Quantification of host proteomic responses to genotype 4 hepatitis E virus replication facilitated by pregnancy serum

All serum samples were collected from patients in Kunming, China. This study was approved by the medical ethics committee of the Medical Faculty, Kunming University of Science and Technology. Patients with HAV, HBV, HCV, and HIV were excluded. Written informed consent was obtained from the patients for the publication of this report and any accompanying images.

HEV-positive swine fecal samples containing HEV genotype 4 (GenBank accession no. KJ155502) were obtained from Kunming, China [20]. The fecal samples were converted into 10% (w/v) suspensions in DEPC–H₂O and centrifuged at 12 000 × g at 4 °C for 10 min, filtered through 0.22 µm microfilters before viral inoculation, and treated with penicillin and streptomycin for 1 h. The suspensions were then stored in liquid nitrogen until use. Viral titers of 1.0 × 10⁶ copies/mL were determined by using quantitative Real-Time PCR (qRT-PCR) [21]. A human hepatoma cell line (HepG2 cells) and a human lung carcinoma cell line (A549 cells) were obtained from the American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium containing 10% (v/v) FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C under 5% CO₂.

HepG2 cells were planted in 12-well microplates, incubated overnight to achieve 70%–80% confluence, washed twice with PBS, and transfected using Lipofectamine 3000 (ThermoFisher Scientific Inc., USA) following the manufacturer's instructions. The shRNA targeting FLNA were constructed according to the previous study [22]. Cells were transfected with shRNA-FLNA-1 plasmid to knock down FLNA.

Thirty serum samples from women in the third trimester of pregnancy that were negative for HEV RNA, HEV IgG and HEV IgM were mixed together, filtered with a 0.22 mm microfilter and heat-inactivated at 56 °C for 30 min, defined as pregnancy serum (PS). Mixed serum samples from thirty healthy non-pregnant women defined as non-pregnant serum (NPS). The cells were planted in six-well microplates for 24 h before virus inoculation and supplemented with 10% FBS (HEV group), 10% NPS (HEV + NPS group), or 10% PS (HEV + PS group) at ~ 50% confluence. The protocol for HEV inoculation was performed in accordance with a previous study [23]. In brief, monolayer cells were washed thrice and inoculated with 0.2 mL of the filtered viral inoculum and 30 mM MgCl₂ (final concentration) for 1 h. The solution was removed after inoculation, and fresh maintenance medium containing 2% FBS, 2% NPS, or 2% PS was added separately. The cells were collected either for proteomic analysis or viral replication determination at 6 days postinoculation (dpi).

Three biological replicates for each group were prepared for the iTRAQ-based proteomics experiments. The cells in Mock, HEV, and HEV + PS groups were suspended in the Lysis buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 40 mM Tris–HCl, pH 8.5, 1 mM PMSF, 2 mM EDTA) and sonicated in ice. The proteins were reduced with 10 mM DTT (final concentration) at 56 °C for 1 h and then alkylated by 55 mM IAM (final concentration) in the darkroom for 1 h. The reduced and alkylated protein mixtures were precipitated by adding 4 × volume of chilled acetone at − 20 °C overnight. After centrifugation at 4 °C, 30,000 g, the pellet was dissolved in 0.5 M TEAB (Applied Biosystems, Milan, Italy) and sonicated in ice. After centrifuging at 30 000 g at 4 °C, an aliquot of the supernatant was taken for determination of protein concentration. The proteins in the supernatant were kept at − 80 °C for further analysis.

Total protein (100 μg) was taken out of each sample solution and then the protein was digested with Trypsin Gold (Promega, Madison, WI, USA) with the ratio of protein: trypsin = 30: 1 at 37 °C for 16 h. After trypsin digestion, peptides were dried by vacuum centrifugation. Peptides were reconstituted in 0.5 M TEAB and processed according to the manufacture’s protocol for 8-plex iTRAQ reagent (Applied Biosystems). Briefly, one unit of iTRAQ reagent was thawed and reconstituted in 24 μL isopropanol. Samples were labeled with the iTRAQ tags. The peptides were labeled with the isobaric tags, incubated at room temperature for 2 h. The labeled peptide mixtures were then pooled and dried by vacuum centrifugation. SCX chromatography was performed with a LC-20AB HPLC Pump system (Shimadzu, Kyoto, Japan). The iTRAQ-labeled peptide mixtures were reconstituted with 4 mL buffer A (25 mM NaH2PO4 in 25% ACN, pH 2.7) and loaded onto a 4.6 × 250 mm Ultremex SCX column containing 5-μm particles (Phenomenex). The peptides were eluted at a flow rate of 1 mL/min with a gradient of buffer A for 10 min, 5–60% buffer B (25 mM NaH2PO4, 1 M KCl in 25% ACN, pH 2.7) for 27 min, 60–100% buffer B for 1 min. The system was then maintained at 100% buffer B for 1 min before equilibrating with buffer A for 10 min prior to the next injection. Elution was monitored by measuring the absorbance at 214 nm, and fractions were collected every 1 min. The eluted peptides were pooled into 20 fractions, desalted with a Strata X C18 column (Phenomenex) and vacuum-dried.

