Kaohsiung Journal of Medical Sciences (2016) 32, 487e493

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ORIGINAL ARTICLE Distinct subpopulations of hepatitis C virus infectious cells with different levels of intracellular hepatitis C virus core

Shu-Chi Wang a,b, Jeng-Fu Yang a, Chao-Ling Wang a,b, Chung-Feng Huang a,b,c,d, Yu-Yin Lin a,b, Yi-You Chen a, Chung-Ting Lo a,b, Po-Yen Lee a, Kuan-Ta Wu a, Chia-I. Lin a,b, Meng-Hsuan Hsieh a,b, Hung-Yi Chuang a,b, Chi-Kung Ho a,b, Ming-Lung Yu b,c,d,e, Chia-Yen Dai a,b,c,d,e,* a Health Management Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan b Department of Occupational and Environmental Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan c Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan d Faculty of Internal Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan e Center for Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung, Taiwan

Received 27 June 2016; accepted 28 July 2016 Available online 14 September 2016

KEYWORDS Abstract Chronic infection by hepatitis C virus (HCV) is a major risk factor for the develop- Core protein; ment of hepatocellular carcinoma (HCC). Despite the clear clinical importance of virus- Fluorescence- associated HCC, the underlying molecular mechanisms remain largely unclarified. Oxidative activated cell sorting; stress, in particular, DNA lesions associated with oxidative damage, plays a major role in carci- Hepatitis C virus; nogenesis, and is strongly linked to the development of many cancers, including HCC. Howev- Quantitative er, in identifying hepatocytes with HCV viral RNA, estimates of the median proportion of HCV- polymerase chain infected hepatocytes have been found as high as 40% in patients with chronic HCV infection. In reaction array order to explore the alternation and association between different viral loads of HCV- infected cells, we established a method to dissect high and low viral load cells and examined the expression of DNA damage-related using a quantitative polymerase chain reaction array. We found distinct expression patterns of DNA damage-related genes between high

Conflict of interest: All authors declare no conflicts of interest. * Corresponding author. Health Management Center, Kaohsiung Medical University Hospital Kaohsiung Medical University, 100 Tzyou Road, Kaohsiung City 807, Taiwan. E-mail address: [email protected] (C.-Y. Dai). http://dx.doi.org/10.1016/j.kjms.2016.08.002 1607-551X/Copyright ª 2016, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 488 S.-C. Wang et al.

