Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients with
Chronic Fatigue Syndrome
Vincent C Lombardi, Francis W Ruscetti, Jaydip Das Gupta, Max A Pfost,
Kathryn S Hagen, Daniel L Peterson, Sandra K Ruscetti, Rachel K Bagni, Cari
Petrow-Sadowski, Bert Gold, Michael Dean, Robert H Silverman, Judy A
Mikovits*
Supporting Online Material
Supporting Online Materials and Methods
Patient samples. Banked samples were selected for this study from patients
fulfilling the 1994 CDC Fukuda Criteria for Chronic Fatigue Syndrome (S1) and
the 2003 Canadian Consensus Criteria for Chronic Fatigue Syndrome/myalgic
encephalomyelitis (CFS/ME) and presenting with severe disability. Samples
were selected from several regions of the United States where outbreaks of CFS
had been documented (S2). These are patients that have been seen in private
medical practices, and their diagnosis of CFS is based upon prolonged disabling
fatigue and the presence of cognitive deficits and reproducible immunological
abnormalities. These included but were not limited to perturbations of the 2-5A
synthetase/RNase L antiviral pathway, low natural killer cell cytotoxicity (as
measured by standard diagnostic assays), and elevated cytokines particularly
interleukin-6 and interleukin-8. In addition to these immunological abnormalities,
the patients characteristically demonstrated impaired exercise performance with
extremely low VO2 max measured on stress testing. The patients had been seen
over a prolonged period of time and multiple longitudinal observations of the
clinical and laboratory abnormalities had been documented.
DNA and RNA isolation. Whole blood was drawn from subjects by
venipuncture using standardized phlebotomy procedures into 8-mL green-
capped Vacutainers containing the anti-coagulant sodium heparin (Becton
Dickinson, Franklin Lakes, NJ). Plasma was collected by centrifugation, aspirated
and stored at -80 ºC for later use. The plasma was replaced with PBS and the
blood resuspended and further diluted with an equal volume of PBS. PBMC were
isolated by layering the diluted blood onto Ficoll-Paque PLUS (GE Healthcare,
Waukesha, WI), centrifuging for 22 min at 800 g, aspirating the PBMC layer and
washing it once in PBS. The PBMC (approximately 2 x 107
cells) were
centrifuged at 500x g for 7 min and either stored as unactivated cells in 90% FBS
and 10% DMSO at -80 ºC for further culture and analysis or resuspended in
TRIzol (Invitrogen, Carlsbad, CA) and stored at -80 ºC for DNA and RNA
extraction and analysis. DNA was isolated from TRIzol preps according the to
manufacturer’s protocol and also isolated from frozen PBMC pellets using the
QIAamp DNA Mini purification kit (QIAGEN, Valencia, CA) according to the
manufacturer’s protocol, and the final DNA was resuspended in RNase/DNase-
free water and quantified using the Quant-iT Pico Green dsDNA Kit (Invitrogen,
Carlsbad, CA). RNA was isolated from TRIzol preps according to the
manufacturer’s protocol and quantified using the Quant-iT Ribo Green RNA kit
(Invitrogen, Carlsbad, CA). cDNA was made from RNA using the iScript Select
cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer’s
protocol.
PCR. To avoid potential problems with laboratory DNA contamination, nested
PCR was performed with separate reagents in a separate laboratory room
designated to be free of high copy amplicon or plasmid DNA. Negative controls
in the absence of added DNA were included in every experiment. Identification
XMRV gag and env genes was performed by PCR in separate reactions.
Reactions were performed as follows: 100 to 250 ng DNA, 2 µL of 25 mM MgCl2,
25 µL of HotStart-IT FideliTaq Master Mix (USB Corporation, Cleveland, OH),
0.75 µL of each of 20 µM forward and reverse oligonucleotide primers in reaction
volumes of 50 µL. For identification of gag, 419F (5’-
ATCAGTTAACCTACCCGAGTCGGAC-3’) and 1154R (5’-
GCCGCCTCTTCTTCATTGTTCTC-3’) were used as forward and reverse
primers. For env, 5922F (5’- GCTAATGCTACCTCCCTCCTGG-3’) and 6273R
(5’-GGAGCCCACTGAGGAATCAAAACAGG-3’) were used. For both gag and
env PCR, 94°C for 4 min initial denaturation was performed for every reaction
followed by 94°C for 30 seconds, 57°C for 30 seconds and 72°C for 1 minute.
