characterized as one of the most frequently mutated or deleted genes in various tumors including HCC (Zhang and Yu, 2010; Song et al., 2012). For instance, three single amino acid mutations in the phosphatase domain have been identified: C124S (lipid and protein phosphatase defective), G129E (lipid phosphatase activity defective), and Y138L (protein phosphatase defective) (Davidson et al., 2010). As protein products translated from alternative
start codons upstream of the ATG initiation sequence of PTEN, four longer isoforms of PTEN, termed PTEN-Long/PTENα, PTEN-M/ PTENβ, PTEN-N, and PTEN-O, respectively, were discovered (Malaney et al., 2017). Whether these translation isoforms have similar or different functions in comparison to canonical PTEN has not been well characterized. PTEN-Long, the focus of the current study, has 173 N-terminal extra amino acids with a poly-arginine region. This region enables PTEN-Long to be exported into extracellular compartments, enter neighboring cells, and induce signaling events in recipient cells (Hopkins et al., 2013; Wang et al., 2015). PTEN-Long possesses comparable or even higher constitutive lipid phosphatase activity than PTEN (Masson et al., 2016; Hopkins et al., 2013; Johnston and Raines, 2015). Similar to the canonical PTEN, PTEN-Long can suppress PI3KAkt activation (Hopkins et al., 2013; Wang et al., 2015; Liang et al., 2017). Interestingly, a G to R point mutation in PTEN-Long (G302R) has the same phenotype as the corresponding G129R mutation in canonical PTEN in regulating Akt activity (Wang et al., 2015; Hopkins et al., 2013), suggesting a conserved functional role for the amino acid residues amongst different PTEN isoforms. Hence, other point mutations of PTEN, such as C124S and Y138L, have also been transferred to the corresponding residues in longer PTEN isoforms in functional studies (Liang et al., 2017).
⁎ Corresponding author at: Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, 120 Veterinary Road, Saskatoon, Saskatchewan, Canada S7N 5E3. E-mail address: [email protected] (Q. Liu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.virol.2017.08.002 Received 23 June 2017; Received in revised form 26 July 2017; Accepted 2 August 2017 0042-6822/ © 2017 Elsevier Inc. All rights reserved.
Virology 511 (2017) 1–8
Q. Wu et al.
for normalization (Qiao et al., 2013). Relative changes in RNA levels were analyzed by the 2-ΔΔct method using the iQ5 program (Bio-Rad Laboratories). The results were analyzed for statistical differences by the Student's t-test. A p value of ≤ 0.05 was considered statistically significant.
We recently demonstrated that PTEN can inhibit HCV replication through interacting with HCV core protein (Wu et al., 2017). Whether PTEN-Long affects HCV replication has not been investigated. In this study, we showed that intracellular expression of or extracellular treatment with PTEN-Long protein can inhibit HCV replication. Furthermore, we showed that PTEN-Long - HCV core protein interaction is involved in the inhibitory effect of PTEN-Long on HCV replication.
2.4. Co-immunoprecipitation (Co-IP) and Western blotting For Flag co-IP experiments, cells were harvested by radioimmunoprecipitation assay (RIPA) buffer and incubated with anti-Flag (SigmaAldrich) antibody at 4 ℃ overnight and then incubated with Protein G Sepharose (GE Healthcare) at 4 ℃ for 4 h. The mixtures were centrifuged at 10,000 rpm for 10 min and the supernatants were removed. The pellets were resuspended in SDS lysis buffer and boiled for 10 min to elute the proteins. For Western blotting, proteins were subjected to SDS-PAGE and then blotted onto nitrocellulose membranes. The membranes were blocked in 5% skim milk in PBS and incubated with a primary antibody overnight at 4 ℃. Membranes were washed and incubated with a secondary antibody for 1 h at room temperature. After a wash with PBST (PBS+0.1% Tween 20), membranes were scanned using Li-Cor Odyssey scanner (ODY-CLx) and band intensities were determined by Quantity One software (Bio-Rad Laboratories). The primary antibodies used were HCV core (Anogen), Flag (Sigma-Aldrich), PTEN and β-actin (Cell Signaling Technology), and PTEN-Long (Gly-2) provided by Ramon Parsons (Hopkins et al., 2013). The secondary antibodies used were IRDye 800CW goat antimouse IgG and IRDye 680RD goat anti-rabbit IgG (Li-Cor Biosciences).