and low viral load cells. This study provides a new method for future study on virus-associated research. Copyright ª 2016, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Introduction Huh-7.5 cells as described previously [9]. The JFH-1 HCVcc particles were incubated with Huh-7.5.1 cells for 6 hours, Hepatitis C virus (HCV) is an important human pathogen, and and then the infection medium was removed and changed the leading infectious cause of hepatic cirrhosis and liver with fresh regular culture medium. The infection pro- cancer in many countries. Suppression of DNA repair mecha- ceeded for 3 days before harvesting for experimental nisms and the induction of DNA breaks caused by viral analysis [11]. Reverse transcription PCR amplification of sense (50-TGAGGAACTACTGTCTTCACG-30) and antisense may enhance the mutation frequency and rear- 0 0 rangements in virus-infected cells that have accumulated (5 -ACTCGCAAGCACCCTATCAGG-3 ) strands were tested quantitatively by real-time PCR. The HCV oligonucleotides genetic alterations and cause hepatocellular carcinoma 0 (HCC). HCV replicates in the cytoplasm and results in mito- were specific for the 5 untranslated region of the HCV chondrial and oxidative injury in liver tissue [1]. HCC may genome. originate from dividing hepatocytes or those that have accu- mulated genetic alterations [2]. The mechanism of HCV Measurement of ROS oncogenesis remains largely unclear. Patients with chronic hepatitis C have more than an 80% chance of developing The amount of ROS generated was estimated by 2,7- chronic diseases compared with patients with hepatitis A, B, dichlorofluorescin diacetate (DCFH-DA). Then, 10mM and E [3]. An increase in the amount of reactive oxygen species DCFH-DA was added to the cell culture medium and incu- (ROS) by two to five orders of magnitude in liver tissue from bated for 15 minutes, and images were captured on a chronic hepatitis C patients have also been reported [4,5]. Nikon Eclipse fluorescence microscope with Spot digital In situ molecular characterization of HCV-infected human camera (Tokyo, Japan) and processed using Adobe Photo- hepatocytes from the liver was performed by Kandathil et al shop 11.0 software (San Jose, CA, USA). Intracellular and [6] to identify hepatocytes with HCV viral RNA and estimated extracellular ROS levels were used to determine the fluo- the median proportion of HCV-infected hepatocytes as 40%. rescence intensity of each well immediately, using a More recently, 2-photon excitation of Q-dot probes was used to microplate fluorescence spectrometer (Thermo Scientific detect hepatic HCV antigens and RNA, suggesting that Multiskan GO, Waltham, MA, USA) reader to set emission of 1.7e21.6% of hepatocytes are infected [6]. In addition, HCV 520 nm. infection affects the ability of the cell to repair DNA damage and increases the frequency of gross chromosomal rear- rangements [7,8]. To shed light on the relationships between Immunofluorescence cell staining viral load and DNA damage of HCV infectious cells, which is important for clarifying the HCV-related carcinogenesis Cells (5 104) were seeded into six-well plates containing mechanism, in the present study, we set a protocol to dissect coverslips coated with 0.1 mg/mL poly-D-lysine (Sigma, St. HCV JFH-1cc particles [9] by different viral load cell pop- Louis, MO, USA) and allowed to attach at 37C for 24 hours ulations in vitro. By using this protocol, we explored the before JFH-1 viral infection. After infection for 3 days, cells distinct DNA damage-related gene expression between cells were washed twice with ice-cold 1 phosphate-buffered with high and low viral loads with quantitative polymerase saline (PBS), fixed with cold 100% methanol for 10 mi- chain reaction (q-PCR) array. nutes at 20C and incubated in blocking buffer [2% bovine serum albumin (BSA) in 1 PBS] for 30 minutes at 37C. Methods Cells were incubated with primary rabbit HCV core mono- clonal antibody (Thermo) diluted 1:100 for 1e2 hours at room temperature. Cells were washed twice with 1 PBS Cell culture and incubated with fluorescein-isothiocyanate (FITC)-con- jugated secondary antibody diluted 1:100 (Sigma) for Human hepatoma Huh-7.5.1 cells were cultured in Dul- 2 hours at room temperature. The slides were washed three becco’s modified Eagle’s medium with 10% heat-inactivated times with 1 PBS and counterstained with 40,6-diamidino- e fetal bovine serum, 5% antibiotic antimycotic, and 5% 2-phenylindole (20 mg/mL) for 15 minutes at room tem- nonessential amino acids [10]. perature. After washing, the slides were mounted onto Colorfrost Plus slides (Fisher Scientific, Waltham, MA, USA) HCV JFH-1 infection assay using VectaShield Hardset (Vector Laboratories, Burlin- game, CA, USA). Images were captured on a Nikon Eclipse The infectious HCV JFH-1 particles were generated by fluorescence microscope with Spot digital camera and transfection of in vitro-transcribed genomic JFH-1 RNA in processed using Adobe Photoshop 11.0 software. Subpopulations of HCV infectious cells 489

