Cells and viruses
The primary isolate (termed SBCMV) was provided by Dr. Ravit Arav-Boger, Johns Hopkins University, and obtained from the urine of a congenitally infected infant with disseminated HCMV disease. Institutional review board (IRB) exemption for the use of this isolate was given by Johns Hopkins Hospital. Primary human brain vascular pericytes and astrocytes were obtained from Cell Systems (Kirkland, WA, USA). Primary BMVEC cells were a kind gift from Dr. Milan Fiala (UCLA, Los Angeles, CA, USA) . All cells were maintained at low passage in complete pericyte medium from ScienCell Corporation (Carlsbad, CA, USA). Cells were trypsinized and plated in uncoated 100 cm2 dishes or uncoated 4.2 cm2/well glass chamber slides at a density 1 × 106 and 2.5 × 105 cells per dish and well, respectively. Heat-killed SBCMV was prepared by heating the viral inoculum to 65°C for 30 minutes in a water bath .
The GeneChip Human Genome U133 Plus 2.0 array (Affymetrix, Santa Clara, CA, USA) with complete coverage of the human genome containing over 47,000 transcripts was used to identify gene expression changes in primary human brain vascular pericytes three days postinfection. Several proinflammatory cytokines that were upregulated after SBCMV infection were selected for further study. A subgroup of genes identified as having the highest fold transcription levels associated with neuroinflammation were selected for qRT-PCR analysis.
Cytomegalovirus infection of pericytes and RNA isolation
Pericytes were infected with SBCMV at a multiplicity of infection (MOI) of 0.1 and virus adsorption was allowed for three hours, then the inoculum was removed and replaced with fresh media. Uninfected pericytes were used as mock-infected controls. Cultures were examined daily for evidence of cytopathology and at three days postinfection, cells were harvested, pelleted, and total RNA extracted at a density of 3 × 106 cells using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Extracted RNA was treated with 0.4 units/ml of RNase-free DNase (Qiagen, Valencia, CA, USA). RNA purity was first determined by spectrophotometric analysis, then resuspended in RNase-free water at a concentration of 200 ng/μl and stored at −80°C. RNA quality was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Three days postinfection, pericytes were washed twice in sodium cacodylate buffer pH 7.4, fixed in 2.5% glutaraldehyde for one hour and held at 4°C for an additional twenty-four hours. Monolayers were rinsed twice with 0.1 M sodium cacodylate buffer (pH 7.4), fixed in 1% osmium tetraoxide for one hour, washed twice for five minutes in cacodylate buffer and dehydrated in graded ethanol. HCMV-infected pericytes were pelleted for 10 minutes at 1500 rpm in a Sorvall Legend RT centrifuge (ThermoFisher Scientific, Waltham, MA, USA). Pellets were embedded in spur resin, sectioned for standard electron microscopy (EM) analysis and visualized on a Phillips CM12 electron microscope (Phillips Research, Eindhoven, The Netherlands).
Cell extracts were prepared using RIPA lysis buffer [50 mM Tris pH 7.5, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA) pH 8.0, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and proteinase inhibitor]. Lysates were placed on ice for 30 minutes and then clarified by centrifugation. Total protein was measured by bicinchoninic acid (BCA) assay (Pierce; ThermoFisher Scientific, Waltham, MA, USA). Fifteen μg of protein lysates from paired, mock and infected samples were fractionated in 4 to 20% SDS-PAGE gels, transferred to nitrocellulose membranes, blocked with 5% milk, 0.1% TBST (0.1% Tween 20, 20 mM Tris, 150 mM NaCl) and incubated at 4°C overnight with a monoclonal antibody to either major immediate early gene (MIE) (mAb 810 recognizes MIE IE1 and IE2, (Millipore, Temecula, CA, USA) or to human cytomegalovirus phosphorylated envelope protein expressed at late times during virus replication (pp65) (Vector Laboratories, Burlingame, CA, USA), both at a 1:2000 dilution. Membranes were washed five times in 0.1% TBST and incubated for one hour followed by incubation with a secondary antibody donkey anti-mouse peroxidase conjugate (Santa Cruz Biotech, Santa Cruz, CA, USA) at a dilution of 1:10,000. Immunoreactive bands were detected with SuperSignal West Dura Extended Substrate (Pierce; ThermoFisher Scientific, Waltham, MA, USA) following exposure to X-ray film.
