JOURNAL OF VIROLOGY, June 2002, p. 6213–6223
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 12
Charting Latency Transcripts in Kaposi’s Sarcoma-Associated
Herpesvirus by Whole-Genome Real-Time
Farnaz D. Fakhari and Dirk P. Dittmer*
Department of Microbiology and Immunology, The University of Oklahoma Health
Science Center, Oklahoma City, Oklahoma 73104
Received 6 December 2001/Accepted 12 March 2002
The division into a latent or lytic life cycle is fundamental to all herpesviridae. In the case of Kaposi’s
sarcoma-associated herpesvirus (KSHV) (human herpesvirus 8), latent genes have been implicated in cell
autonomous transformation, while certain lytic genes procure a tumor friendly milieu through paracrine
mechanism. To query KSHV transcription, we devised and validated a high-throughput, high-specificity,
high-sensitivity, real-time quantitative reverse transcription-PCR array. This novel methodology is applicable
to many human pathogens. Its first use demonstrated that the mRNA levels for KSHV LANA, v-cyclin, and
v-FLIP do not increase at any time after viral reactivation. The mRNA for LANA-2/vIRF-3 is similarly resistant
to viral reactivation. In contrast, every other latent or lytic message was induced. Hence, LANA, v-FLIP,
v-cyclin, and LANA-2 constitute a group of uniquely regulated transcripts in the KSHV genome.
By using representational difference analysis, Chang et al.
(9) demonstrated the presence of a novel human virus in
Kaposi’s sarcoma (KS) biopsies: Kaposi’s sarcoma-associ-
ated herpesvirus (KSHV) (human herpesvirus 8). Solid epi-
demiological evidence has since been assembled, which links
KSHV to KS (20, 21, 32, 40, 44, 73). Classic KS was first
described in 1872 as a rare, disseminated sarcoma of the skin
(reviewed in reference 1). KS is endemic in parts of equatorial
Africa, where it is responsible for an estimated 1% of all adult
tumors. In these regions, evidence for KSHV infection can be
observed even before puberty (reviewed in reference 72). Con-
comitant, widespread human immunodeficiency virus type 1
(HIV-1) infection has since turned KS into an epidemic dis-
ease. For instance, the seroprevalence levels for KSHV reach
30% in black South African HIV patients (66), and childhood
KS has become the most common neoplasm in this part of the
continent. KS has also been documented in solid organ and
bone marrow transplant recipients, in whom it can comprise up
to 3% of all tumors depending on the regional prevalence of
KSHV (38, 39, 43), and in 1981 KS was recognized as a signa-
ture pathology of AIDS. Recently, mucosal shedding of KSHV
was verified for a group of men in the United States (49, 71)
demonstrating that KSHV, like other members of the herpes-
viridae, is transmitted by saliva. KSHV can be detected in all
manifestations of KS, and within the lesions all tumor cells, but
not infiltrating lymphocytes, carry the viral genome (4, 68). The
virus is also present in multifocal Castleman’s disease (48, 67)
and in primary effusion lymphoma (PEL) (8). A number of cell
lines have been established from these PEL patients. Since
KSHV otherwise replicates extremely poorly in culture, PEL
cell lines constitute the primary system to investigate KSHV
biology. Based on the complete sequence of the 137-kbp
unique region, KSHV is classified as a gamma-2-herpesvirus, a
member of the lymphotropic subgroup of the Herpesviridae
family (25, 47, 60).
All members of the Herpesviridae display a tightly regulated
program of gene expression (reviewed in reference 59). KSHV,
too, conforms to this paradigm and can enter one of two modes
of infection: (i) latent infection, in which the viral genome
persists in its host cell but with dramatically restricted gene
expression and without cell destruction (75), and (ii) lytic in-
fection, which generates infectious progeny and destroys the
host cell. Lytic infection, in turn, can be divided into alpha or
immediate-early, beta or early, and gamma or late stages. By
using genomic KSHV DNA fragments as probes, Sarid et al.
defined three classes of differentially transcribed messages
in a KSHV-infected PEL cell line (63). Class I mRNAs can
be detected in untreated cultures and are not increased
after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment,
which reactivates the KSHV lytic replication cycle. Class II
mRNAs can be detected in untreated cultures but are greatly
increased by TPA treatment. Finally, class III mRNAs can only
be detected in TPA-treated cultures. Translated into the cus-
tomary herpesvirus classification (59), class III mRNAs corre-
spond to lytic transcripts (alpha, beta, and gamma) and class I
correspond to latent transcripts. Class II mRNAs present a
conundrum, since it is not clear whether the mRNAs detected
in untreated cultures stem from the few percent of cells that
undergo spontaneous lytic reactivation in culture (56). These
data were recently confirmed and expanded upon by using
hybridization KSHV-specific cDNA arrays (28, 50). In Epstein-
Barr virus, a related human gammaherpesvirus, latency-associ-
ated genes are essential for episome maintenance, as well as
for host cell transformation (reviewed in reference 57). This
also holds true for at least one KSHV type one latency gene:
the latency-associated nuclear antigen, LANA (2, 12, 19, 53).
