Cells
The human endometrial HEC-1A cell line (ATCC HTB-112) was used for infection experiments. Cells were propagated in McCoy’s medium (ATCC 30-2007), supplemented with 10% fetal calf serum (FCS) (complete McCoy’s medium).
The human JEG-3 choriocarcinoma cell line (ATCC HTB-36) was used for spheroid assays. Cells were cultured in RPMI 1640 medium (ThermoFisher Scientific, Milan, Italy) supplemented with 10% FCS (complete RPMI medium).
Virus
Cell-free HHV-6A inocula were obtained by density gradient purification from culture supernatant and cell lysate of HHV-6A infected J-Jhan T cells, as previously described [13, 16, 17]. Virus inocula were maintained in phosphate buffered saline (PBS) with 5% bovine serum albumin (BSA) at − 80 °C until use, and quantified by quantitative real-time PCR (qPCR) targeting U94 virus gene [14]. All the experiments were performed using the same virus inoculum, containing 1010 genome copies/ml, corresponding to about 109 infecting particles/ml, as previously described [17]. UV-inactivated virus inocula were obtained as previously described [18].All experiments involving virus production and infection were performed under the standard BLS-2 biosafety level.
Endometrial Cell Infection
HEC-1A cells were seeded in 6-well plates (5 × 105 cells/well) 24 h before infection, to obtain optimal density, and infected with gradient-purified cell-free HHV-6A at a 10:1 multiplicity of infection (MOI, virus genomes:cell ratio), as previously described [17]. Virus adsorption was carried out in 2% FCS medium for 3 h; then, cells were washed with PBS to eliminate unbound virus, and fresh complete McCoy’s medium was added. Control cells were uninfected or treated with UV-inactivated virus inoculum. At 1, 4, 8, 14, and 21 days post infection (d.p.i.), aliquots of cell cultures were collected and analyzed for virus DNA presence and transcription, and for expression of miRNAs. Evaluation of cell viability was performed by cell counting after Trypan Blue exclusion test.
Analysis of Virus Replication
Virus presence and transcription in HEC-1A cells were evaluated by analyzing DNA and RNA from infected cells. Briefly, 100 ng of total extracted DNA was analyzed by specific qPCR targeting U94 gene, and 200 ng of total extracted RNA was retrotranscribed using the RT2 First Strand kit (Qiagen, Hilden, Germany) and analyzed by two qPCRs targeting U94 and U42 viral genes, as previously described [14, 16]. The human RNaseP housekeeping gene was amplified as a control. In addition, viral antigen production was analyzed by immunofluorescence, using an anti-HHV-6 p41-PE IgG (SantaCruz Biotechnology, Heidelberg, Germany) and Hoechst (ThermoFisher Scientific, Milan, Italy) for nucleus staining. Images were obtained with Nikon Eclipse TE2000S, equipped with a digital camera.
miRNA Analyses
For miRNA analyses, total extracted RNA was retrotranscribed using the miScript RT kit (Qiagen, Hilden, Germany), and 100 ng of obtained cDNA were analyzed by the “Human Inflammatory Response & Autoimmunity” microarray (Qiagen, Hilden, Germany), able to detect and quantify 84 different miRNAs simultaneously. In addition, 29 further individual assays were performed, including miRNAs specifically involved in pregnancy and not included in the array: miR18, miR21-5p, miR22, miR24, miR30b-5p, miR31, miR92, miR125-3p, miR125b-5p, miR145-5p, miR155_1, miR155-2, miR196b-5p, miR199b-5p, miR200_1, miR200_2, miR222, miR374a-5p, miR378a-3p, miR422, miR423-5p, miR424-5p, miR449a, miR517, miR572, miR575, miR1207-5p, miR3663-3p, miR4306 and miR5739. miRTC_1, and SNORD11 were used as controls (Qiagen, Hilden, Germany). Amplification results were analyzed and normalized by a specific Qiagen software, to obtain comparable values between control and infected cells at each time post infection.
Trophoblast Attachment Assay
Based on preliminary experiments set up to optimize the kinetics of JEG-3 spheroid growth, JEG-3 cells at 80% confluence were seeded at 3 × 104 cells/ml in complete RPMI medium additioned with 1.5% agarose [19], allowing to obtain spheroids of 100–250 μm in diameter at 2–4 days of culture. JEG-3 spheroid size was measured using an optic microscope equipped with a calibrated eyepiece reticule. Cell viability was measured by DAPI stain.
For attachment assays, HEC-1A cells were seeded in 12-well plates (Nunc), at 1.6 × 106 per ml/well, and cultured for 2 days. Then, JEG-3 spheroids were transferred onto HEC-1A monolayers one by one with a fine Pasteur pipette. After 0.5, 1, and 2 h of incubation, the attachment rate was calculated by the NucSpot Live 488 test (Biotium, Fremont, CA, USA), under fluorescence microscopy, and expressed as the rate between attached and seeded spheroids.
Statistical Analysis
Statistical analysis was performed by Student’s t test for comparison between infected and control cells, with Bonferroni correction for multiple comparisons (microarray data). Linear regression analysis was conducted to evaluate the time dependence of the sphere attachment rate. A corrected p (pc) value ≤ 0.05 was considered significant.