2. Materials and methods 2.1. Plasmids and in vitro transcription Plasmids containing HCV-2a J6/JFH-1(p7-RLuc-2A) or HCV-2a J6/JFH-1(p7-RLuc-2A) ΔE1E2 sequences were provided by Charles Rice (Jones et al., 2007). Plasmid expressing Flag-PTEN was provided by Jack Dixon (Maehama and Dixon, 1998). Plasmids expressing PTEN-Long-V5-His6 (Addgene plasmid #49417) and PTEN-V5-His6 (Addgene plasmid #49420) in the bacterial JpExpress404 vector or in the pcDNA3 vector were obtained from Addgene or Ramon Parsons (Hopkins et al., 2013). Bacterial or eukaryotic expression plasmids encoding red fluorescent protein (RFP) fusion proteins with PTEN or PTEN-Long were constructed as described (Hopkins et al., 2013). Plasmids expressing PTEN-Long mutants with single amino acid substitutions C297S, G302E, or Y311L were generated in reference to the corresponding mutations of PTEN (Peyrou et al., 2013). His6-blue fluorescent protein (BFP)-expressing plasmid in the pT7 His6-SUMO vector (Lucigen) was described previously (Hoffman et al., 2015). Plasmid expressing a fusion protein between HCV-2a J6 core and enhanced green fluorescent protein (EGFP) was also constructed. All plasmids were generated using standard methods and confirmed by DNA sequencing when necessary. HCV RNA was generated by in vitro transcription using the MEGAscript T7 In Vitro Transcription reagents (Thermo Fisher Scientific).
2.5. Purification of recombinant proteins The expression of recombinant proteins in E. coli was induced by IPTG (Thermo Fisher Scientific). His6-tagged PTEN, PTEN-Long, BFP, PTEN-RFP, PTEN-Long-RFP, and RFP proteins were purified by NiNTA agarose (Qiagen) as previously described (Hoffman et al., 2015).
2.2. Cell culture, transfection, fluorescent microscopy, and HCV infection
3. Results
Huh-7 and Huh-7.5 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) fetal bovine serum (FBS). Huh-7 cells with replicating HCV-2a J6 core-Flag/JFH-1(p7-RLuc-2A) or HCV-2a J6 core R50A-Flag/JFH-1(p7-RLuc-2A) RNAs were maintained with culture medium plus G418 (Enzo Life Sciences) as described previously (Wu et al., 2017). Cells were transfected with plasmid DNA or HCV RNA using the calcium phosphate precipitation method (Jackel-Cram et al., 2007) or the jet-PEI transfection reagent (Polyplus Transfection) (Shi et al., 2016). Cells transfected with fluorescent protein-expressing plasmids were visualized with appropriate settings under a Leica TCS SP5 confocal microscope (Leica Microsystems). HCV-2a JFH-1 rLuc subgenomic replicon cells and infection with HCV-2a J6 core-Flag/JFH-1(p7-RLuc-2A) virus were described previously (Wu et al., 2017).