The cycle was repeated 45 times followed by final extension at 72°C for 2
minutes. Six microliters of each reaction product was loaded onto 2% agarose
gels in TBE buffer with 1 kb+ DNA ladder (Invitrogen, Carlsbad, CA) as markers.
PCR products were purified using Wizard SV Gel and PCR Clean-Up kit
(Promega, Madison, WI) and sequenced.
PCR amplification for sequencing full-length XMRV genomes was
performed on DNA amplified by nested or semi-nested PCR from overlapping
regions from PBMC DNA. For 5’ end amplification of R-U5 region, 4F (5’-
CCAGTCATCCGATAGACTGAGTCGC-3’) and 1154R was used for first round
and 4F and 770R (5’-TACCATCCTGAGGCCATCCTACATTG-3’) was used for
second round. For regions including gag-pro and partial pol, 350F(5’-
GAGTTCGTATTCCCGGCCGCAGC-3’) and 5135R (5’-
CCTGCGGCATTCCAAATCTCG-3’) was used for first round followed by second
round with 419F and 4789R (5’-GGGTGAGTCTGTGTAGGGAGTCTAA-3’). For
regions including partial pol and env region, 4166F (5’-
CAAGAAGGACAACGGAGAGCTGGAG-3’) and 7622R (5’-
GGCCTGCACTACCGAAAT TCTGTC-3’) were used for first round followed by
4672F (5’GAGCCACCTACAATCAGACAAAAGGAT-3’) and 7590R (5’-
CTGGACCAAGCGGTTGAGAATACAG-3’) for second round. For the 3’ end
including the U3-R region, 7472F (5’-TCAGGACAAGGGTGGTTTGAG-3’) and
8182R (5’-CAAACAGCAAAAGGCTTTATTGG-3’) were used for first round
followed by 7472F and 8147R (5’-CCGGGCGACTCAGTCTATC-3’) for second
round. The reaction mixtures and conditions were as described above except for
the following: For larger fragments, the final extension was done at 68°C for 10
min instead of 72°C for 2 min. All second round PCR products were column
purified as described above and overlapping sequences were determined with
internal primers.
Nested RT-PCR for gag sequences was done as described (5) with
modifications. GAG-O-R primer was used for 1st strand synthesis; cycle
conditions were 52o
C annealing, for 35 cycles. For second round PCR,
annealing was at 54o
C for 35 cycles.
PCR analysis performed on 20 of the identical patient PBMC DNA
specimens stored at the NCI (Frederick, MD) since 2007 confirmed nearly
identical gag sequences, thereby diminishing the possibility of laboratory
contamination as a source of XMRV.
Phylogenetic Analysis. Sequences were aligned using ClustalX (S3). Clustal
alignments were imported into MEGA4 to generate neighbor-joining trees using
the Kimura 2-parameter plus Γ distribution (K80+Γ) distance model (S4). Free
parameters were reduced to the K80 model, and α values were estimated from
the data set using a maximum likelihood approach in PAUP*4.0 (Sinauer
Associates, Inc. Publishers, Sunderland, MA, USA). The bootstrap consensus
tree inferred from 1000 replicates is taken to represent the evolutionary history of
the taxa analyzed. Accession numbers were acquired from GenBank.
(
http://www.ncbi.nlm.nih.gov/Genbank): FLV (NC_001940), MoMLV
(NC_001501), XMRV VP35 (DQ241301), XMRV VP42 (DQ241302) XMRV VP62
(EF185282). Genomic Nonecotropic MLV Provirus Sequences were downloaded
from PLOS Genetics (S5).
