Significant Association of Multiple Human Cytomegalovirus Genomic
Loci with Glioblastoma Multiforme Samples
Padhma Ranganathan,a,bPaul A. Clark,eJohn S. Kuo,c,d,e,fM. Shahriar Salamat,gand Robert F. Kalejtaa,b,c,d
Institute for Molecular Virology,aMcArdle Laboratory for Cancer Research,bCarbone Comprehensive Cancer Center,cStem Cell and Regenerative Medicine Center,dand
Departments of Neurological Surgery,eHuman Oncology,fand Pathology and Laboratory Medicine,gUniversity of Wisconsin—Madison, Madison, Wisconsin, USA
counts for nearly half of all central nervous system (CNS) malig-
nancies in adults. Equivalent to a grade IV diffuse astrocytoma,
GBMs consist primarily of neoplastic astrocytes, the most abun-
plastic cell types, including neurons, oligodendrocytes, macro-
phages, and glial and neural stem cells (42). The heterogeneous
nature of these tumors may at least partially explain why they are
refractory to current therapeutics and has led to the hypothesis
although exposure to ionizing radiation, electrical, or magnetic
fields has been proposed as a risk factor (22). Recently, several
reports have detected a potential association between GBMs and
human cytomegalovirus (HCMV), a common betaherpesvirus.
Viruses are causative agents of human cancers (21). At least
15% of all human tumors have a viral etiology. Human cancer
human T-lymphotropic virus type 1 (HTLV-1), human papillo-
mavirus (HPV), hepatitis C virus (HCV), Kaposi’s sarcoma-
associated herpesvirus (KSHV), and Merkel cell polyomavirus
the entire genome is maintained, as well as abortive infections
to cellular transformation and cancer development. Not surpris-
of the molecular hallmarks of cancer (10) that promote cellular
plasticity (through genomic instability, inflammation, deregula-
tion of cellular energetics, and induction of angiogenic and met-
astatic processes), proliferation (by sustaining proliferative
signaling, evading growth suppressors, and enabling replicative
lioblastoma multiforme (GBM) is an aggressive and malig-
nant tumor of glial origin with a grim prognosis (43). It ac-
immortality), and survival (avoidance of immune detection and
inhibition of apoptosis). However, many other viruses not yet
lecular events, leading to speculation that more human cancers
have a viral etiology or association than is currently appreciated.
receiving increased examination is that of HCMV and GBM.
HCMV asymptomatically infects the majority of the human
population, and virus-induced sequelae are generally observed
only under conditions of insufficient host immune function (7).
Examples include birth defects in congenitally infected neonates,
retinitis and blindness in AIDS patients, and graft rejection in
transplant patients receiving immunosuppressive therapy. How-
ever, an emerging concept hypothesizes that chronic conditions,
although perhaps not directly caused by HCMV, are likely to be
to HCMV infection include immunosenescence (40), certain car-
diovascular diseases (36), and cancer (34). Either HCMV infec-
duce all of the molecular hallmarks of cancer (13). For example,
viral infection or HCMV-encoded proteins promote genomic in-
stability (8, 30), inflammation (2), angiogenesis (4), and cell mi-
and life span (35), and inactivate cellular immune functions (25)
Received 22 August 2011 Accepted 3 November 2011
Published ahead of print 16 November 2011
Address correspondence to Robert F. Kalejta, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.org 0022-538X/12/$12.00Journal of Virologyp. 854–864
and apoptotic pathways (3). Therefore, HCMV represents an in-
triguing candidate human cancer virus.
The major experimental method identifying HCMV in GBM
samples has been immunohistochemical (IHC) detection of the
72-kDa viral immediate-early 1 (IE1) protein. Individual studies
20 out of 21 (31), or 8 out of 49 (16). The viral phosphoprotein of
uct of the 28th gene in the unique short region of the genome
(US28) was found in 20 out of 21 GBM samples (31). In situ
hybridization (ISH) for either the HCMV IE locus (5), total viral
genomic DNA (5, 20), or an undisclosed region(s) of the viral
genome (29) has also been used to score for the presence of
HCMV, obtaining positive results in every (29 out of 29) GBM
unique long section of the genome (UL55 that encodes glycopro-
tein B [gB]) in 7 out of 9 (5) or 21 out of 34 (20) GBM specimens.