3.1. Intracellular PTEN-Long inhibits HCV replication We recently showed that ectopic expression of PTEN inhibits HCV replication (Wu et al., 2017). Whether PTEN-Long, a longer isoform of PTEN, can also regulate HCV replication has not been studied. To investigate the effect of PTEN-Long on HCV replication, we transfected Huh-7 cells harboring HCV-2a J6 core-Flag/JFH-1(p7-rLuc-2A) replicating RNA with vector, plasmids expressing PTEN or PTEN-Long and measured HCV replication by luciferase assay. We also used a set of plasmids expressing fusion proteins with RFP, which will enable subsequent experiments. Consistent with our previous results, overexpression of PTEN significantly inhibited HCV replication in comparison to vector (Fig. 1A). Expression of the fusion protein PTEN-RFP also inhibited HCV replication as effectively as PTEN itself, while RFP expression had no effect (Fig. 1A). Overexpression of PTEN-Long or PTEN-Long-RFP could reduce HCV replication to the same extent as PTEN (Fig. 1A). The intracellular expression of PTEN and PTEN-Long proteins was demonstrated by Western blotting using a PTEN-specific antibody (Fig. 1B). It is interesting to point out that while the endogenous PTEN protein could be readily detected in the Huh-7 cells as shown in Fig. 1B, the endogenous PTEN-Long protein was not visible in Western blotting using a PTEN-Long-specific antibody (Gly2) (Hopkins et al., 2013) (data not shown). These results indicated that both PTEN and PTEN-Long can inhibit HCV replication when expressed intracellularly. Because Huh-7 cells harboring HCV-2a J6 /JFH-1(p7-rLuc-2A) or HCV-2a J6 core-Flag/JFH-1(p7-rLuc-2A) replicating RNA have been shown to produce infectious virus particles (Jones et al., 2007; Wu
2.3. Luciferase and RT-qPCR assays Cells were lysed in Passive Lysis Buffer (Promega) and the firefly or renilla luciferase activities were measured by Luciferase Assay reagents (Promega) in a TD 20/20 Luminometer (Turner Designs). Luciferase levels were normalized to the protein concentrations determined by the Bradford assay (Bio-Rad Laboratories). To determine the HCV RNA levels, total RNA was extracted by Trizol (Thermo Fisher Scientific) and reverse transcribed into cDNA by SuperScript II Reverse Transcriptase (Thermo Fisher Scientific). Real time PCR was performed using primers HCV-FD (5`-AGAGCCATAGTGGTCTGCGGAAC-3`) and HCV-rev (5`-CCTTTCGCAACCCAACGCTACTC-3`) (Lim and Hwang, 2011). The transcript levels of GUSB, a house keeping gene, were used 2
Virology 511 (2017) 1–8
Q. Wu et al.
Fig. 1. Intracellular expression of PTEN-Long inhibits HCV replication. (A and B) Huh-7 cells harboring HCV-2a J6/JFH-1(p7-rLuc-2A) replicating RNA were transfected with plasmid vector, or plasmids expressing RFP (red fluorescent protein), PTEN-RFP, PTEN-Long-RFP (PTEN-L-RFP), PTEN, or PTEN-Long (PTEN-L). All proteins have a C-terminal V5-His6 tag. (A) At 48 h after transfection, luciferase assay was performed and the statistical differences between samples were demonstrated as ** if p ≤ 0.01, or NS for not significant. (B) The expression of PTEN and PTEN-Long proteins was demonstrated by Western blotting using an anti-PTEN antibody. The levels of β-actin were also determined as loading controls. (C and D) Huh-7.5 cells were co-transfected with HCV ΔE1E2 or HCV full-length genomic RNA together with plasmid vector or a plasmid expressing PTEN-Long with a C-terminal V5-His6 tag. (C) At 48 h after transfection, luciferase assay was performed and the statistical differences between samples were demonstrated as * if p ≤ 0.05. (D) The expression of PTEN-Long protein was demonstrated by Western blotting using an anti-V5 antibody. The levels of β-actin were also determined as loading controls. (E and F) Huh-7.5 cells were transfected with plasmid vector or plasmid expressing PTEN-Long. At 48 h after transfection, cells were infected with a cell culture derived HCV-2a J6 core-Flag/JFH-1(p7-RLuc-2A) virus. At 72 h after infection, luciferase activity (C) and HCV RNA levels (D) were determined. * = p ≤ 0.05.
deleted (Jones et al., 2007) in a co-transfection together with plasmid vector or a plasmid expressing PTEN-Long. We also used the wild-type HCV RNA as a control. In both cases, PTEN-Long expression resulted in significantly lower RLuc activities than vector control (Fig. 1C). The
et al., 2017), HCV RNA levels measured at 48 h after transfection with PTEN-Long expressing plasmid may not only reflect the degree of HCV replication. To explicitly determine the effect of PTEN-Long on HCV replication, we used an HCV genomic RLuc RNA with the E1 and E2
3
Virology 511 (2017) 1–8
Q. Wu et al.
and used to infect naïve Huh-7.5 cells. At 72 h after infection, luciferase activity was determined. As shown in Fig. 2F, significantly lower luciferase activity was detected after PTEN-Long-RFP treatment in comparison to RFP. These results indicated that extracellular PTENLong treatment inhibits HCV propagation. Taken together, these results demonstrated that extracellular PTEN-Long protein can enter the cells and inhibit HCV replication.