Isolation, separation and culture of primary cells. Leukopaks of peripheral
blood from healthy donors were collected according to a NIH approved IRB #99-
CC-0168 protocol. Patients’ peripheral blood and plasma samples were from
frozen banked samples obtained under NIH exempt status. Mononuclear
leukocytes from both normal and patients’ cells were isolated by Ficoll-Hypaque
gradient centrifugation. The light density fraction (buffy coat) was collected and
washed twice with PBS. PBMC were activated by 1 µg/mL PHA (Abbott
Diagnostics, Abbott Park, IL) and after 72 hours the cells were cultured with 20
units/mL of IL-2 (Zeptometrix, Buffalo, NY) and subcultured every 3-5 days. For
isolation of CD4+T cells, CD8, CD11b, CD14, CD19, CD33 and CD56 positive
cells were removed using magnetic activated cell sorting (MACs) methods
according to the manufacturer’s instructions (Miltenyi Biotec, Inc., Auburn, CA).
After isolation, the CD3+, CD4+ T cells (>95% pure) were cultured in RPMI-1640
medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 1 mM
sodium pyruvate and antibiotics. CD4+
T cells were activated by culturing with 20
units/mL of IL-2 and 1 µg/mL PHA.
In vitro expansion of primary B-cells. NIH 3T3 cells transduced with a
retroviral vector expressing CD40L (gift of Eugene Barsov, NCI-Frederick) were
maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen,
Carlsbad, CA) supplemented with 10% calf serum (CS) (Lonza, Basel,
Switzerland) and 1% penicillin, streptomycin and L-glutamine (Invitrogen,
Carlsbad, CA) at 37°C with 5% CO2. To stimulate B cell expansion, ~3.5 x 106
NIH3T3-CD40L cells were trypsinized (0.25% trypsin with EDTA )(Invitrogen,
Carlsbad, CA), resuspended in 3 mL medium and irradiated with an absorbed
radiation dose (rad) of 9600 using a Cesium137 irradiator. Cells plus 7 mL medium
were added to a T25 cell culture flask (Corning, Corning, NY) and allowed to
adhere (2-3 h) to the flask surface (optimal density ~50%).
CD19+ B cells were isolated from PBMC using immunomagnetic bead
technology (Miltenyi Biotec, Auburn, CA). CD19+ cells were separated from 108
freshly isolated PBMC by positive selection according to the manufacturer’s
protocol. After magnetic separation, CD19+ B cells (>95% pure) were added to
an irradiated NIH3T3-CD40L monolayer and incubated at 37 °C with 5% CO2.
Cultures were monitored for B cell proliferation and split 1:5 every 72-96 hr onto
freshly irradiated NIH 3T3-CD40L monolayer. CD19+ primary B cells were
cultured and expanded in primary B cell expansion media: Iscove’s Modified
Dulbecco’s Medium (IMDM) (Invitrogen, Carlsbad, CA) + 10% FCS (Atlanta
Biologicals, Lawrenceville, GA), 1% penicillin, streptomycin and L-glutamine
(Invitrogen, Carlsbad, CA), 40 ng/mL interleukin 4 (IL-4) (PeproTech, Inc., Rocky
Hill, NJ), 50 µg/mL holo-transferrin (Sigma, St. Louis, MO) and 5 µg/mL insulin
(Invitrogen, Carlsbad, CA).
Cell culture and reagents. Raji, SupT1 and LNCaP were obtained from
American Type Culture Collection (ATCC, Manassas, VA). The cells were
maintained in RPMI-1640 supplemented with L-glutamine (2 mM), penicillin (100
U/mL), streptomycin (100 ng/mL), and FCS (10%) and subcultured 1:5 every 4-5
days. HCD-57 cells are a mouse erythroleukemia cell line that expresses both
ecotropic and polytropic MLVs; HCD-57/SFFV are HCD-57 cells infected with
SFFV. BaF3-ER cells are a murine pro B cell line engineered to express the
erythropoietin receptor. BaF3ER-SFFV Env cells were derived and maintained
as described (S6).