Many interpret these results as solid evidence indicating that
HCMV is present in GBMs.
detect HCMV in GBM samples. PCR and IHC for IE1 or pp65
failed to detect the presence of HCMV in 22 GBM samples (24).
Likewise, an independent study (15) that used IHC for pp65, ISH
samples. An additional investigation (28) detected IE1 by IHC in
only 9 out of 81 GBMs and by ISH in only 7 out of 81 GBMs,
cating issues have created uncertainty about the results of the
above studies that detected a strong association of HCMV with
GBM tumors. For example, the IHC images presented are very
difficult for all but a trained pathologist to decode, and thus, it
could be argued that the conclusions drawn from such experi-
ments are more subjective than objective. Furthermore, the viral
antigens are detected only when ultrasensitive IHC methods are
employed. Perhaps most troubling, the normally nuclear IE1 and
pp65 proteins detected with this technique are almost invariably
cytoplasmic when detected in GBM tissue, calling into question
the assay’s specificity. Thus, any association of HCMV with GBM
is considered with a healthy skepticism.
The previous studies discussed above often suffer from small
sample sizes, low numbers of HCMV loci analyzed, subjective
assays, and insufficient quantitative analysis. Thus, we sought to
independently determine whether HCMV was statistically more
or noncancerous brain tissues by testing for the presence of mul-
tiple viral genomic loci by PCR in GBM specimens. Our results
lead us to conclude that all regions of the HCMV genome are
present in the vast majority of GBM samples but that only a small
minority of cells in any individual sample harbors HCMV DNA.
MATERIALS AND METHODS
Sample collection and DNA extraction. This retrospective study was
conducted in agreement with the terms of a University of Wisconsin—
Madison (UW-Madison) institutional review board (IRB) protocol (M-
2009-1420). Tumor samples were obtained with prior patient consent.
The samples were deidentified and are untraceable to individual patients.
Only the date of collection of the sample and diagnosis information were
available. The samples were destroyed during the course of the analysis.
Sample preparation was performed in clean laboratories never before
used for HCMV research in order to prevent spurious contamination.
Paraffin-embedded archived astrocytoma grade 4 (GBM), meningioma,
schwannoma, oligodendroglioma, or nonneoplastic epileptic brain sam-
slides were examined. Sections containing the highest percentage of tu-
mor cells and the least amount of necrosis were chosen. The 75 GBM
samples analyzed here represent essentially the entire collection of usable
samples collected from 1994 through 2009 at UW-Madison. To purify
DNA from paraffin-embedded tissues, 10-?m sections were extracted
TABLE 1 PCR primer sequences used in this studya
Gene Forward primerReverse primer
aNucleotide sequences for primer pairs used to amplify segments of the indicated HCMV gene are shown. The expected size of the amplified product is listed in base pairs. Also,
the sequences of the primers (named “IE1”) employed in the comparative PCR (Fig. 1) and copy number (Fig. 10) studies are indicated.
HCMV Genome in Glioblastoma (GBM)
January 2012 Volume 86 Number 2jvi.asm.org 855
twice with xylene and treated with DNA lysis buffer (50 mM KCl, 10 mM
Tris-HCl [pH 8.3], 2.5 nM MgCl2, 100 ?g/ml gelatin, 0.45% IGEPAL
nase) at 55°C for 3 h and then at 95°C for 10 min. DNA was subsequently
precipitated from supernatants with sodium acetate and ethanol and fi-
nally resuspended in deionized/distilled water. For nonarchived tissue
samples, ?1 mg of frozen but not fixed tissue was minced and DNA was
manufacturer’s protocol. Positive-control samples were generated by in-
fecting primary human foreskin fibroblasts with HCMV strain TB40/E at
multiplicities of infection (MOIs) of 10?5, 10?4, 10?3, 10?2, or 10?1
PFU/cell for 18 h and preparing DNA with the Qiagen DNeasy kit. It is
suspected that little to no viral DNA replication takes place within this
time frame, so the amount of viral DNA present reflects input viral ge-
nomes. DNA was quantitated by absorbance at 260 nm.