expression of PTEN-Long protein was demonstrated by Western blotting (Fig. 1D). These results indicated that intracellular PTENLong can inhibit HCV replication. To determine whether PTEN-Long regulates HCV RNA replication after infection, Huh-7.5 cells transfected with plasmid vector or plasmid expressing PTEN-Long were infected with a cell culture derived HCV-2a J6 core-Flag/JFH-1(p7-RLuc-2A) virus. HCV replication was analyzed by luciferase assay (for luciferase activity) and RTqPCR (for HCV RNA level). Results of both assays demonstrated a significantly lower replication after PTEN-Long expression in comparison to control (Figs. 1E and F). These results indicated that intracellular PTEN-Long can inhibit HCV replication after viral infection.
3.3. PTEN-Long interacts and co-localizes with HCV core We recently showed that HCV core protein interacts with PTEN through the arginine residue at position 50 (R50) and the interaction is required for the inhibitory effect of PTEN on HCV replication (Wu et al., 2017). To determine whether HCV core also interacts with PTEN-Long and the role of R50, we performed Flag co-immunoprecipitation experiment after transfecting PTEN-Long expressing plasmid into HCV-2a J6 core-Flag/JFH-1(p7-rLuc-2A) and HCV-2a J6 core R50A-Flag/JFH-1(p7-rLuc-2A) replicon cells. As shown in Fig. 3A, PTEN-Long was detected in the immunoprecipitates using lysates containing wild-type, but not R50A, HCV core. Since PTEN is translated (internally) from the PTEN-Long coding sequence, PTEN protein was also present/absent in the immunoprecipitates (Fig. 3A). These results indicated that HCV core interacts with PTEN-Long and requires R50. To determine whether HCV core and PTEN-Long proteins colocalize in the cell, Huh-7 cells were co-transfected with plasmids expressing fluorescent fusion proteins HCV core-EGFP and PTENLong-RFP. Plasmids expressing EGFP, RFP, or PTEN-RFP were included for control purposes. Consistent with the literature (Liang et al., 2017), confocal microscopic images in Fig. 3B showed mostly cytoplasmic localization of PTEN-Long and both nuclear and cytoplasmic distribution of PTEN. Substantial co-localization of HCV core with both PTEN and PTEN-Long could be observed (Fig. 3B). Protein expression was demonstrated by Western blotting (Fig. 3C).
3.2. Extracellular PTEN-Long protein treatment inhibits HCV replication In comparison to the canonical PTEN, a unique feature of PTENLong lies in its ability to enter cells as a secreted protein (Hopkins et al., 2013). We therefore were interested in determining whether PTEN-Long as an extracellular protein regulates HCV replication. To this end, we purified His6-tagged PTEN and PTEN-Long proteins with or without fusion with RFP as previously described (Hopkins et al., 2013). Huh-7 cells harboring HCV-2a J6 core-Flag/JFH-1(p7-RLuc2A) replicating RNA were treated with increasing amounts of PTEN or PTEN-Long protein and HCV replication quantified. PBS and BFP protein were used as controls. As shown in Fig. 2A, while HCV replication was not affected by treatment with PBS, BFP, or PTEN proteins in three escalating doses, HCV replication was reduced upon PTEN-Long protein treatment in a dose-dependent manner. These results indicated that extracellular PTEN-Long protein can inhibit HCV replication. We previously showed that the lipid phosphatase deficient G129E mutant of PTEN can inhibit HCV replication as effectively as the wildtype PTEN, whereas the protein phosphatase deficient Y138L and the lipid and protein phosphatase deficient C124S mutants have no effect (Wu et al., 2017). Next, we