Flow cytometry for viral proteins. Adherent cells were incubated in trypsin for
10 minutes at 37o
C. After additional washes, adherent and suspension cells
were incubated for 15 min at RT in 1 mL of paraformaldehyde (4% w/v in PBS),
washed in permeabilization wash buffer (0.5% saponin 0.1%, sodium azide, 2%
human AB sera in PBS) (PWB), and resuspended in 300 µL of permeabilization
buffer (PBS with 2.5% saponin) (PB). After incubating at 22o
C for 20 min, 5 mL of
human AB sera and either rat anti-MLV p30 mAb, rat anti-SFFV Env mAb, goat
anti-Rauscher MLV gp70 Env, p30 Gag, or p10 Gag, or the appropriate isotype
control (anti-rat IgG, rat myeloma supernatant, or preimmune goat serum) were
added, and the cells incubated at 4o
C for an additional 30 min. Cells were then
washed in PWB, resuspended in 100 µL of PB with 3 µL (0.6 µg) of FITC-
conjugated goat anti-rat IgG or rabbit anti-goat antibody (BD PharMingen, San
Jose, CA) and incubated for 20 min at 4o
C. The efficiency of permeabilization
was determined using a FITC-conjugated anti-actin antibody. Cells were then
washed twice in PB, resuspended in 500 µL of sheath fluid (BD PharMingen, San
Jose, CA) to prevent clumping and analyzed by flow cytometry. For experiments
in which purified cell populations were examined, cells were stained with an anti-
CD3 or anti-CD19 antibody prior to permeabilization, and analyzed by gating on
the CD3+
or CD19+ subsets.
Western Blot (WB) analysis. Cells were pelleted, washed twice with PBS, and
lysed for 30 min on ice in RIPA lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl,
0.25% deoxycholate,1% NP-40, and protease inhibitor cocktail (Sigma, St. Louis,
MO). Debris was removed by centrifugation for 15 min at 21,000x g at 4o
C.
Protein concentration was determined with the Bio-Rad Protein Assay reagent
and equal amounts of protein (70–200 ug) were separated by SDS-PAGE
electrophoresis on 4-20% Tris-Glycine gels (Invitrogen, Carlsbad, CA) and then
transferred to Immobilon-P membranes (Millipore, Billerica, MA). The
membranes were blocked with 5% non-fat dry milk/1x TBST (Tris-buffered saline
with 0.1% Triton X-100) for 1 h at room temperature, hybridized with the
appropriate antiserum diluted in 5% non-fat dry milk/1xTBST for 2 h at room
temperature or overnight at 4o
C, washed twice with 1xTBST, hybridized with the
appropriate horseradish-peroxidase conjugated antibody diluted 1:5000 for 1 h at
room temperature, and washed three times with 1xTBST. Hybridized bands
were visualized using HyGlo chemiluminescent HRP antibody detection reagent
(Denville Scientific, Metuchen, NJ) and exposed to film (Kodak, Rochester, NY).
Antibodies used were a rat monoclonal Ab to SFFV gp55 Env (7C10), diluted
1:100 and detected with peroxidase-labeled anti-rat secondary antibody
(Amersham, Waukesha, WI); goat anti-Rauscher MLV gp70 Env, p30 Gag and
p10 Gag (provided by NCI); and goat anti-NZB xenotropic MLV (provided by
NCI), all diluted 1:2500 and detected with peroxidase labeled anti-goat
secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
Viral transmission. Frozen cell-free plasma and 0.22 µm filtered cell free
supernatants from PBMC and T cell cultures were diluted 1:1 with tissue culture
media and 600 µL aliquots were added to a six-well culture plate with the LNCaP
cell line (50% confluent) or a million primary activated CD4+ T cells isolated from
healthy donors. The plates were centrifuged for 5 min at 1500 RPM, rotated 180o
and centrifuged again for 5 min. The entire cycle was repeated once and cells
were then diluted in their growth media. For cell-cell transmission, 1 x 106 T cells
or PBMC without any IL-2 in the growth media were added to a six-well culture
plate with the LNCaP cell line (50% confluent) in 1 mL of media for 3 h. After 1
hr, T cells in suspension were removed and the LNCaP cells were grown for
several passages in the absence of IL-2 which caused any remaining T cells to
die. At the times after transmission indicated, protein analysis was done by
western blot and flow cytometry.