Experimental and statistical analyses. Samples were analyzed with
PCRs and agarose-ethidium bromide gel electrophoresis in clean labora-
tories never before used for HCMV research in order to prevent spurious
contamination. For conventional PCRs, 100 ng template DNA and Taq
polymerase (New England BioLabs) along with nucleotides and buffers
provided by the polymerase manufacturer were utilized in 25-?l reaction
mixtures that were run for 35 cycles. Primer sequences are presented in
Table 1. Primer pair fidelity was confirmed by sequencing positive-
control and paraffin-embedded GBM tumor DNA reaction products. Se-
separated by electrophoresis, and bands of the expected size were quanti-
tated with densitometry using Image J software. Quantitative real-time
PCR (Q-PCR) methods have been previously described (12). Statistical
analyses were performed with Graphpad prism. Linear graphs were cre-
ated with Microsoft Excel, and heat maps were created with Heatmap
Semiquantitative PCR as an objective assay for HCMV se-
quences in GBM samples. In previous studies where PCR was
used to probe for HCMV genomes in GBM samples (5, 20, 29), a
ably, on visual examination of reaction products separated on
agarose gels. We sought a more quantitative and objective
method, and thus, we compared the efficiency with which quan-
titative real-time PCR (Q-PCR) and semiquantitative (conven-
genomes in complex samples. Templates consisted of a series of
DNA preparations from primary human fibroblasts (HFs) in-
fected with 10-fold dilutions of HCMV strain TB40/E for 18 h.
These samples also served as positive controls during the analysis
viral genomes should be complete but viral DNA replication not
yet initiated. Thus, the level of DNA detected should be directly
FIG 1 Comparison of Q-PCR and conventional PCR for the detection of an
HCMV genomic sequence. (A) Quantitative real-time PCR detection of the
HCMV UL123 (IE1) genomic locus from human fibroblasts infected at MOIs
panel A. (C) Agarose gel detection of the HCMV UL123 (IE1) genomic locus
amplified by conventional PCR using the same primers and templates as in
panel A. (D) Linear plot of Image J quantification of the gel shown in panel C.
FIG 2 HCMV genomic regions surveyed. Schematic representation of the genomic loci amplified by the PCR primers used in this study. Primer pair locations
(e.g., UL82) amplified by the indicated primer pair.
TABLE 2 P values from Wilcoxon rank sum test
P value for comparisona
replicates GBM vs epilepsy
GBM vs control
aP values for the indicated comparisons of the levels of PCR amplification products for
the indicated viral genes are displayed. P values that indicate statistical significance are
shown in boldface type.
Ranganathan et al.
jvi.asm.org Journal of Virology
related to input viral genomes. For this test comparison, primers
(Table 1) that could be used for both Q-PCR and traditional PCR
amplification of the IE1 locus were employed.
Q-PCR was performed (Fig. 1A), and threshold values were
determined and plotted (Fig. 1B) as a function of the multiplicity
of infection (MOI). Regression analysis demonstrated the linear-
ity of the assay (coefficient of determination [R2] ? 0.93). Con-
ventional PCR was also performed, and reaction products were
separated by agarose gel electrophoresis (Fig. 1C), quantitated by
Image J software, and plotted (Fig. 1D) as a function of MOI.
Regression analysis demonstrated the linearity of this assay (R2?
0.97). Thus, for this primer set, conventional PCR (yielding the
larger R2value) would have comparable (in fact, superior) pre-
dictive value for future samples than Q-PCR. Therefore, we
FIG 3 Mann-Whitney U/Wilcoxon rank sum test of paraffin-embedded samples. DNAs from tissue samples were used as templates for conventional PCR
amplification with primers detecting the indicated HCMV genomic locus. Bands on agarose gels were quantitated by Image J software, and arbitrary units are
plotted. Samples from individual classes (epilepsy, GBM, and other brain tumors [OBT]) were compared with the Mann-Whitney U/Wilcoxon rank sum test.
Statistically significant differences are indicated with asterisks as follows: ???, P value ? 0.0001; ?, P ? 0.05.
HCMV Genome in Glioblastoma (GBM)
January 2012 Volume 86 Number 2jvi.asm.org 857
judged that conventional PCR would be sufficient to obtain the
semiquantitative data that would allow for an objective evalu-
ation of the presence of HCMV DNA in a given sample. Due to
the large number of samples to be examined and primer pairs
to be employed, we chose to use conventional PCR for most of
Detection of HCMV sequences in paraffin-embedded sam-
ples. Twelve primer pairs for the examination of paraffin-
embedded samples (Table 1) were developed based on the se-
quence of the cloned TB40/E strain (GenBank accession no.