Total RNA was extracted from SBCMV-infected pericytes and controls using a Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA). RNA was DNase-treated prior to elution on the column according to the manufacturer’s recommendations. Messenger RNA in 1 μg of each sample was primed using oligo-dT and reverse transcribed with a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Gene-specific primer pairs included C chemokine (C-C motif) ligand 5/Rantes (CCL5/Rantes), chemokine (C-X-C motif) ligand 11/I-TAC (CXCL11/I-TAC), and (CXCL8/IL-8) and 10 to 200 ng of cDNA for RT-PCR amplification, using PuReTaq Ready-To-Go PCR beads (GE Healthcare, Buckinghamshire, UK). PCR was carried out in a MJ Mini thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) in a final volume of 25 μl. The cycling protocol used was 95°C for five minutes, 55°C for thirty seconds, and 72°C for one minute for thirty-six cycles, with a final extension at 72°C for ten minutes. PCR products were electrophoresed in 1.5% agarose and DNA bands visualized by ethidium bromide. Primers for RT-PCR amplification were designated as: HCMV MIE 1 forward 5′-CCAAGCGGCCTCTGATAACCAAGCC-3′, reverse 5′-CAGCACCATCCTCCTCTTCCTCTGG3′ (exon 4, 435 bp); radical S-adenosyl methionine domain-containing protein 2 (RSAD2) forward 5′-CTTTGTGCTGCCCCTTGAGGAA- 3′, reverse 5′CTCTCCCGGATCAGGCTTCCA3′ (256 bp); CXCL8/IL-8 forward 5′-TGGGTGCAGAGGGTTGTG-3′, reverse 5′-CAGACTAGGGTTGCCAGATTTA-3′ (532 bp); CXCL11/I-TAC forward 5′- TTAAACAAACATGAGTGTGAAGGG-3′, reverse 5′- CGTTGTCCTTTATTTTCTTTCAGG-3 (228 bp); and CCL5/Rantes forward 5′- GGCAGCCCTCGCTGTCATCCTCA-3′, reverse 5′ –CTTGATGTGGGCACGGGGCAGTG-3′. Gyceralaldehyde phosphate dehydrogenase (GAPDH) forward 5′- TGATGACATCAAGAAGGTGGTGAA-3′, reverse 5′-TCCTTGGAGGCCATGTGGGCCAT-3′ (256 bp) was amplified in mock and infected cells as a loading and quality control.
Real-time PCR was performed in 96 well optical plates (Sorenson Bioscience, Inc., Salt Lake City, UT, USA) with cDNA using the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) in 25 μl reaction volumes. A master mix was made according to manufacturer’s instructions using SYBR green supermix (Bio-Rad Laboratories, Hercules, CA, USA) or VeriQuest master mix (Affymetrix, Santa Clara, CA, USA) for amplification of high GC-content cDNAs. Forward and reverse primers were used at a concentration of 250 nM per well, made in RNase/DNase-free H2O. Primer sequences for qPCR were as follows: TNF-alpha forward 5′- CAGAGGGAAGAGTTCCCCAG -3′, reverse 5′-CCTTGGTCTGGTAGGAGACG- 3′; IL-1beta forward 5′-CAAATTCGGTACATCCTCGAC-3′, reverse 5′- GTCAGGGGTGGTTATTGCATC-3′); and IL-6 forward 5′- AAACAGATGAAGTGCTCCTTCCAGG-3′, reverse 5-TGGAGAACACCACTTGTTGCTCCA- 3′. The cDNAs from mock-infected and SBCMV-infected pericytes were diluted 1:3 using RNase/DNase-free H2O; 3 μl of this dilution was added to each well, and control wells substituted water for cDNA. The cycling sequence included 95°C for three minutes, 95°C for fifteen seconds, 60°C for one minute, 95°C for one minute, 55°C for one minute, and 55°C for thirty seconds for eighty-one total cycles. A GAPDH primer set was included for normalization. Data analysis was performed using Bio-Rad iQ5 optical system software version 2.0.