Flow cytometric analysis HCV viral load detection

Cells (105) were fixed by 4% Paraformaldehyde (PFA) for Sorted cells were suspended in supernatant and the cell 20 minutes at room temperature and permeabilized by 0.5% HCV RNA was determined by qualitative PCR (Cobas Triton-X100 for 10 minutes. Cells were blocked in 5% normal Amplicor Hepatitis C Virus Test, V.2.0; Roche Diagnostics, goat serum in PBS for 15 minutes at room temperature, Branchburg, NJ, USA; detection limit: 50 IU/mL). followed by incubation with human monoclonal FITC- conjugated HCV core antibody in 1% BSA with 2.5% goat q-PCR array serum for 45 minutes on ice. Cells were washed with 1% BSA and analyzed by fluorescence cell-activated sorting (FACS). The complementary DNA (cDNA) was synthesized using 1-mg m Cells were filtered through a 30- m nylon mesh before final RNA and the High-capacity cDNA Reverse Transcription kit centrifugation, and resuspended in PBS containing 1% fetal (Applied Biosystems, Foster City, CA, USA). To assay the bovine serum for sorting. oxidative DNA damage signaling pathway, the q-PCR array used RT2 Profiler PCR Array profiles (Qiagen, Valencia, CA, USA) for the expression of 84 genes involved in DNA damage- Distinguishing high and low viral loads cells by FACS signaling pathways. The PCR array is a set of optimized real- time PCR primers on 96-well plates. One microliter of 10-fold Huh-7.5.1 cells were infected with JFH-1 particles (HCV cc) dilution cDNA template was added to the ready-to-use Power for 6 hours and then fresh Dulbecco’s modified Eagle’s SYBR Green PCR Master Mix (Thermo Fisher), then equal medium was exchanged for 72 hours before harvesting for volumes were aliquoted to each well of the same plate, and the experimental analysis. The JFH-1-infected cells were then the real-time PCR cycling program by ABI Prism 7900 fast labeled with FITC using HCV core antibody (Clone C7-50, detection system was run. The housekeeping genes were used MA1-080; Thermo Fisher, Waltham, MA, USA). The JFH-1- to normalize the data before the amplified transcripts were infected cells were detected in the FL1 channel with un- analyzed by the threshold cycle (Ct) method using ABI 7900 infected Huh-7.5.1 cells used as the control. The low viral system software (Applied Biosystems, Foster City, CA, USA). load cells were gated at lowest FITC intensity of 20% pop- ulation and high viral load cells were gated at highest FITC Statistical analysis intensity of 20% population of total infected cells by his- togram. After sorting, fluorescence intensity of HCV core The statistical analysis was performed using Student t test protein was used to confirm the viral load levels and the significant difference was set at p < 0.05 or immediately. p < 0.01.

Figure 1. Reactive oxygen species (ROS) increasing at 48 hours after JFH-1 infection. (A) Cell light and fluorescence microscopy image with the ROS fluorescent probe, 2,7-dichlorofluorescin diacetate (10mM) on Huh-7.5.1 cells or JFH-1 at 48 hours postinfection in six-well plates; (B) quantification of ROS fluorescence intensity by emission in 520 nm. 490 S.-C. Wang et al.

Results

ROS level in JFH-1 infectious cells

To confirm that ROS generation was associated with viral infection, we used ROS indicator DCFH-DA to detect intra- cellular ROS level in JFH-1-infected cells and uninfected Huh-7.5.1 cells. We cultured 106 cells in six-well plates for infection. After 6-hours infection, viral supernatant was removed and fresh culture medium was changed for 48 hours. Following usage of a DCFH-DA fluorescence indi- cator, we confirmed by cell imaging that JFH-1-infected cells generated high ROS levels (Figure 1A). In addition to intracellular ROS generation, we found that JFH-1-infected cells also generated high levels of ROS in culture medium. The quantification result presented six-fold ROS generation (p < 0.01) in JFH-1-infected cells (Figure 1B). These ROS data validated that both intracellular and extracellular ROS generation occurred by HCV viral infection.