EF999921.1) that spanned 229 kb of the HCMV genome at ap-
proximately 20-kb intervals (Fig. 2). Primers within genes encod-
used whenever possible. Primer specificity was confirmed by se-
quencing PCR amplification products generated with positive-
control templates (data not shown). Paraffin-embedded tissue
sections were obtained from the Pathology archives of the Hospi-
tal & Clinics at the University of Wisconsin—Madison and con-
sisted of samples from patients diagnosed with GBM (n ? 75),
meningioma (n ? 6), schwannoma (n ? 5), oligodendroglioma
(n ? 5), or epilepsy (n ? 15). Healthy (nondiseased) brain sam-
ples were rare in the collection, so nonneoplastic brain samples
sample from the collection. DNA preparation and PCR analysis
were carried out in clean rooms not previously used for HCMV
mixtures. The total reaction products were separated by agarose
gel electrophoresis, and bands of the appropriate size were quan-
titated with Image J software (data not shown). Background val-
ues were subtracted from individual band intensities. Where in-
dicated (Table 2), technical replicates were averaged. Data and
statistical analyses are described below.
Mann-Whitney U/Wilcoxon rank sum test. The levels of
HCMV sequences in GBM, epilepsy, or other brain tumor
paraffin-embedded samples were compared using the Mann-
Whitney U test (also called the Wilcoxon rank sum test), a non-
parametric statistical examination applicable to arbitrary sample
an asterisk in Fig. 3) to indicate that GBM samples were more
likely to contain larger amounts of HCMV DNA than the com-
parison cohort and comparisons with P ? 0.0001 (denoted by
three asterisks in Fig. 3) to indicate that GBM samples were much
more likely to contain HCMV DNA than the comparison cohort.
Our analysis indicated that 8 out of 12 HCMV genomic loci
tested were statistically more likely to be present in GBM samples
2). The eight loci were UL17, UL27, UL69, UL82, UL96, UL122,
US11, and US28. Of these loci, three (UL96, US11, and US28)
were also statistically overrepresented in GBM samples compared
to other brain tumors. The HCMV UL55 locus was statistically
more likely to be present in GBM samples compared to other
brain tumors, but not compared to brain samples from patients
represented in control samples (epilepsy or other brain tumors)
compared to GBM samples. We also corrected for multiple com-
parisons by estimating false discovery rates (FDR) by calculating
Q values (1). We considered comparisons with Q ? 0.05 to indi-
cate that HCMV DNA was more likely to be found in GBM sam-
ples than in the comparison cohort and comparisons with Q ?
0.0001 to indicate that HCMV DNA was much more likely to be
of FDR analysis echoed our initial analysis (Fig. 4 and Table 2).
From these two tests, we conclude that multiple HCMV genomic
to objectively classify each sample as positive or negative for the
viral genomic region amplified by each primer pair. To obtain
objectivity for the complete data set (Fig. 5), we used a set of
while some primer sets (e.g., UL17) can be used to predict un-
known values very efficiently, others (e.g., UL55) do so less effec-
tively. At the y intercept, the value of x (the MOI of HCMV) is
Therefore, samples with band intensities greater than the
y-intercept value were considered positive, and those with band
intensities equal to or less than the y-intercept value were consid-
ered negative. Heat maps depicting either positive (red) or nega-
tive (green) results for each sample and each primer pair were
generated (Fig. 6).
fication of HCMV DNA sequences from paraffin-embedded tis-
sue was less likely in brain samples from patients with epilepsy
(Fig. 6A) and other brain tumors (Fig. 6B) than from GBM sam-
ples (Fig. 6C). By using this method of data analysis, which is
independent of the statistical one described above, we reached a
similar conclusion, namely, that multiple HCMV genomic loci,
and very likely the entire genome, are found in GBM tumors.
FIG 4 False discovery rate analysis. P values (Table 2) were converted to Q
the Q value for each primer pair is plotted for two comparisons, GBM versus
epilepsy and GBM versus OBT. Lines are drawn at P values of 0.05 and 0.001.
Values between the lines are considered to represent significant differences
between the compared cohorts. Values above both lines are considered highly
loci surveyed is shown for illustrative purposes (not to scale).