Raw data were processed with Proteome Discoverer 2.1 (PD, Thermo Fisher Scientific Inc.) and submitted to iProx database (https://​www.​iprox.​cn/​page/​home.​html, IPX0004999000). Proteins identification were performed by using Mascot search engine (Matrix Science, London, UK; version 2.3.02). Proteins identified with p < 0.05 and fold changes (FC) > 1.2 were considered as DEPs. DEPs were hierarchically clustered with log2 fold-change (FC) log2(FC) values. Regulations of filamin-A (FLNA), thioredoxin (TXN) and cytochrome (CYCS) in HEV-infected HepG2 cells at 6 dpi were shown in Table 1 with fold changes.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to analyze the function of proteins (biological process, molecular function, and cellular component) and the involved pathway [24, 25]. The protein–protein interaction (PPI) networks of DEPs were analyzed by using STRNG database to obtain the global profile of HepG2 cells in response to HEV replication facilitated by PS [26]. Then, Cytoscape software version 3.0 was used to visualize PPIs [27].

The expression levels of five key proteins in HepG2 cells were detected through Western blot analysis. Cells were collected at indicated times and lysed in radio-immunoprecipitation assay buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, pH 8.0, 30 mM NaF, 1 mM Na₃VO₄, 40 mM β-glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride, protease inhibitors, 10% glycerol, and 1% Nonidet-P40). Proteins were analyzed through 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane. Nonspecific binding sites were blocked with 5% skimmed milk, and the membrane was separately incubated with primary antibodies, HEV ORF2 (Millipore, 1:1000 dilution), FLNA, CYCS, or TXN (Bioworld, 1:1000 dilution) at 4 °C overnight. Horseradish peroxidase-conjugated IgG was used as the secondary antibody (Promega, 1:10,000 dilution). The GAPDH protein served as the loading control. Bands were exposed to X-ray films by using an Immobilon ECL kit (Millipore).

Total RNA was isolated from cells by Trizol (Invitrogn, America) in accordance with the manufacturer's instructions. cDNA was prepared by using AMV Reverse Transcriptase XL (Takara, Japan) in accordance with the provided directions. The copy number of HEV was quantified through SYBR green-based qRT-PCR with HEV-specific primers as described in our previous study [21]. The expression levels of RIG-1 were quantified by using specific primers described in previous studies [28]. GAPDH was applied as the housekeeping control. Relative gene expression was calculated through the 2^{−(△Ct of gene − △Ct of GAPDH)} method, where Ct is the threshold cycle. qRT-PCR was performed with a Bio-Rad CFX96TM Real-Time PCR System.

All experiments were performed at least three times. Data were presented as mean ± SD. Statistical analysis was performed on Western blot analysis results by using GraphPad Prism software version 8.0, and p values were calculated by using Student’s t-test to determine the significance of differences between two or more groups. p < 0.05 was considered to indicate statistical significance (Additional file 1).

Hormones, estradiol, and progesterone play vital roles in maintaining pregnancy. The viral titer in pregnant women or pregnant rhesus macaques is significantly higher than that in nonpregnant ones [14]. In vitro, HEV replication is significantly facilitated by PS, especially PS from women in the third trimester of pregnancy [10]. However, the mechanism underlying this enhancement remains unclear. HEV-infected hepatoma cells (HepG2) and lung cancer cells (A549) were supplemented with 10% FBS, 10% NPS, or 10% PS from women in the third trimester of pregnancy to explore the enhancement in HEV infection under PS supplementation. The capsid protein of HEV was detected by Western blot analysis at 6 dpi. Notably, viral replication in cells supplemented with PS was significantly enhanced relative to that in cells supplemented with FBS and NPS (Fig. 1A and B). HepG2 cells derived from the liver were chosen as the candidate cells for HEV infection to explore HEV pathogenesis through proteomics analysis.