Protocol for dissecting high and low viral load cells by FACS

To study the viral load effects, we established a protocol to dissect two populations of cells by intracellular high and low viral load. The protocol schema is shown in Figure 2. Cells were collected from cells transfected with JFH-1 and inoculated into uninfected Huh-7.5.1 cells for 3 days. The cells were conjugated with HCV core antibody by the intracellular staining method. The HCV-core-positive cells were directly analyzed via HCV-core-conjugated fluores- Figure 2. Schematic representation of fluorescence- cence intensity. The cytometry histogram showed that two activated cell sorting by hepatitis C virus (HCV) core protein populations of HCV core protein fluorescence intensity on staining. The intracellular HCV core florescence intensity of Day 3 post-infection. To sort high and low viral load cells, the infected cells was immediately analyzed by flow cytom- we determined the lowest core-intensity of 20% population etry. The histogram parameter displaying FL1-fluorescein iso- for HCV-low infected cells and highest core-intensity of 20% thiocyanate (FITC) lasers on the x axis and cell numbers on the population for HCV-high infected cells by histogram. Sorted y axis. For the cell sorting, we gated the lowest 20% of the FL1 cells were collected in fetal bovine serum and the HCV core fluorescent intensity for the low-viral load cells and gated the intensity was confirmed immediately. highest 20% of the FL1 fluorescent intensity for the high-viral load cells. Cell were sorted in tubes containing fetal bovine serum. Different viral load cell population on JFH-1 infectious in vitro mode Alteration of DNA-damage-related genes between high- and low-viral load cell populations After Day 3 post-JFH-1 infection, immunocytochemistry by FITC-conjugated HCV core antibody staining clearly showed HCV core protein impairs DNA repair [7], therefore, we that only some of the cells were HCV core positive used the DNA Damage Signaling Pathway q-PCR array to (Figure 3A). The image indicated that HCV core protein was screen the gene alterations between high- and low-viral distributed throughout the cytoplasm. Indeed, HCV infec- load cells. We selected 48 genes to perform the gene tion clustered and spread outwardly from the central expression profile. The genes included gathering. Sorted cells confirmed FITC intensity as shown in genes: APEX2, CCNO, LIG3, MPG, MUTYH, NEIL2, NYHL1, Figure 3B where the sorting efficiency was 94% in HCV low- OGG1, PAPR1, PAPR3, POLB, SMUG1, TDG, and UNG; viral load cells and 89% in HCV high-viral load cells. The nucleotide excision repair genes: ATXN3, BRIP1, CCNH, high-viral load cells presented a six-fold higher viral load DDB1, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERCC6, compared to the low-viral load cells (p < 0.01; Figure 3C). ERCC8, LIG1, MMS19, PNKP, POLL, RAD23A, RPA1, RPA3, These results indicated that this protocol efficiently and SLK, XAB2, XPA, and XPC; and other damage-repair genes accurately discriminated between high- and low-viral load included: CDK7, EXO1, MGMT, NEIL3, RAD18, RFC1, TOP3A, cell populations infected with JFH-1 in an in vitro model for TOP3B, XRCC6BP1, and GPX2. We set 1 0.3 as the immediate dissection. threshold and RPLP0 as the housekeeping gene. The array Subpopulations of HCV infectious cells 491