Chamber slides containing either infected or uninfected cells were washed twice with phosphate buffer saline (PBS) pH 7.4, air dried, and fixed in absolute methanol for 10 minutes. Cells were air dried for fifteen minutes, hydrated in Tris saline (pH 7.4) for five minutes, and then incubated for one hour with monoclonal antibodies to the HCMV MIE at a 1:50 dilution (MIE, mAb 810, Millipore, Temecula, CA, USA) or the HCMV tegument protein pp65 (UL83) at 1:50 (Vector Laboratories, Burlingame, CA, USA). Dual-labeled immunofluorescence was performed using a mouse monoclonal to HCMV MIE and goat polyclonal antibodies at 1:100 dilution to human CXCL8/IL-8, CXCL11/I-TAC, and CCL5/Rantes purchased from R&D Systems (Minneapolis, MN, USA). Glial fibrillary acid protein (GFAP), (Millipore, Temecula, CA, USA), von Willebrand factor (VWF) (Millipore, Temecula, CA, USA) and neuron-glial antigen 2 (NG2) proteogylcan (Santa Cruz Biotech, Santa Cruz, CA, USA) were used at a 1:50 dilution for immunofluorescent staining of astrocytes, brain microvascular endothelial cells (BMVEC) and pericytes, respectively. Cells were washed three times with Tris saline and then incubated at 37°C for thirty minutes with a combination of secondary donkey anti-mouse Immunoglobulin G (IgG) antibodies conjugated with rhodamine isothiocyanate (RITC), and donkey anti-goat antibody conjugated with fluroescein isothiocyanate (FITC), (Jackson ImmunoResearch, West Grove, PA, USA) at a 1:100 dilution in PBS. Cells were washed another three times in Tris saline and mounted with Vectashield mounting media (Burlingame, CA, USA) containing 1.5 μg/ml of 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence was photographed with a Nikon TE 2000 S fluorescent microscope mounted with a charge-coupled device (CCD) camera (Nikon, Tokyo, Japan).
Human cytokine ELISA
The effects of HCMV exposure on pericyte expression of cytokines was measured with a human angiogenesis cytokine ELISA assay kit, profiling angiogenic cytokines (Signosis Sunnyvale, CA, USA). Mock-infected, pericytes exposed to heat-killed virus and SBCMV-infected pericytes were plated at a density of 5 x 105 cells in 10 cm dishes and grown to 90% confluence. Cell supernatants were then assayed for TNF-alpha, IL-6 and tumor growth factor-beta (TGF-beta), according to the manufacturer’s protocol. Quantitative analysis of IL-6 and TNF-alpha secreted from SBCMV-infected pericyte supernatants after 24 hours were monitored using human TNF-alpha and human IL-6 cytokine ELISA assays from Signosis according to the manufacturer’s protocol.
Dual-labeled immunohistochemistry (IHC) was performed as previously described  Archival brain tissue from an HIV-infected patient was obtained from the Texas Repository for AIDS Neuropathogenesis Research (Galveston, TX, USA). Specimen use was HIPPA compliant and approved by the Meharry Medical College IRB. Brain tissue was histologically identified as positive for HIV and HCMV. Tissue was mounted on chemate slides, paraffin embedded and dual stained by IHC for HCMV MIE in combination with the pericyte marker NG2 proteoglycan (Abcam, Cambridge, MA, USA) .
Experiments presented in this study were performed in triplicate (CMV-exposed brain pericytes, astrocytes and BMVEC cell pellets were used for qRT-PCR, while parallel supernatants were pooled for the ELISA assay). Quantitative real-time PCR experiments were replicated three times and normalized to GAPDH.