wanted to determine whether these mutations in PTEN-Long as an extracellular protein will have a similar effect. We therefore transferred these point mutations of PTEN to the corresponding positions in PTEN-Long and purified these proteins for use in the treatment. Fig. 2B showed that while PTEN-Long proteins with C297S and Y311L mutations no longer inhibited HCV replication, the G302E mutant protein had the same inhibitory effect as the wildtype PTEN-Long. These results indicated that the functions of the three amino acids are conserved between PTEN and PTEN-Long in inhibiting HCV replication. To confirm that PTEN-Long but not PTEN protein can enter the cells, we fused PTEN-Long or PTEN with RFP and detected red fluorescence after treatment. As shown in Fig. 2C, red fluorescence was observed after treatment with PTEN-Long-RFP in more than 60% of the cells (50 positive cells out of 80 in total), but not with PTEN-RFP or RFP, indicating that only extracellular PTEN-Long protein can enter the cells whereas PTEN-RFP or RFP cannot. Fusion with RFP did not affect the function of PTEN and PTEN-Long in regulating HCV replication (compare Figs. 2D and 1A). To rule out the possibility that the changes in Renilla luciferase activity observed after PTEN-Long protein treatment was due to an effect of PTEN-Long on the Renilla luciferase itself, we measured HCV RNA levels by RT-qPCR. As shown in Fig. 2E, treatment with PTENLong-RFP protein resulted in a significantly lower HCV RNA level than RFP treatment. These results indicated that PTEN-Long protein indeed regulates HCV RNA replication. To determine whether extracellular treatment with PTEN-Long downregulates HCV propagation, Huh-7 cells harboring HCV-2a J6 core-Flag/JFH-1(p7-RLuc-2A) replicating RNA were once again treated with RFP or PTEN-Long proteins. The supernatants containing infectious HCV reporter viruses were collected at 24 h after treatment
3.4. PTEN-Long inhibits HCV replication through interaction with HCV core Next, we wanted to investigate the role of HCV core - PTEN-Long interaction in regulating HCV replication. HCV-2a J6 core R50A-Flag/ JFH-1(p7-rLuc-2A) replicating Huh-7 cells were treated with 24 nM of BFP, PTEN, PTEN-Long and phosphatase mutant proteins, and PBS before HCV replication measured by luciferase assay for luciferase activity (Fig. 4A) or RT-qPCR assay for HCV RNA levels (Fig. 4B). No effects on HCV replication were observed. These results indicated that PTEN-Long interacts with HCV core, which is required for HCV replication inhibition. To corroborate these findings, we treated HCV rLuc reporter subgenomic replicon cells that do not express HCV structural proteins with PTEN-Long-RFP protein or RFP protein as a control. When HCV replication was measured by both luciferase and RT-qPCR assays, we found that PTEN-Long could no longer inhibit, but instead increased, HCV RNA replication in comparison to RFP treatment (Figs. 4C and D). These results once again indicated that the inhibition of HCV replication by PTEN-Long requires the core protein. 4. Discussion Viruses are the etiologic agents for more than 10% of tumors in human (Mirvish and Shuda, 2016). However, the mutual interactions between PTEN-Long and oncogenic viruses have yet to be characterized. As an extension to our recent research on the effect of the canonical PTEN on HCV replication (Wu et al., 2017), we characterized the role of PTEN-Long in this study. We showed that intracellular expression of PTEN-Long could inhibit HCV replication as effectively as the PTEN protein (Fig. 1). More importantly, we found that HCV 4