Genotyping. The rs486907 R462Q SNP was genotyped using Applied
Biosystems’ Taqman® 5' nucleotidase assays, Taqman® Universal PCR Master
Mix: No AmpErase UNG, and 5 ng of genomic DNA. The thermal cycling
conditions consisted of an initial hold at 95o C for 10 minutes followed by 50
cycles of a 15 second 95o C denaturation step and a one minute 60o C annealing
and extension step. A 7900HT instrument was used to detect fluorescent
probes, and Sequence Detection Software (SDS) v2.2 was used to discriminate
alleles and call genotypes (Applied Biosystems, Foster City, CA). The variant is
in Hardy-Weinberg equilibrium in both cases and controls. A Chi square test was
performed for both genotypes and alleles of RNASEL comparing XMRV negative
and XMRV positive controls. Both tests were not significant and the allele test is
displayed in Table S2. Homozygous R462Q variant of RNASEL is represented in approximately 13% of the human population (S6, S7).
Flow cytometry for detection of antiviral antibodies in CFS plasma. The
murine cell lines BaF3ER and BaF3ER-SFFV Env (S8) were grown in 2 units/ml
of Epo in RPMI 1640 plus 7% FCS. 500,000 cells per sample in log phase were
used as targets for direct staining. Cell lines were first washed in wash buffer (2%
FBS, 0.02% Na Azide, PBS) and resuspended in 200 µL of BSA staining buffer
(BD PharMingen, San Jose, CA). Patient plasma was thawed rapidly and used at
20 µL or 2 µL per tube (1:10 and 1:100 respectively) and incubated at 4o
C or on
ice for 30 minutes. Cells were then washed with 0.5mL of wash buffer. Tubes
were centrifuged at 800 rpm for 5 minutes, the supernatant was removed and
tubes blotted on a towel. Next, 100 µL of the following working solution was
added: 5 µL human A/B sera, 1 µL biotin labeled anti-human IgG (for human
plasma) or biotin-labeled anti-rat IgG (for SFFV Env mAb)(Ebioscience, San
Diego, CA), 1 µL of strep/avidin phycoerythrin (PE), 94 µL cold staining buffer.
Samples were then incubated at 4o
C for 20 minutes, washed with 0.5 mL of wash
buffer, and spun at 800 rpm for 5 minutes before being analyzed by flow
cytometry. For the competition experiments, 100 µL of cold staining buffer and 10
µL of human plasma were added to each tube prior to addition of either anti-
SFFV Env mAb (7C10) or Y3 myeloma supernatant (control). Samples were
incubated at 4o
C or on ice for 20 minutes, washed with 0.5 mL of wash buffer
and spun at 800 rpm for 5 minutes before being analyzed by flow cytometry.
Legends:
Figure S1. Gag sequences of XMRV in CFS patients. Partial sequences (nt 649-
1017) from CFS XMRV strains WPI-1130, WPI-1138 and WPI-1169 in
comparison to XMRV strains VP35, VP42 and VP62 derived from prostate
cancer patients. The yellow highlighting denotes the differences from the
reference strain (VP62).
Figure S2. Phylogenetic Analysis of XMRV in CFS patients. Neighbor-joining
analysis of CFS XMRV strains WPI-1104, WPI-1106 and WPI-1178 with
previously identified XMRVs and the nonecotropic MLVs xenotropic (Xmv),
polytropic (Pmv) and modified polytropic (Mmpv) (S5). Ecotropic MLVs FLV and
MoMLV were included to root the tree as outgroups in the phylogenetic
reconstruction. Bootstrap supports of >70% are shown next to branch nodes.
The scale measures evolutionary distance in substitutions per nucleotide.