In fact, LANA was the first latent protein of KSHV to be
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, The University of Oklahoma Health Science
Center, 940 Stanton L. Young Blvd., Oklahoma City, OK 73104.
Phone: (405) 271-2690. Fax: (405) 271-3117. E-mail: dirk-dittmer
identified based on its immunoreactivity with patient sera (31,
33, 54). We therefore sought to determine whether there are
other type one latency mRNAs in the KSHV genome and, if
so, how many?
To address this question, we developed a novel methodology
for scrutinizing transcription. Our genome scan is based on
real-time quantitative reverse transcription-PCR (RT-PCR)
(26). RT coupled to amplification by PCR is widely recognized
as the most sensitive method for detecting the presence of
specific RNAs. Unlike conventional DNA arrays, RT-PCR can
distinguish between various spliced mRNAs through exon-
specific primers. Real-time quantitative PCR measures the
amount of PCR product at each cycle of the reaction ei-
ther by binding of a fluorescent, double-strand-specific dye
(SYBRgreen) or by hybridization to a third sequence-specific,
dual-labeled fluorogenic oligonucleotide (Molecular Beacon
or TaqMan). By evaluating primers specific for every predicted
open reading frame (ORF) in the KSHV genome, we found
that, in addition to LANA, v-cyclin, and v-FLIP—all of which
are under control of a common cis-regulatory element (15, 64,
70)—LANA-2/vIRF-3 is encoded by a second latency type one
mRNA in the KSHV genome. No other KSHV mRNA dis-
plays a comparable pattern of regulation. This observation
establishes LANA, v-cyclin, v-FLIP, and LANA-2/vIRF-3 as
markers for latent KSHV infection and implicates them in viral
persistence and tumorigenesis.
MATERIALS AND METHODS
Cell lines and induction conditions. BCBL-1 cells were cultured in RPMI
1860, 25 mM HEPES (pH 7.55), 15% fetal bovine serum, 0.05 mM 2-mercap-
toethanol, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.05 ?g of penicillin/ml,
and 50 U of streptomycin/ml (all reagents from Life Technologies, Rockville,
Md.) at 37°C and in 5% CO2. Cells were split every 5 days to 2 ? 105cells/ml,
and a fresh aliquot was thawed after 30 passages. Where indicated, the cells were
reseeded to 2 ? 105cells/ml and induced 24 h later with 40 ng of TPA/ml
(?10,000 stock in dimethyl sulfoxide; Calbiochem, San Diego, Calif.), 1 ?M
ionomycin (?2,000 stock in ethanol; Sigma, St. Louis, Mo.), or 0.8 mM sodium
butyrate (?1,000 stock in ethanol; Sigma) for 24 h. Afterward the cells were
collected by centrifugation (200 ? g for 10 min at 4°C in a Hereaus Megafuge)
and resuspended in an equal amount of fresh medium. Cells were collected at the
indicated times after induction by centrifugation, washed once in phosphate-
buffered saline, pelleted, and resuspended in 500 ?l of RNAzol (Tel-Test, Inc.,
Friendswood, Tex.) per 107cells.
RNA isolation and RT. RNA was isolated by using RNAzol according to the
supplier’s protocol. Poly(A) mRNA was prepared by using dT-beads (Qiagen,
Inc., Valencia, Calif.) according to the manufacturer’s recommendations, and
500 ng of mRNA was reverse transcribed in a 20-?l reaction with 100 U of
Moloney murine leukemia virus reverse transcriptase (Life Technologies), 2 mM
deoxynucleoside triphosphates, 2.5 mM MgCl2, 1 U of RNasin (all from Applied
Biosystems, Foster City, Calif.), and 0.5 ?g of random hexanucleotide primers
(Amersham Pharmacia Biotech, Piscataway, N.J.). The RT reaction was sequen-
tially incubated at 42°C for 45 min, 52°C for 30 min, and 70°C for 10 min. The
reaction was stopped by heating it to 95°C for 5 min, 0.5 ?l of RNase H (Life
Technologies) was added, and the reaction was incubated at 37°C for an addi-
tional 30 min. Afterward, the cDNA pool was diluted 25-fold with diethyl pyro-
carbonate-treated distilled H2O and stored at ?80°C.