HepG2 cells were collected at 6 dpi and analyzed by iTRAQ to identify the DEPs in mock and HEV-infected cells supplemented with FBS or PS. iTRAQ is a powerful technology for protein quantification that exhibits higher sensitivity and precision than conventional proteomics methods especially when used to quantify proteins with low abundance [29, 30]. iTRAQ has been widely used to explore the interactions between viruses and their hosts [30‐32]. In the present study, the DEPs in cells with or without HEV infection and supplemented with FBS or PS were identified by using iTRAQ. A total of 18 909 spectra and 5322 peptides were identified with 4847 unique peptides and 1511 proteins. Among the 1511 proteins, 14%, 12%, 12%, 11%, 10%, and 18% had weights of 10–20, 20–30, 30–40, 40–50, 50–60, and > 100 KDa, respectively. Most of the identified proteins comprised less than 10 peptides. Additionally, 42.03% of the identified proteins showed more than 10% sequence coverage. The possible functions of these proteins were predicted and classified in accordance with the COG database. The five top functional categories identified through general functional prediction were post-translational modification; protein turnover and chaperones; translation, ribosomal structure, and biogenesis; energy production and conversion; and carbohydrate transport and metabolism.

In consideration of the alteration in host proteomic responses to HEV replication facilitated affected by PS, a Venn diagram displayed the differentially and coincidentally dysregulated proteins in HepG2 cells supplemented with PS (Fig. 2A). Among the total 1511 identified proteins, 548 were defined as DEPs. On the basis of the analysis, 60, 38, and 112 DEPs were exclusively identified with stage specificity in the HEV versus Mock, HEV + PS versus Mock, and HEV + PS versus HEV groups, respectively. In addition, a total of 128 proteins were found to be simultaneously regulated significantly. Interestingly, PPI analysis with STRING revealed that 118/128 DEPs formed a tight interaction network (Fig. 2B). Furthermore, clustering analysis was used to assess the relationships among 128 DEPs in HepG2 cells with or without PS supplementation. Notably, most of the DEPs were down-regulated in cells infected with HEV (Fig. 2C, HEV vs. Mock), whereas the inverse was observed in HEV-infected cells supplemented with PS (Fig. 2C, HEV + PS vs. mock). Significant changes occurred in HEV-infected cells supplemented with PS or FBS (Fig. 2C, HEV + PS vs. HEV). GO enrichment analysis was conducted to further predict the functions of 128 DEPs (Fig. 2D). Extracellular exosome was the most significantly enriched GO cellular component term, and protein binding was the most significantly enriched GO molecular function term. Meanwhile, biological process terms were mostly enriched in cell–cell adhesion. Notably, the identified DEPs were found to play important roles in viral processes, viral transcription, and complement activation. KEGG pathway analysis was also performed to investigate the significant pathways wherein the 128 DEPs were enriched (Fig. 2E). Most enriched KEGG pathways were mainly composed of metabolic pathways. The DEPs also played important roles in human diseases, such as legionellosis and Parkinson’s disease. Complement and coagulation cascades and antibiotic biosynthesis were also associated with the DEPs.

A total of 328 proteins (66 up-regulated proteins and 262 down-regulated proteins) were significantly changed in HEV-infected HepG2 cells relative to in the Mock group. Significant changes in DEPs were found in HEV-infected HepG2 cells supplemented with PS (201 up-regulated proteins and 63 down-regulated proteins, Fig. 3A). Clustering analysis was performed to assess the expression profiles of 548 DEPs in HepG2 cells with or without PS treatment. Notably, HEV-infected HepG2 cells supplemented with PS showed significant up-regulation compared with mock or HEV-infected cells supplemented with FBS (Fig. 3B). Meanwhile, the PPI networks of DEPs were constructed to analyze the interactions of these proteins. The PPIs in the HEV versus Mock, HEV + PS versus Mock, and HEV + PS versus HEV groups consisted of 285 proteins and 2997 interactions (Fig. 3C), 240 proteins and 2227 interactions (Fig. 3D), 375 proteins and 4491 interactions (Fig. 3E), respectively. Up-regulated or down-regulated proteins tended to cluster together because of their PPIs.