Subgroup designations are marked with arrows. XMRVs (WPI and VPs) form a
distinct clade clustering together and separately from the xenotropic (Xmv)
proviruses (shown in red).
Figure S3. Detection of cloned XMRV-VP62 using a rat mAb to SFFV Env and a
goat antiserum to mouse NZB xenotropic MLV. A. Lysates were prepared from
XMRV-VP62-infected Raji (lane1), LNCaP (lane 2) or Sup-T1 (lane 3). Positive
controls used were HCD-57 cells, a mouse erythroleukemia cell line expressing
polytropic MLV gp70 Env (lane 4), and HCD-57 cells infected with SFFV, which
also express SFFV gp55 Env (lane 5). WB analysis was carried out using rat
anti-SFFV Env mAb 7C10. Molecular weight markers in kD are shown on the
left. B. Lysates were prepared from XMRV-VP62-infected Raji (lane 1), LNCaP
(lane 2) or Sup-T1 (lane 3). Lysates from SFFV-infected mouse HCD-57 cells
(lane 4) and from uninfected Raji, LNCaP and Sup-T1 are shown in lanes 5-7,
respectively. WB was carried out using goat antiserum to mouse NZB xenotropic
MLV. Molecular weight markers in kD are shown on the left.
Figure S4. Expression of XMRV proteins in PBMC from CFS patients. A.
Activated B cells from CFS patient WPI-1125, activated T cells from CFS patient
WPI-1105 or normal activatedT cells were incubated with goat antisera (black
area) against Rauscher MLV gp70 Env (top), p30 Gag (middle) and p10 Gag
(bottom) and analyzed by IFC. Preimmune goat serum (light area) was used as a
control. B. A B cell line from a CFS patient was incubated with rat anti-SFFV Env
mAb (right panel) or control myeloma supernatant (left panel) and then analyzed
by IFC. C. Lysates were prepared from B cells (lane 1) or T cells (lanes 2 and 3)
from CFS patients that had been grown for 42 days on CD40L or IL-2
respectively were analyzed by WB using rat anti-SFFV Env mAb (top panel) or
goat anti-NZB xenotropic MLV serum (bottom panel). Lane 4: normal T cells;
Lane 5: mouse HCD-57 cells; Lane 7: SFFV-infected HCD-57 cells. Molecular
weight markers in kD are shown on the left.
Figure S5: Infectious XMRV in CFS patients’ PBMC and plasma. A. The
indicated T-cell cultures from CFS patients were co-cultured with LNCaP as
described in the Methods. XMRV p30 Gag expression was detected in the
LNCaP cells using a rat anti-MLV p30 Gag mAb and IFC. Bottom panel: LNCaP
co-cultured with normal T cells. B. Plasma from the indicated CFS patients was
co-cultured with LNCaP. At the second passage, XMRV p30 Gag expression in
the LNCaP cells was detected by flow cytometry using a rat anti-MLV p30 Gag
monoclonal Ab. Co-culture with plasma from a normal healthy donor is shown in
the bottom panel.
Figure S6: Presence of antibodies in CFS plasma that recognize the cell
surface of SFFV Env expressing BAF3ER cells. A. Plasma from CFS patients
or normal healthy controls was diluted 1:10, reacted with BaF3-ER or BaF3ER-
SFFV Env cells and analyzed by IFC. Shown is the difference in mean
fluorescence intensity (MFI) between CFS and control plasma direct binding to
BaF3ER-SFFV Env cells versus BaF3ER (control) cells. B. Competition
experiment, carried out as described in the Methods, showing that plasma from a
CFS patient can block binding of a rat anti-SFFV Env mAb to BaF3ER-SFFV Env
cells. Left panel: CFS plasma diluted 1:10 (white area) eliminates most of the
anti-SFFV Env binding (striped area) and overlaps with the negative control
(black area). Right panel: CFS plasma diluted 1:100 (white area) eliminates less
of the anti-SFFV Env binding (striped area) and overlaps much more with the
positive than the negative control (black area).