Primer design. The predicted KSHV ORFs were extracted from the complete
KSHV sequence as described by Russo et al. (60). Quantitative real-time PCR
primer pairs were designed by using Prime Express 1.5 (Applied Biosystems) to
comply with TaqMan criteria (5), namely, a Tmof 59 ? 2°C for each primer, a
maximal Tmdifference for both primers of ?2°C, a GC content of 20 to 80%, no
GC clamp, a primer length of between 9 and 40 nucleotides, fewer than four
repeated G residues per primer, no hairpins with a stem size of ?4, and a
amplicon size 50 to 150 bp. Where applicable, TaqMan probes were designed to
have a Tmto be greater than 10°C compared to the flanking primer pair and no
G at the 5? end. The top 100 potential primer pairs for each KSHV ORF were
visually inspected, and the highest ranking primer-probe combination was se-
lected, unless a primer-probe combination with similar characteristics but closer
to the 3? end of the ORF could be identified. In cases where the predicted ORFs
overlap (orfK3 exon 1 and orf70; orf17 and orf18; orf19, orf20, and orf21; as well
as spliced forms of orfK8), primers were selected outside the region of overlap.
In the absence of a complete transcript map for this virus, we cannot, however,
exclude the possibility that some primer combinations were located in regions in
which the 3? untranslated region (3?UTR) or the 5?UTR segments overlap.
Primer-probe combinations for known spliced transcripts were designed by using
relaxed criteria in order to span splice junctions. The primer sequences are
available online (http://w3.ouhsc.edu/mi/frame_faculty/dittmer.html).
Real-time quantitative PCR. The set of forward primers was synthesized on a
separate plate then the reverse primers were synthesized by using a MerMade
96-well synthesizer (BioAutomation Corp., Plano, Tex.) and stored in distilled
H2O at ?80°C at a concentration of 100 pmol/?l (100 ?M). Primer length and
purity was verified by using a P/ACE MDQ Capillary Electrophoresis System
(Beckman Coulter, Inc., Fullerton, Calif.). Forward and reverse primers were
combined from the master plate to yield enough primer pairs for 20 assays at a
time, and their concentrations were adjusted to 1 pmol/?l (1 ?M). The final PCR
contained 2.5 ?l of primer mix (final concentration, 166 nM), 7.5 ?l of 2? SYBR
PCR mix (Applied Biosystems), and 5 ?l of sample. For TaqMan PCR, FAM-
TAMRA-labeled probe (Applied Biosystems) was stored at 100 ?M for long-
term at ?80°C and added to the primer mix to yield a 1 ?M stock and a 166 nM
final concentration. In this case the PCR contained 2.5 ?l of primer-probe mix
(final concentration, 166 nM), 7.5 ?l of 2? TaqMan PCR mix (Applied Biosys-
tems), and 5 ?l of sample. PCR was set up in a segregated, locked, “white” room
(using designated pipettes and filtered tips), in which only primers and PCR mix
was handled. Here all surfaces and tools were treated with 10% bleach monthly
and exposed to ceiling UV lights overnight. Designated gowns, gloves, and face
masks were required for all work. Samples were prepared and added to the PCR
mix in a second, “gray” room, in which no cloned KSHV plasmids were present,
by using a designated set of pipettes and filtered tips. Real-time PCR was
performed by using an ABI PRIZM5700 or ABI PRIZM7700 machine (Applied
Biosystems) and universal cycle conditions (2 min at 50°C and 10 min at 95°C and
then 40 cycles of 15 s at 95°C and 1 min at 60°C) (26). CTvalues were determined
by automated threshold analysis by using the ABI PRIZM software. Here, the
threshold was set to five times the standard deviation (SD) of the non-template
control. Dissociation curves were recorded after each run, and the amplified
products were routinely analyzed by agarose gel electrophoresis.