Ranganathan et al.
jvi.asm.org Journal of Virology
repeated our analysis restricting samples to those collected within
16 non-GBM brain tumors. Counting all PCRs, we found the
GBM samples to be 55% positive, non-GBM brain tumors to be
7) agreed well with the previous analysis (Fig. 3 and Fig. 4), indi-
cating that multiple regions of the genome are statistically more
likely to be found in GBMs than in other brain tumors or brain
samples from patients with epilepsy.
Sequencing confirms PCR product identity. Amplified PCR
labeling a sample as HCMV positive or HCMV negative. To con-
firm that they actually represented HCMV DNA, selected PCR
FIG 5 y-intercept analysis of individual paraffin-embedded samples. PCR data from GBM (gray squares), epilepsy (black triangles), meningioma (crosses),
schwannoma (large gray asterisks), and oligodendroglioma (gray circles) samples (Fig. 3) are plotted along with values from positive-control samples (black
diamonds) that consisted of human fibroblasts infected with HCMV at MOIs of 0.01, 0.001, 0.0001, and 0.00001. Linear regression analysis with the positive-
control samples allowed for extrapolation to the y-axis intercept, denoted as a horizontal line.
HCMV Genome in Glioblastoma (GBM)
January 2012 Volume 86 Number 2jvi.asm.org 859
products resulting from amplifications using template DNA de-
rived from paraffin-embedded GBM samples were sequenced
(Table 4). Seventy-three individual PCR bands representing 10
distinct HCMV loci were sequenced. Internal regions were com-
HCMV strains in our laboratory, AD169 (GenBank accession no.
AC146999.1) and TB40/E (GenBank accession no. EF999921.1).
Seventy-one (97%) of the sequences matched the predicted
HCMV locus (Table 4). One sample amplified with UL144 prim-
uct with limited identity (82% over 47 nucleotides) to the human
SLC25A16 gene (GenBank accession no. AL713888.10) and vari-
ous human and primate bacterial artificial chromosome (BAC)
clones. Only 11 of the HCMV sequences (15%) were 100%
bands were unlikely to result from sample contamination. Se-
quence confirmation strengthens our conclusion that HCMV is
associated with GBM tumors.
samples prompted us to examine newer, nonarchival GBM sam-
12 frozen GBM specimens and analyzed by conventional PCR
other viral genomic loci (UL1, UL18, UL19, UL36, UL44, UL97,
and UL98). The positive control was HFs infected with HCMV
PCR was separated by agarose gel electrophoresis (Fig. 8), and
bands of the appropriate size were quantitated with Image J soft-
ware. Bands with intensities more than twice that of the water
negative reactions, are displayed in heat map format (Fig. 9).
Overall, 70% of the individual PCRs with GBM sample templates
positive. These data support our previous conclusion that multi-
ple HCMV genomic loci, and very likely the entire genome, are
found in GBM tumors. Unfortunately, the small sample number
and close collection dates (2006 to 2008) prevent us from deter-
mining whether HCMV sequences are more likely to be detected
in newer frozen samples than in older ones, as they are for the
One primer pair (UL18) produced only negative PCRs of the
GBM samples but was able to amplify the positive control. This
could indicate that this region of the genome may be missing in
otherwise HCMV-positive GBM samples. However, adjacent
primer sets (UL17 and UL19) showed overwhelmingly positive
fragment spanning UL17, UL18, and UL19, all were positive for
negative results with the UL18 primer pair likely represents inef-
ficient amplification rather than the absence of those genomic
regions in HCMV-positive GBM specimens.
Cellular genomes outnumber viral genomes in HCMV-
5, 8, and 10) seems to indicate that while the majority of GBM
samples are positive for multiple regions of the viral genomes, the
or are carrying viral DNA sequences. If this were true, cellular
genomes might outnumber viral genomes in GBM samples.
To directly examine whether cellular genomes outnumber vi-
ral genomes, we used quantitative real-time PCR to determine
exact amounts of viral IE1 and cellular actin sequences present in
HCMV-positive frozen GBM specimens and used those values to
develop a ratio between cellular and viral DNA sequences. We
found that, on average, there were approximately 160 copies of
nature of the HFs means that if every infected cell retained only a
single copy of the HCMV genome, only 1 in 80 cells within an
average HCMV-positive GBM specimen would carry viral se-
quences. As many virus-infected cancer cells harbor more than
able to establish the approximate MOIs of the infected samples.
as expected is substantially higher than those predicted from the
analysis of paraffin-embedded samples (Table 3). In total, the re-
sults of our analysis indicate that the majority of the HCMV ge-
nome is present in the majority of GBM samples, but only in a
minority of the cells of any individual tumor specimen.