Virology 511 (2017) 1–8
Q. Wu et al.
Fig. 2. Extracellular treatment with PTEN-Long protein inhibits HCV replication. Huh-7 cells with HCV-2a J6/JFH-1(p7-rLuc-2A) replicating RNA were treated with BFP, RFP, PTEN, PTEN-Long, PTEN-RFP, or PTEN-Long-RFP proteins or equal volume of PBS (phosphate-buffered saline). All proteins had a His6-tag and were purified from E. coli through affinity chromatography. In (A), 1.5, 6, and 24 nM of PTEN or PTEN-Long proteins and 24 nM of BFP protein were used. In (B, C, D and E), cells were treated with 24 nM of proteins. At 24 h after treatment, luciferase (A, B and D) or RT-qPCR (E) assays were performed to determine HCV replication. The statistical differences between samples were demonstrated as * if p ≤ 0.05, ** if p ≤ 0.01, *** if p ≤ 0.001, or NS for not significant. In (C), cells were stained with DAPI at 6 h after treatment. Images were collected by confocal microscopy and analyzed by the Image J software. In (F), the supernatants containing infectious HCV reporter viruses were collected at 24 h after treatment with RFP or PTEN-Long-RFP proteins and used to infect naïve Huh-7.5 cells. At 72 h after infection, luciferase assay was performed. ** = p ≤ 0.01.
5
Virology 511 (2017) 1–8
Q. Wu et al.
Fig. 3. PTEN-Long interacts and co-localizes with HCV core. (A) Huh-7 cells harboring HCV-2a J6 core-Flag/JFH-1(p7-rLuc-2A) or HCV-2a J6 core R50A-Flag/JFH-1(p7-rLuc-2A) replicating RNAs were transfected with plasmid expressing PTEN-Long. Forty eight hours after transfection, cell lysates were subjected to co-immunoprecipitation (co-IP) using an antiFlag antibody. Input and co-IP products were analyzed by Western blotting (WB) using anti-PTEN and anti-HCV core antibodies, respectively. (B and C) Huh-7 cells were co-transfected with plasmids expressing EGFP, HCV core-EGFP, RFP, PTEN-RFP, or PTEN-Long-RFP as indicated. At 48 h after transfection, cells were stained with DAPI and observed under a confocal microscope with appropriate settings (B). Co-localization sites were indicated by a square. Protein expression was demonstrated in Western blotting (C).
replication was inhibited by incubation with purified PTEN-Long protein in a dose-dependent manner (Fig. 2). This inhibition is due to the permeability feature of the PTEN-Long protein because we
showed that PTEN-Long in the form of an extracellular protein, but not the PTEN protein, can enter HCV replicating cells (Fig. 2). As for the mechanisms, we found that the C297S and Y311L 6
Virology 511 (2017) 1–8
Q. Wu et al.
Fig. 4. PTEN-Long protein inhibits HCV replication by interacting with HCV core. (A and B) Huh-7 cells harboring HCV-2a J6 core R50A-Flag /JFH-1(p7-rLuc-2A) replicating RNA were treated with 24 nM of BFP, PTEN, PTEN-Long proteins or equal volume of PBS. Luciferase (A) or RT-qPCR (B) assays were performed at 24 h after treatment to determine HCV RNA replication. The statistical differences between samples were demonstrated as NS for not significant. (C and D) Huh-7 cells harboring HCV-2a JFH-1 subgenomic replicating RNA were treated with 24 nM of RFP or PTEN-Long-RFP proteins. Luciferase (C) or RT-qPCR (D) assays were performed at 24 h after treatment to determine HCV RNA replication. * = p ≤ 0.05.
consideration, these results suggest that PTEN-Long, once present inside the cell, very likely exerts its inhibitory effects on the replication of HCV full genomic RNA through similar mechanisms as the canonical PTEN. However, given the fact that the canonical PTEN has no effect on the replication of subgenomic HCV RNA (Wu et al., 2017), PTENLong may have additional mechanisms to regulate HCV RNA replication in the absence of the core protein. In conclusion, we demonstrated that PTEN-Long inhibits HCV replication. To the best of our knowledge, this is the first report on characterizing the effect of PTEN-Long on a viral replication. In addition, although direct-acting antivirals (DAAs) are very effective in achieving sustained virological responses in hepatitis C patients, the long-term effects of this therapy on liver diseases, especially hepatocellular carcinoma (HCC), in these patients are controversial and remain to be characterized (Janjua et al., 2017; van der Meer and Berenguer, 2016). The fact that PTEN-Long exerts its inhibitory function on HCV replication (this study) and on tumor growth (Hopkins et al., 2013) in the form of an extracellular protein should provide an additional avenue for developing therapeutics for HCV infection and the resultant HCC.