GO and KEGG annotation and enrichment analysis were performed on the DEPs to explore the functional changes between different groups. Remarkably, cargo receptor activity, channel inhibitor activity, peroxiredoxin activity, enzyme inhibitor activity, and translation regulator activity were significant changed in HEV-infected groups (supplemented with FBS or PS) compared with mock uninfected cells (HEV vs. Mock, Fig. 4A; HEV + PS vs. Mock, Fig. 4B), which indicated that HEV infection activate host defense reactions. Notably, a large number of DEPs involved in host immune responses, including activation of immune, regulation of immune system process, immune system development and leukocyte activation, were activated in HEV-infected cells supplemented with PS compared with those supplemented with FBS (HEV + PS vs. HEV, Fig. 4C), which implied that the changes of host immune responses during pregnancy may facilitate HEV replication.

KEGG pathway analysis suggested that the signaling networks in HEV-infected cells supplemented with PS had increased compared with those supplemented with FBS. Compared with those in the uninfected Mock cells, these DEPs in HEV-infected cells supplemented with FBS were mainly involved in diseases, such as Parkinson’s disease, coronavirus disease, and amoebiasis, as well as in complement and coagulation cascades (HEV vs. Mock, Fig. 5A). These proteins in HEV-infected cells supplemented with PS also participated in some metabolic networks, such as the citrate cycle, glutathione metabolism, and glycolysis (HEV + PS vs. Mock, Fig. 5B). Once the HEV-infected cells were supplemented with PS, additional pathways, including human diseases, metabolic networks, and immune responses, became involved (HEV + PS vs. HEV, Fig. 5C). These results suggested that supplementation with PS accelerated virus–host interactions and regulated additional signaling pathways.

Three key proteins, including FLNA, TXN, and CYCS, were subjected to Western blot analysis at 0, 4, and 24 h post infection (hpi) to validate the iTRAQ process. The expression levels of the three proteins quantified by iTRAQ in cells infected with or without HEV are listed in Table 1. NPS isolated from healthy uninfected women was used as the control to further identify the effects of PS on HEV-infected cells. Notably, FLNA, an actin-binding protein that plays a primary role in signal transduction by linking the actin cytoskeleton to various transmembrane proteins to facilitate intracellular communication [33], was activated once HEV bound to the receptors on the host membrane at 4 hpi but was inhibited at 24 hpi after the complete entry of HEV (Fig. 6A and 6B), except cells supplemented with PS. FLNA has been reported to regulate the actin cytoskeleton to facilitate HCV [34] and HIV-1 [35] infection. To clarify the interactions between HEV replication and FLNA expression, FLNA were knockdown by shRNA (Fig. 6C). Notably, the down regulation of FLNA significantly suppressed the expression of retinoic acid-inducible gene I (RIG-I)-like receptors, the most important pathogen recognition receptors (PRR) during viruses infection (Fig. 6D). As a consequence, the suppression of RIG-I by FLNA inhibition facilitate HEV replication (Fig. 6E). Thus, PS supplementation maintained the expression of FLNA, which may be responsible for the promotion of viral replication (Fig. 6A, B and C).

TXN is a key component in the link between redox regulation and disease pathogenesis [36]. It mainly participates in the regulation and activation of NF-κB and other transcription factors and also displays antiviral, anti-inflammatory, antiapoptotic, and cell proliferative activities [37]. Nakamura [38] found that TXN plays a protective role against the influenza virus. The protective mechanism of TXN can be attributed to its potent antioxidative and anti-inflammatory activities. In the present study, TXN significantly increased in the HEV-infected HepG2 cells supplemented with PS at 4 and 24 hpi (Fig. 6A and B, Additional file 1). The increase in TXN expression in the HEV-infected cells revealed that TXN may be involved in early antiviral or anti-inflammatory responses. However, this involvement needs to be further identified.

CYCS regulates the intrinsic apoptotic pathway and is altered during viral infections. CYCS oxidase (COX) VIC is activated in the early stage of influenza virus infection [39]. Moreover, the X protein of HBV impairs the mitochondrial respiration chain and energy metabolism through interactions with COX subunit III [40]. HIV infection results in the dysregulation of CYCS [41]. The interaction of CYCS with the HEV capsid protein has been confirmed by using a yeast two-hybridization system [42]. In the present study, CYCS significantly decreased in HEV-infected HepG2 cells supplemented with NPS or PS, but increased in cells supplemented with FBS at 4 hpi, the early stage of infection (Fig. 6A and 6B, Additional file 1). Significantly increased CYCS were observed in HEV-infected HepG2 cells supplemented with PS, but significantly decreased in HEV-infected HepG2 cells supplemented with FBS or NPS at 24 hpi (Fig. 6A and B, Additional file 1). HEV ORF3 interacts with CYCS to protect against mitochondrial depolarization and death [43]. The promotion of CYCS in HEV-infected cells supplemented with PS may contribute to mitochondrial protection.