Data we present here clearly demonstrate that HCMV genomes
the majority of studies that have investigated this potential inter-
action (5, 16, 20, 29, 31, 35). Our results also indicate that primer
selection and sample quality (age or method of preservation) can
genomes inside cells become refractory to PCR amplification due
to physical damage (19) and that HCMV genomes in vivo show
sequence variability that mirrors that of RNA viruses (26). All of
TABLE 3 Estimated MOIs and parameters of semiquantitative analysisa
Gene Slope y-axis interceptR2
4.5 ? 10?7
2.7 ? 10?5
3.6 ? 10?5
1.06 ? 10?6
9.7 ? 10?4
1.2 ? 10?6
4.7 ? 10?7
5.3 ? 10?7
4.7 ? 10?6
3.3 ? 10?5
7.6 ? 10?6
8.9 ? 10?6
aSlope, y-axis intercept, and correlation coefficient (R2) values for standard curve
analysis with the indicated PCR primer set are shown. These parameters were used to
estimate the multiplicity of infection (MOI) with HCMV within tumor samples based
on the MOIs of the standards.
Ranganathan et al.
jvi.asm.orgJournal of Virology
these factors may contribute to the few negative studies that have
of the association of HCMV with GBMs.
genomes exist in these tumors. Deep sequencing analysis should
give a more definitive answer as to the extent of viral sequences
FIG6 Heat map representation of HCMV-positive and HCMV-negative PCRs from paraffin-embedded samples. HCMV genomic loci are indicated across the
top of the heat maps. Unique samples (including the year in which they were collected) are displayed on individual rows. (A) Brain samples from patients with
than the y-intercept value are considered negative and denoted as a green square. (B and C) OBT samples (B) and GBM samples (C) are displayed as in panel A.
HCMV Genome in Glioblastoma (GBM)
January 2012 Volume 86 Number 2jvi.asm.org 861
present in GBMs. Our data would strongly argue for the use of
recently sampled tumors for such experiments. Should entire ge-
nomes be observed in GBMs, determining their structure (likely
circular episomes or integrated, linear units) could provide clues
in these tumors.
The results of our quantitative analysis indicated that cellular
genomes greatly outnumber viral genomes in the GBMs we ana-
lyzed (Fig. 11). This likely means that not every cell within the
sample is HCMV positive and probably explains why viral ge-
nomes were not detected in a genomic sequencing study (23) of
GBM tumors (Charles A. Whittaker, personal communication).
Therefore, if HCMV is contributing to the oncogenicity of the
GBM tumor (see below), it likely does so through mechanisms
distinct from other classical cancer viruses (such as EBV and
HPV), where full genomes or genomic fragments are found in
essentially every cell within a virally induced tumor.
cell types within the tumor are preferentially (or exclusively) in-
CD11b-positive cells of the monocyte lineage, but not in CD11b-
stem cells harbor the virus. The theory that pluripotent stem cells
or stem-like cells are uniquely able to maintain and propagate at
least a subset of human tumors is gaining widespread acceptance.
explain our results indicating that very few cells within a tumor
FIG 7 False discovery rate analysis of recently collected paraffin-embedded
the multiple comparisons performed. The negative log of the Q value for each
primer pair is plotted for two comparisons, GBM versus epilepsy and GBM
diagram of the viral genome labeled with the genomic loci surveyed is shown
below the graph for illustrative purposes (not to scale).