Calculations. In order to evaluate the significance of the real-time quantitative
PCR array data, some theoretical observations seem in order. (i) Prior to reach-
ing saturation (due to exhaustion of primers and nucleotides, loss of polymerase
activity, etc.), PCR amplification proceeds exponentially and can be described by
Ni? N0? (1 ? k)i, where N0represents the number of molecules in the original
sample and Niis the number of mRNA molecules at cycle i (i ? 0 . . . 40). During
the exponential phase the amplification efficiency k (0 ? k ? 1) of a given primer
pair is constant. Real-time quantitative PCR visualizes the progressive amplifi-
cation at each cycle and measures the amount of product under exponential
conditions, exclusively. (ii) The amount of PCR product at each cycle is quan-
tified by the incorporation of a fluorescent dye (SYBRgreen) or the cleavage of
a TaqMan oligonucleotide (26). By either method the fluorescence intensity Rn
has a logarithmic dependence on fluorophor concentration, yielding Rn ?
log(Ni) ? log[N0? (1 ? k)i]. Real-time quantitative PCR compares two samples
with the target concentrations Naand Nbby recording the cycle numbers (CT) for
a and b at which the amplification product yields enough fluorescence to cross an
operator determined threshold, T (set at five times the SD of the non-template
control). Consequently, Rna? Rnband log[Na? (1 ? k)a] ? log[Nb? (1 ? k)b]
or log(Na) ? log(Nb) ? log(1 ? k)b ? log(1 ? k)a ? log(1 ? k)(b ? a) (for i ? 0,
Ni ? 0? N0? (1 ? k)0, i.e., Ni ? 0? N0). Ideally, k ? 1 and (1 ? k) ? 2, i.e.,
at each cycle two reactions products are produced per target molecule. This leads
to Ni? N0? (1 ? 1)i? N0? 2i. Assuming that log ? log2, Na/Nb? 2(b ? a),
where Na/Nbrepresents the fold difference in mRNA levels of two samples, CT
? a and CT? b. The algorithm used here was designed to identify primer pairs,
which under universal cycling conditions (94°C for 30 s and 60°C for 60 s [40
cycles]) have an amplification frequency of 1.8 ?(1 ? k) ? 2. This is achieved by
restricting the amplicon length to 100 ? 50 bp and the primer Tmto 60 ? 1°C for
each primer. Hence, it is possible to extract the relative ratio of abundance in two
samples based on this calculation. (iii) By comparison, solution hybridization-
based DNA arrays have similar characteristics, since the color intensity ratio in
a fluorescent Cy3/Cy5 DNA array also exhibits a logarithmic dependence on the
amount of hybridized probe. This is simply the nature of fluorescent absorption
(7). Analogous to the amplification efficiency k for PCR, a hybridization-effi-
ciency K0applies to DNA arrays, which is a function of the length and base
6214FAKHARI AND DITTMERJ. VIROL.
composition of the particular cDNA fragment at a given hybridization temper-
ature. For the purpose of this study, we therefore applied similar, rank-based
statistics and cluster algorithms (17) to compare the relative ratios of the mRNA
levels between different samples as determined by real-time quantitative RT-
Quantitative real-time PCR primers pairs were designed for
each KSHV ORF as described in Materials and Methods.
Table 1 lists a subset of primer pairs for which we generated
additional, sequence-specific TaqMan probes. This list com-
piles primers for spliced mRNAs of each KSHV kinetic class
(latent, alpha, beta, and gamma). To determine the specificity
of each primer pair, we analyzed the amplification products by
gel electrophoresis. Figure 1 shows an ethidium bromide-
stained agarose gel of the amplification products after 40 cycles
with cDNA from TPA-induced BCBL-1 cells as a template. No
primer dimers are visible in any of the reactions. The majority
of amplicons are of a similar size (ca. 50 to 100 bp) as predicted
by the design. Only a single band is amplified in each case even
after 40 cycles. Exceptions with regard to size include some of
the amplicons, which were designed to cross splice junctions or
which required special design, namely, orf57 (115 bp), orf72/
v-cyclin (192 bp), orf73/LANA 5?UTR splice (71 bp spliced
and 174 bp unspliced), LANA-2/vIRF-3spliced (174 bp spliced
and 268 bp unspliced), K9/vIRF-1 (141-bp latent start site and
61-bp lytic start site ), K6 (172 bp), K8.1 (255 bp unspliced
and 161 bp spliced), actin (295 bp), GAPDH (for glyceralde-
hyde-3-phosphate dehydrogenase) (225 bp), K14/vGPCR (234
bp spliced), orf22 (123 bp), orf26 (223 bp), orf41 (324 bp
spliced and 452 bp unspliced), and orf50 (111 bp). These re-
sults imply that unprocessed RNA or DNA species, which
contain small introns (?500 bp) can be amplified under uni-
versal cycle conditions, even though this is less efficient com-
FIG. 1. Ethidium bromide-stained 2% agarose gel of the PCR products for each KSHV ORF after 40 cycles. The template was reverse
transcribed poly(A) mRNA from BCBL-1 cells 48 h after TPA induction. Most amplicons are of the same size; exceptions are noted by name.