Figure 3. Different viral load cell distribution on JFH-1 infection in vitro cell model. (A) Immunocytochemical staining for hepatitis C virus (HCV) core (green) and DNA counterstaining with 40,6-diamidino-2-phenylindole (DAPI; blue) at 72 hours post-JFH-1 infection; (B) the histogram parameter of fluorescein isothiocyanate (FITC) core protein intensity on high- and low-viral load cells. The number indicates the percentage of cell population in R2 and R3 region; (C) cell HCV RNA was determined by qualitative polymerase chain reaction. data (Figure 4) showed that low-viral load cells had 32 distinct cell damage-repair gene regulation and response genes (ATXN3, CCNH, ATM, DDB1, ERCC1, ERCC2, ERCC3, modalities. ERCC4, ERCC5, ERCC6, LIG1, MMS19, MRE11, NTHL1, PAPR3, PNKP, OGG1, POLL, RAD18, RAD23A, RFC1, RPA1, RPA2, RPA3, SLK, TOP3B, XAB2, XPA, XPC, XRCC6BP1, GPX2, Discussion and POLB; 66.7%) upregulated; five genes (APEX1, CCNO, MPG, TDG, and UNG; 10.4%) downregulated; and 11 genes Researchers have reported that in chronic hepatitis, im- (BRIP1, ERCC8, EXO1, RPLP0, LIG3, MGMT, MUTYH, NEIL2, munity initiates the production of ROS [11] and nitric oxide PAPR1, SMUG1, and TOP3A; 22.9%) constantly expressed. In [12].Inanin vitro model, we found ROS generation in JFH- contrast, high-viral load cells had only six genes (ATXN3, 1-infected cells, even though HCV persistence does not ERCC3, ERCC6, NTHL1, RPA3, and SLK; 12.5%) upregulated; include integration into the host genome and the virus 14 genes (APEX1, BRIP1, CCNO, ERCC4, LIG1, MPG, PAPR1, maintains itself as an endoplasmic reticulum-associated PAPR3, OGG1, SMUG1, TDG, UNG, GPX2, and POLB; 29.2%) episome. However, it is generally accepted that HCV downregulated; and 28 genes (CCNH, ATM, DDB1, ERCC1, replication induces DNA damage stress and activates DNA ERCC2, ERCC5, ERCC8, EXO1, RPLP0, LIG3, MGMT, MMS19, damage signaling pathways. Reports have shown that HCV MRE11, MUTYH, NEIL2, PNKP, POLL, RAD18, RAD23A, RFC1, core proteins diminish DNA repair [6] and HCV E2 protein RPA1, RPA2, TOP3A, TOP3B, XAB2, XPA, XPC, and and CD81 interact to induce double-stranded DNA breaks XRCC6BP1; 58.3%) constantly expressed. Nineteen of a total [7]. Moreover, HCV NS5A proteins induce chromosome 48 genes (ATM, DDB1, ERCC1, ERCC2, ERCC5, MMS19, instability [8]. It was further reported by Farinati et al [4] MRE11, PNKP, POLL, RAD18, RAD23A, RFC1, RPA1, RPA2, that HCV produced more ROS than other hepatitis viruses TOP3B, XAB2, XPA, XPC, and XRCC6BP1) were significantly [4]. Therefore, HCV induces ROS and causes cellular DNA upregulated only in low-viral load cell populations, and five damage. of the total 48 genes (ERCC4, LIG1,PAPR3, OGG1, and HCV replicon cell lines are the most used in vitro models GPX2) were significantly expressed in high- and low-viral to study the gene regulatory mechanisms of HCV-specific load cell populations. This q-PCR screening result demon- polyproteins [6,8]. In this study, we used JFH-1 in vitro strated that different viral load cell populations present viral infection models to isolate infected cells because the 492 S.-C. Wang et al.

HVL 123456 A APEX2 ERCC2 LIG1 NTHL1 RFC1 TOP3B 0.60 1.07 0.72 1.78 1.24 1.25

B ATXN3 ERCC3 LIG3 PAPR1 RPA1 UNG 2.15 1.42 1.09 0.75 0.85 0.03 B C BRIP1 ERCC4 MGMT PAPR3 PRA2 XAB2 0.67 0.56 0.85 0.40 0.82 0.77

D CCNH ERCC5 MMS19 PNKP RPA3 XPA 1.16 1.27 0.93 1.18 5.03 1.29 A E CCNO ERCC6 MPG POLB SLK XPC 0.16 0.60 1.43 1.05 1.42 1.05

F CDK7 ERCC8 MUTYH POLL SMUG1 XRCC6BP1 1.24 0.89 0.81 0.85 0.27 1.27

G DDB1 EXO1 NEIL2 RAD18 TDG GPX2 1.18 0.78 0.29 1.06 0.20 0.10

H ERCC1 RPLP0 NEIL3 RAD23A TOP3A OGG1 1.23 1.00 1.12 0.85 1.00 0.10

LVL 123456 A APEX2 ERCC2 LIG1 NTHL1 RFC1 TOP3B 0.47 1.52 1.56 1.51 2.07 1.51

B ATXN3 ERCC3 LIG3 PAPR1 RPA1 UNG 2.33 1.93 1.17 0.95 1.48 0.07 B C BRIP1 ERCC4 MGMT PAPR3 RPA2 XAB2 1.04 2.04 1.24 1.58 1.31 1.54