mutants of PTEN-Long do not inhibit HCV replication (Fig. 2), suggesting the involvement of these two amino acid residues of PTEN-Long in this process. The corresponding amino acid residues in canonical PTEN, namely C124 and Y138, are both involved in regulating the protein phosphatase activity of PTEN (Davidson et al., 2010). If the functions of these amino acids are conserved, our data would suggest that the protein phosphatase activity of PTEN-Long is required for inhibiting HCV replication. This is consistent with a previous study where the protein phosphatase activity was found to be involved in regulating HCV egress (Peyrou et al., 2013). It has been shown that the protein phosphatase mutant of PTEN induced larger lipid droplets and increased cholesterol esters biosynthesis (Peyrou et al., 2013). Furthermore, we showed an interaction and co-localization between PTEN-Long and HCV core proteins (Fig. 3). We also found that the arginine residue at position 50 of the HCV core protein is critical for the interaction and PTEN-Long could no longer inhibit HCV RNA replication in the absence of this R50 residue, suggesting a role for protein interaction (Figs. 4A and B). In an effort to further confirm these findings, we used an HCV subgenomic replicon that does not express the structural proteins, including core. PTEN-Long indeed could not inhibit the replication of the subgenomic HCV RNA. Somewhat to our surprise, we found that PTEN-Long could increase HCV subgenomic RNA replication (Figs. 4C and D). Taking our recent study on how PTEN inhibits HCV replication (Wu et al., 2017) into
Acknowledgements This work was supported by grants from Canadian Institutes of 7
Virology 511 (2017) 1–8
Q. Wu et al.
PTENbeta is an Alternatively Translated Isoform of PTEN That Regulates rDNA TranscriptionNat. Commun. 8, 14771. Lim, Y.S., Hwang, S.B., 2011. Hepatitis C virus NS5A protein interacts with phosphatidylinositol 4-kinase type III alpha and regulates viral propagation. J. Biol. Chem. 286, 11290–11298. Maehama, T., Dixon, J.E., 1998. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5trisphosphate. J. Biol. Chem. 273, 13375–13378. Malaney, P., Uversky, V.N., Dave, V., 2017. PTEN proteoforms in biology and disease. Cell Mol. Life Sci. 74, 2783–2794. Masson, G.R., Perisic, O., Burke, J.E., Williams, R.L., 2016. The intrinsically disordered tails of PTEN and PTEN-L have distinct roles in regulating substrate specificity and membrane activity. Biochem. J. 473, 135–144. Messina, J.P., Humphreys, I., Flaxman, A., Brown, A., Cooke, G.S., Pybus, O.G., Barnes, E., 2015. Global distribution and prevalence of hepatitis C virus genotypes. Hepatology 61, 77–87. Mirvish, E.D., Shuda, M., 2016. Strategies for human tumor virus discoveries: from microscopic observation to digital transcriptome subtraction. Front Microbiol. 7, 676. Moriya, K., Fujie, H., Shintani, Y., Yotsuyanagi, H., Tsutsumi, T., Ishibashi, K., Matsuura, Y., Kimura, S., Miyamura, T., Koike, K., 1998. The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat. Med. 4, 1065–1067. Peyrou, M., Clement, S., Maier, C., Bourgoin, L., Branche, E., Conzelmann, S., Kaddai, V., Foti, M., Negro, F., 2013. PTEN protein phosphatase activity regulates hepatitis C virus secretion through modulation of cholesterol metabolism. J. Hepatol. 59, 420–426. Qiao, L., Wu, Q., Lu, X., Zhou, Y., Fernandez-Alvarez, A., Ye, L., Zhang, X., Han, J., Casado, M., Liu, Q., 2013. SREBP-1a activation by HBx and the effect on hepatitis B virus enhancer II/core promoter. Biochem. Biophys. Res. Commun. 432, 643–649. Scheel, T.K., Rice, C.M., 2013. Understanding the hepatitis C virus life cycle paves the way for highly effective therapies. Nat. Med. 19, 837–849. Shi, Q., Hoffman, B., Liu, Q., 2016. PI3K-Akt signaling pathway upregulates hepatitis C virus RNA translation through the activation of SREBPs. Virology 490, 99–108. Song, M.S., Salmena, L., Pandolfi, P.P., 2012. The functions and regulation of the PTEN tumour suppressor. Nat. Rev. Mol. Cell Biol. 13, 283–296. van der Meer, A.J., Berenguer, M., 2016. Reversion of disease manifestations after HCV eradication. J. Hepatol. 65, S95–S108. Wang, H., Zhang, P., Lin, C., Yu, Q., Wu, J., Wang, L., Cui, Y., Wang, K., Gao, Z., Li, H., 2015. Relevance and therapeutic possibility of PTEN-long in renal cell carcinoma. PLoS One 10, e114250. Wu, Q., Li, Z., Mellor, P., Zhou, Y., Anderson, D.H., Liu, Q., 2017. The role of PTEN – HCV core interaction in hepatitis C virus replication. Sci. Rep. 7, 3695. Zhang, S., Yu, D., 2010. PI(3)king apart PTEN's role in cancer. Clin. Cancer Res. 16, 4325–4330.