TABLE 1. Primer and probe sequences for selected KSHV latent and lytic mRNAs
VOL. 76, 2002CHARTING LATENCY TRANSCRIPTS IN KSHV6215
pared to the smaller, correctly spliced variant. Significant
amounts of the unspliced form only accumulated at higher
cycle numbers (compare the band intensities for the spliced
and unspliced forms in the case of orf73 5?UTR, vGPCR, and
orf41). Since quantification by real-time PCR is obtained from
the earlier, exponential phase of the reaction, these products
did not affect the outcome. In the case of the LANA/orf73 we
previously cloned two isoforms of its mRNA: one spliced and
another unspliced in the 5?UTR (15). Therefore, both PCR
bands stem from authentic mRNAs rather than from genomic
DNA. With regard to orf74/vGPCR a novel mRNA was re-
cently found which is unspliced and originates within the K14
ORF (46). This mRNA species can be amplified with our
primers and might explain the upper band on the agarose gel.
Large introns (?500 bp) were never amplified under universal
cycling conditions, as evidenced by the absence of any high-
molecular-size band in the case of orf29 (3,251-bp intron) and
v-cyclin (4,037-bp intron). Primer pairs for K1 were directed
against the intracellular domain of the ORF, which is con-
served for all clades of KSHV (51). The primer pair for K15
did not work in any of the assays, since primer sequences were
based on a BC-1-derived isolate (60) rather than BCBl-1 iso-
late, which differs in the K15 region (11, 24, 30, 51, 77) (a
separate effort is currently under way to design primers that
are specific for the different K15 spliced isoforms and different
clades). Similarly, primers for K12/kaposin were omitted from
the array, since kaposin mRNA varies widely between isolates
(45, 61, 75). In contrast to the CTvalues determined during
exponential amplification by real-time quantitative PCR, any
differences in the amounts of product seen by gel electrophore-
sis were coincidental. Overall, primers that were designed
based only on TaqMan criteria yielded the expected products
and did not display cross-reactivity to cellular mRNAs.
To obtain an independent measure of primer specificity, we
performed melting curve analysis after the final amplification
cycle. The reaction was gradually heated and actual Tmvalues
were obtained since SYBRgreen fluorescence is lost during
duplex to single-strand phase transition. Figure 2 shows the
melting curves for 10 representative amplicons. In each case
the single peak melting curve indicated that only a single am-
plification product was present in the reaction. Individual Tm
values were not identical, since they depended on the length
and base composition of the entire amplified sequence rather
than those of the flanking primers. Every amplicon in this study
yielded similar single peak melting curves (data not shown).
Taken together, the results obtained by these analyses instill
confidence in the specificity of the KSHV real-time quantita-
tive PCR array.
Genome scan by real-time quantitative RT-PCR. To dem-
onstrate that the PCR signal is a measure of the amount of
reverse-transcribed mRNA rather than contaminating geno-
mic DNA, we compared RT-positive to RT-negative cDNA
pools from TPA-induced BCBL-1 cells. Equal amounts of
total poly(A) mRNA (as determined by absorption spectros-
copy at 260 nm) served as starting material. Figure 3A plots the
unmanipulated CTvalues for each KSHV-specific primer pair
obtained under either condition (as mentioned above, CTval-
ues represent the amount of target on a log2scale). In this case,
primer pairs were sorted based on the CTof the RT-positive
reaction rather than their coordinates on the KSHV genome.
For each individual primer pair, RT-negative samples con-
tained much less target (as indicated by the higher CTvalue)
than the RT-positive samples. In all subsequent experiments
we adjusted the amount of input cDNA so that no amplifica-
tion could be observed with RT-negative samples. This dem-
onstrates that our assay measures KSHV mRNA levels and not
contaminating viral DNA.
Figure 3 also introduces the first layer of our quantitative
analysis. CTvalues are not a linear measure of target quantity,
since they depend logarithmically on the amplification effi-
ciency k for each primer pair, as well as the initial target copy
number N0. Although absolute copy numbers can be deter-
mined through dilution of an external standard, this is not
practical for a 96-primer pair array. Nor is it necessary for the
purpose of a genome scan experiment, which is—just like hy-
bridization-based DNA arrays—concerned only with the dif-
ferences between different treatments. Those were calculated
as follows: dCT? CT(mock) ? CT(treatment) for each primer
At no point in this study did we attempt to compare CT
values between two different primer pairs. However, we calcu-
lated the mean difference over all primer pairs between the
RT-positive and RT-negative sample. Figure 3B shows the
relative frequency of dCT? CT(RTneg) ? CT(RTpos). The
dCTs are normally distributed with mean difference of 13.25
and an SD of ?2.66. Based on an amplification efficiency of 1.8
? (1 ? k) ? 2 (213.25? 9,742 and 1.813.25? 2,412, respective-
ly), the fraction of contaminating DNA accounted for no more
than 0.05% of the signal. Figure 3B also superimposes the
normal distribution based on these parameters, which fits the
experimental data. The fact that CTvalues have a logarithmic
dependence on the mRNA concentration is extremely benefi-
cial for our statistical analysis, since the data are inherently
variance stabilized (17, 23).