D CCNH ERCC5 MMS19 PNKP RPA3 XPA 2.86 2.41 2.26 1.31 3.27 2.81 A E CCNO ERCC6 MPG POLB SLK XPC 0.46 1.84 2.12 5.74 2.76 3.12

F CDK7 ERCC8 MUTYH POLL SMUG1 XRCC6BP1 2.31 2.17 1.04 2.76 0.78 2.46

G DDB1 EXO1 NEIL2 RAD18 TDG GPX2 2.43 1.16 0.46 2.3 0.36 2.51

H ERCC1 RPLP0 NEIL3 RAD23A TOP3A OGG1 2.73 1 1.08 1.78 1.25 1.4

Figure 4. DNA damage- and repair-related gene expression on high- and low-viral load cells by quantitative polymerase chain reaction array. The 48 genes were analyzed by the DNA Repair RT2 Profiler Polymerase Chain Reaction Array kit in high-viral load (HVL) cells and in low-viral load (LVL) cells. The data are normalized to RPLP0 housekeeping gene levels and presented as the fold change in number with the gene name. specific HCV core protein marker clearly identified them. [14]. The relationship of viruses to the DNA-damage Using this protocol, we successfully and efficiently sorted response is now of particular and increasing scientific in- cells with different viral loads. terest. Numerous studies have shown that HCV not only To isolate sorted cell RNA was the major application of damages DNA but also inhibits DNA repair [15]. However, in this study. However, co-culture sorted cells was the limi- screening of the repair genes expression by q-PCR, we tation because the intracellular immunofluorescence la- found 19 of the DNA damage-repair genes (CCNO, ATM, beling with antibody causes cell decomposition and reduces DDB1, ERCC1, ERCC2, ERCC5, MMS19, MRE11, PNKP, POLL, viability. For further study on the characteristics of high- RAD18, RAD23A, RFC1, RPA1, RPA2, TOPB3, XAB2, XPA, XPC, and low-viral load cells, construction of the viral particles and XRCC6BP1) were upregulated in the low-viral load cells, might need to contain a detection marker in this protocol. but exhibited no change in the high-viral load cells. This There is evidence for an association between the peak finding indicated that the different HCV viral loads in the viral RNA in a cluster and the number of cells that constitute cells might have different DNA repair ability. Therefore, the cluster [13], suggesting a dependence of adjacently infected cells recognize viral replication as a DNA damage infected hepatocytes on the robustness of viral replication stress and elicit DNA damage signal transduction, which in the hepatocytes that are most permissive to viral repli- ultimately induces apoptosis as part of the host immune cation [6]. This phenomenon was also observed in our JFH-1- surveillance. In contrast, the cells surrounding the cells infected in vitro model. After 72 hours infection, only 35% of with high viral replication may initiate the cell repair ca- infected cells were positive for HCV core protein, which pacity to protect cells from oxidative stress damage; how- clustered but did not randomly distribute on the cell culture ever, the detailed mechanism of low-viral load cell gene well. Even though HCV core intensity was not detected, HCV expression needs clarification. RNA was detected in low-viral load cell populations. Base on In conclusion, we established a cell sorting protocol to this finding, we confirmed that high- and low-viral load cells study the effects of viral loads in HCV-infected cell pop- are all infected with HCV viral particles. ulations. The results demonstrated different capacities of Defects in DNA repair genes cause genetic instability, damage-related gene expression. Our findings highlight the gross chromosomal rearrangements, and accumulation of important role of viral load in the study of gene expression mutations, leading ultimately to neoplastic transformation in research in viral infection. Subpopulations of HCV infectious cells 493

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