TABLE 4 Sequence confirmationsa
Region analyzed (nt)
225 (116334–116559) 95.6
160 (133375–133531) 94.5
100 (151643–151743) 96.4
210 (173778–173988) 98.3
100 (214774–214874) 95
aThe indicated loci were amplified by PCR and sequenced, and the indicated internal
region (in nucleotides [nt]) was compared to the NCBI nucleotide database. Average
percent identities for each locus compared to clinical (TB40/E) and laboratory (AD169)
strains of HCMV are shown. AD169 lacks the UL144 locus, so this comparison is not
FIG 8 PCR analysis of frozen GBM tumors. DNA extracted from 12 individ-
ual frozen GBM tumors (lanes 1 to 12) was analyzed by PCR with primers
detecting the indicated HCMV genomic locus. Reaction products were sepa-
rated by agarose gel electrophoresis and visualized with ethidium bromide
0.1) human fibroblasts (HCMV strain TB40/E), as well as water (W) served as
Ranganathan et al.
jvi.asm.org Journal of Virology
sample are infected and why cultured GBM cell lines (that are
clearly not neural stem cells) are invariably HCMV negative. In-
terestingly, HCMV antigens were recently found to be expressed
in cultured neural stem cells (6). The primary location of neural
stem cells is the subventricular zone in the CNS (9), which is also
latency (37). Thus, a more thorough examination of natural
HCMV infections of neural stem cells appears warranted.
The question remains as to what, if anything, HCMV is doing
within virus-positive GBMs. It is certainly possible that the mi-
croenvironment created by the tumor may provide a favorable
HCMV-infected cells. Were this the case, the virus might serve as
biology, but it would be unlikely that a virus-based treatment
would be an effective cancer therapy. Clinical data for the useful-
ness of anti-HCMV therapy for the treatment of GBM patients is
currently being collected, and while positive results have been re-
HCMV has been described as oncomodulatory (17, 18), at this
time, it is perhaps more appropriate to describe it as oncoacces-
sory, since the current evidence supports its presence in GBM
tually modulate tumor cell biology in vivo or in vitro.
However, if antiviral treatments prove effective therapies for
GBMs, it may indicate that viral infection is providing some
growth or survival advantage to the tumor. Clearly, HCMV en-
codes activities capable of eliciting all of the hallmarks of human
have been identified, determining which portions of the viral ge-
nome are expressed in GBM tumors should provide clues as to
how viral infection may be associated with oncogenesis.
That viruses can cause cancer is a well-accepted paradigm.
However, the heterogeneity of human tumors and the substan-
tially different ways in which known tumor viruses are oncogenic
make it challenging to establish a set of diagnostic experiments
that can prove or disprove a causative role for a virus in a neo-
plasm. A major confounding issue is that most infections even
with known cancer viruses do not result in oncogenic events. De-
number of human tumors than is currently appreciated. Previ-
indicate that GBMs, at the very least, are virus-associated tumors.
We thank Phil Balandyk for expert technical assistance, Norman Drink-
water for guidance during the statistical analysis, Charlie Whittaker
(David H. Koch Institute for Integrative Cancer Research, Massachusetts
members of our laboratories for helpful comments.
This work was supported by NIH grants AI080675 to R.F.K.,
T32AG027566 (P.A.C.) to the University of Wisconsin—Madison Stem
Cell Training Program, and CA014520 (J.S.K.) to the University of Wis-
consin—Madison Carbone Cancer Center. J.S.K. was partially supported
by the HEADRUSH Brain Tumor Research Professorship and the Roger
Loff Memorial Fund for GBM Research. R.F.K. is a Burroughs Wellcome
Fund Investigator in the Pathogenesis of Infectious Disease.
P.R. and R.F.K. designed the experiments. P.R. performed the exper-
iments. P.A.C., J.S.K., and M.S.S. provided materials. P.R. and R.F.K.
analyzed the data. P.R. and R.F.K. wrote the manuscript with input from
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FIG 9 Heat map representation of HCMV-positive and -negative PCRs from
frozen GBMs. Amplified bands (Fig. 8) were quantitated with Image J soft-
ware. Values greater than twice the water control for each individual PCR are
considered positive and denoted as a red square. Values equal to or less than
found on each individual row along with their year of collection.
FIG 10 Long-range PCR. Randomly selected DNA templates prepared from
the indicated GBM and control samples (from Fig. 8) were amplified with
agarose-ethidium bromide gel electrophoresis. HFF, human foreskin fibro-
plates prepared from the indicated GBM and control samples (from Fig. 8)
were analyzed by quantitative real-time PCR for the viral UL123 (IE1) locus
DNA in each sample is presented. Error bars represent the standard errors of
HCMV Genome in Glioblastoma (GBM)
January 2012 Volume 86 Number 2jvi.asm.org 863
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