What is the measurement error and linear range in these
experiments? To gain an understanding of the limitations of
the real-time quantitative RT-PCR array, two kinds of repli-
cate experiments were conducted. (i) We previously published
the dilution profiles for several primer pairs (29). This empha-
sized the extraordinarily robustness of real-time quantitative
PCR, since the CTvalues were linear dependent on input
FIG. 2. Melting curve profile for selected KSHV ORFs after 40
cycles (amplification products for orf26, K9spl, LANA-2spl, actin,
KbZIP, orf40spl, K9, LANA-2unspl, v-cyc, orf40unspl, orf57spl,
orf57unspl). The template was reverse transcribed poly(A) mRNA
from BCBL-1 cells 48 h after TPA induction. The temperature is
shown on the horizontal axis. The vertical axis shows the value of the
first derivative of the change in fluorescence intensity at each temper-
ature. Thus, the peak identifies the phase transition.
6216FAKHARI AND DITTMERJ. VIROL.
cDNA over more than 4 orders of magnitude, with as little as
103BCBL-1 cell equivalents as input. We have obtained sim-
ilar performance data for the primer pairs used here (data not
shown). All data in this particular study are based upon input
mRNA amounts, which were poly(dT) purified from ?104
BCBL-1 cells per primer pair (or 106, i.e., 1 ml of culture per
96-well array). Hence, we were in the middle of the linear
performance range of the assay. (ii) To determine the com-
bined handling and instrument error, we performed triplicate
measurements with a 22-primer-pair subset. Figure 3C com-
pares the standard deviation (SD) values for each triplicate
measurement (on the horizontal axis) to the mean CTvalue.
No correlation between the dCTand the SD is evident (R ? 0.4
by linear regression analysis). This establishes that the exper-
imental error is independent of the target concentration N0or
amplification efficiency k of a given primer pair.
Biological variation accounts for the overwhelming majority
of error in most genome array applications. To determine the
contribution of changes in culture conditions to our array anal-
ysis, we prepared mRNA from mock-treated BCBL-1 cells
cultured for 24, 48, or 72 h and determined the abundance of
KSHV mRNAs by using the real-time RT-PCR array. Initially,
we normalized each RT reaction according to the total amount
of input poly(A) mRNA. Under these circumstances the level
of GAPDH mRNA did not change significantly over time
(CTGAPDH ? 12.71 at 24 h, CTGAPDH ? 13.58 at 48 h, and
CTGAPDH ? 12.05 at 72 h). Since GAPDH mRNA was also the
most abundant mRNA species, we normalized all individual
CTvalues according to dCTtest ? CTtest ? CTGAPDH. Figure 4
shows the resulting dCTvalues for each of the KSHV primer
pairs at indicated times in culture. As a result of this normal-
ization, higher dCTvalues represent lower target concentra-
tions relative to the level of GAPDH mRNA. Primer pairs
were sorted according to dCTvalues at t ? 72 h. The dCT
values at 24 and 48 h are virtually identical for every mRNA. In
contrast, all values were increased after 72 h in the same
culture medium, i.e., expression levels were lower due the cells
entering saturation phase. Interestingly, this result was ob-
served after we adjusted for fluctuations in GAPDH mRNA
levels. This implies that the KSHV transcripts levels were de-
pleted in the stationary phase relative to GAPDH. An excep-
tion were the mRNA levels for K3, K5, orf55, and K11 which
were identical at all three time points (SD ? 0.5). RT-PCR was
able to detect a signal for lytic KSHV mRNAs even in the
absence of induction, since on average 3% of the BCBL-1 cells
undergo spontaneous reactivation in culture (56). If we assume
a total input of 106cells, that amounts to ?104cells, which is
well within the sensitivity of RT-PCR. To describe this result in
quantitative terms, the normal distributions for the relative
differences, ddCT? dCT(t ? 72) ? dCT(t ? 24, t ? 48), were
calculated for each primer pair and used to construct fre-
quency histograms. The mean ddCT for 48 and 24 h for all 96
primer pairs is ddCT? ?0.30 ? 1.4 compared to ddCT? 2.44
? 1.6 for 72 and 24 h and ddCT? 2.74 ? 1.3 for 72 and 48 h.