Health Research (CIHR) (FRN# 109427), Saskatchewan Health Research Foundation, and Natural Sciences and Engineering Research Council of Canada to QL. The research leading to these results has received funding from CIHR (FRN# NHC-142832) and the Public Health Agency of Canada (PHAC) in the form of a Ph.D. scholarship to QW. ZL is a recipient of a University of Saskatchewan Vaccinology and Immunotherapeutics Graduate Student scholarship. This article is published with the permission of the Director of VIDOInterVac, journal series no. 790. References Davidson, L., Maccario, H., Perera, N.M., Yang, X., Spinelli, L., Tibarewal, P., Glancy, B., Gray, A., Weijer, C.J., Downes, C.P., Leslie, N.R., 2010. Suppression of cellular proliferation and invasion by the concerted lipid and protein phosphatase activities of PTEN. Oncogene 29, 687–697. Hoffman, B., Shi, Q., Liu, Q., 2015. Arginine 112 is involved in HCV translation modulation by NS5A domain I. Biochem. Biophys. Res. Commun. 465, 95–100. Hopkins, B.D., Fine, B., Steinbach, N., Dendy, M., Rapp, Z., Shaw, J., Pappas, K., Yu, J.S., Hodakoski, C., Mense, S., Klein, J., Pegno, S., Sulis, M.L., Goldstein, H., Amendolara, B., Lei, L., Maurer, M., Bruce, J., Canoll, P., Hibshoosh, H., Parsons, R., 2013. A secreted PTEN phosphatase that enters cells to alter signaling and survival. Science 341, 399–402. Jackel-Cram, C., Babiuk, L.A., Liu, Q., 2007. Up-regulation of fatty acid synthase promoter by hepatitis C virus core protein: Genotype-3a core has a stronger effect than genotype-1b core. J. Hepatol. 46, 999–1008. Janjua, N.Z., Chong, M., Kuo, M., Woods, R., Wong, J., Yoshida, E.M., Sherman, M., Butt, Z.A., Samji, H., Cook, D., Yu, A., Alvarez, M., Tyndall, M., Krajden, M., 2017. Long-term effect of sustained virological response on hepatocellular carcinoma in patients with hepatitis C in Canada. J. Hepatol. 66, 504–513. Johnston, S.B., Raines, R.T., 2015. Catalysis by the tumor-suppressor enzymes PTEN and PTEN-L. PLoS One 10, e0116898. Jones, C.T., Murray, C.L., Eastman, D.K., Tassello, J., Rice, C.M., 2007. Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus. J. Virol. 81, 8374–8383. Kao, C.C., Yi, G., Huang, H.C., 2016. The core of hepatitis C virus pathogenesis. Curr. Opin. Virol. 17, 66–73. Leslie, N.R., Batty, I.H., Maccario, H., Davidson, L., Downes, C.P., 2008. Understanding PTEN regulation: PIP2, polarity and protein stability. Oncogene 27, 5464–5476. Liang, H., Chen, X., Yin, Q., Ruan, D., Zhao, X., Zhang, C., McNutt, M.A., Yin, Y., 2017.
8