The difference between the 72 h and 24 h time points was
significant to P ? 5 ? 10?7based on a one-way analysis of
variance (ANOVA), whereas the differences in KSHV mRNA
levels between 48 and 24 h were insignificant. The data pre-
FIG. 3. (A) CTvalues for each primer pair in the KSHV array.
Closed circles indicate the CTvalues if poly(A) mRNA without RT was
used (RTneg); open circles indicate the CTvalues if the mRNA was
subjected to RT (RTpos). The vertical axis indicates the CTvalue, and
the horizontal axis indicates the percentile of primers. (B) Frequency
histogram of the dCTdifference between RTposand RTnegreactions
for each primer pair. Superimposed is the expected normal distribu-
tion. The relative frequency is shown on the vertical, and the dCTis
shown on the horizontal axis. (C) SD (on the horizontal scale) and
mean (n ? 3) CTvalues for a group of amplicons. The dotted line
indicates the mean of means.
FIG. 4. dCTvalues, which were normalized to CTGAPDH for each
primer pair in the KSHV array. The template was poly(A) mRNA
from untreated BCBL-1 cells held in culture for 24, 48, and 72 h. The
vertical axis indicates the dCTvalue, and the horizontal axis indicates
the percentile of primers.
VOL. 76, 2002CHARTING LATENCY TRANSCRIPTS IN KSHV6217
sented henceforth were obtained from BCBL-1 cells, which
were under experimental conditions for 48 h or less. The data
in Fig. 4 were used to establish the average margin of error for
further studies, i.e., the minimal biologically meaningful dif-
ference, which is significant to dCT? 1.50 or 21.5?3-fold (5 ?
the mean ddCT? 0.30 for the 48- and 24-h replicate measure-
ment of n ? 96 primers).
Identification of type one latency mRNAs in the KSHV ge-
nome. We and others previously identified a cluster of KSHV
latent mRNAs, based upon in situ hybridization of KS tumor
biopsies (13, 15, 54, 70). These experiments showed that the
LANA/orf73, v-cyclin/orf72, and v-FLIP/orf71 mRNAs—un-
like that of the latent mRNA for kaposin—were not induced
upon reactivation of BCBL-1 cells. The mRNAs encoding
LANA, c-cyc, and v-FLIP are coterminal, and the common
promoter for these mRNAs, likewise, was resistant to TPA
stimulation (29). Another latent mRNA, encoding LANA-2/
vIRF-3, has recently been described, but conflicting data have
been reported with regard to its expression profile (37, 58). We
therefore applied the KSHV real-time quantitative PCR array
to distinguish between the hypotheses (i) that LANA-1 was the
only type one latent mRNA in the KSHV genome, (ii) that
LANA-2 also belonged to this class, and (iii) that there might
be additional type one latent messages.
First, poly(A) mRNA was isolated either from TPA-treated
or mock-treated BCBL-1 cells at 48 h after induction and
subjected the real-time quantitative RT-PCR. Figure 5A shows
the outcome of such an experiment. In this representation dCT
values (normalized relative to GAPDH) are used as x and y
coordinates and are plotted for every primer pair of the KSHV
array. Hence, GAPDH marks the origin at 0,0. If two dCT
values are identical under either condition, they specify a point
on the 45° axis (slope of 1). If a given mRNA is induced by
TPA (lower dCT), the point is above, and if it is inhibited, the
point is below the 45° axis. orfK8.1 illustrates this projection:
mock-treated BCBL-1 cells yielded a dCT? 7.5, which is
indicated on the y axis. TPA-treated BCBL-1 cells yielded a
dCT? 2.5, which is indicated on the x axis. As before, a lower
dCTcorresponds to higher target concentrations. Assuming an
amplification efficiency of (1 ? k) ? 2, the induction can be
approximated to 2(7.5 ? 2.5 ? 5)? 64-fold, which agrees with
existing Northern blot and protein data (36, 52, 76). Almost
every KSHV mRNA is induced at 48 h after TPA treatment, as
evidenced by the location of their corresponding dCTabove
the 45° line. Several primer pairs in the array are specific for
the two latent mRNAs encoding LANA, v-cyclin, and v-FLIP.
All fall on the 45° axis, as indicated by the slope of regression
m ? 0.958 with R2? 0.967. The “LANA” primer pairs are
located within ORF LANA or in the LANA 5?UTR, primer
pair “v-FLIP” is located within ORF v-FLIP, primer pair “cyc”
is located within ORF v-cyclin, and primer pair “cyc-spl” spans
the v-cyclin splice site. The different amplicons do not have
identical dCTs, because the dCTvalues depend on (i) the level
of either the tricistronic 5,300-nucleotide mRNA encoding
LANA, v-cyclin, and v-FLIP or the bicistronic 1,700-nucleotide
mRNA encoding v-cyclin and v-FLIP, as well as (ii) the indi-
vidual amplification efficiencies k for each primer pair. The
dCTvalues for LANA-2/vIRF-3 (two primer pairs) also fall
into this group. Hence, LANA-2/vIRF-3 mRNA is another
latency type one mRNA in the KSHV genome (as represented
on our array). In addition to LANA and LANA-2/vIRF-3,
there are other primer pairs on the 45° line, as well some
primer pairs the target mRNA of which seemed inhibited by
TPA (below the 45° line). However, these mRNAs increased at
later time points after induction as discussed below.
To gain a better perspective of the significance of these
observations, we calculated the ddCTas dCTGAPDH (mock) ?
dCTGAPDH (TPA). The result is graphed in Fig. 5B. ddCTfor
GAPDH is located at 0; a positive ddCTindicates induction,
and a negative ddCTindicates inhibition. The ddCTfor
GAPDH marks the 25th percentile of KSHV mRNAs on the
FIG. 5. (A) Plot of the dCTvalues (normalized to gapdh) for each
primer pair in the KSHV array (circles). The vertical axis indicates the
dCTvalue from mock-treated BCBL-1 cells, and the horizontal axis
indicates the dCTvalue from 48-h-TPA-treated BCBL-1 cells. Also
shown is the regression line, equation, and R2value through the values
for latency type one mRNAs (solid dots). The dotted line traces the
values for orfK8.1 under either condition. The “LANA” primer pairs
are located either within the LANA ORF or within the LANA 5?UTR
Similarly, for LANA-2/vIRF-3 we designed two primer pairs, one per
exon. (B) Plot of the difference for each primer pair in the KSHV array
between mock- and TPA-treated BCBL-1 cells at 48 h after induction.
CTvalues were normalized to GAPDH to yield dCT, which were then
compared to yield ddCT. The vertical axis indicates the ddCTvalue, the
horizontal axis the percentile of the total distribution. Note that the
ddCTvalues represent a log transformation of the relative abundance
of the target mRNA. A value of ddCT? 0 indicates identical abun-
dance of the target mRNA under each condition. A positive ddCT
value indicates induction, and a negative ddCTvalue indicates sup-
pression. Arrows indicate the values of latency type one genes.
6218FAKHARI AND DITTMER J. VIROL.
array; in other words, 75% of the KSHV primer pairs are
induced relative to GAPDH. For instance, orfK8.1 represents
one of the most highly altered KSHV mRNAs. In contrast,
each primer pair that measures the abundance of a latent
mRNA has (i) a negative ddCT, (ii) differs by ?1 ddCTunit
from GAPDH, and (iii) is located in the lower 25th percentile
of all data. Other mRNAs with a ddCTvalue in the lower 25th
percentile were the ORFs 19, 21, 40, 67, 68, K5, K9, and K4.
However, their ddCTvalues increased at later time points (see
below). This demonstrates that LANA and LANA-2 are resis-
tant to TPA induction, which is a novel, unique property
among KSHV mRNAs.
To further corroborate this result, we diluted the input
cDNA pool 10-fold and subjected it again to real-time quan-
titative PCR analysis. A similar picture ensued (data not
shown) verifying that relative induction ratios did not depend
upon total mRNA levels. We then calculated the difference
ddCT? dCTGAPDH (mock) ? dCTGAPDH (TPA) for each
mRNA. Next, ddCTvalues were rank ordered and split into
two groups: group 1 contained the results of primer pairs for
the predicted latency type one genes (n ? 8, since multiple
primer pairs for the each latency type one mRNA were used),
and group 2 contained all other KSHV primer pairs (n ? 85).
Actin, c-myc, and GAPDH were excluded. Next, the ANOVA
based on ranks was calculated, which showed that both groups
differed significantly (P ? 0.0004). This demonstrates that la-
tency type one mRNAs are a separately regulated class of
KSHV mRNAs and that both LANA and LANA-2/vIRF-3
belong to this class.
It is important to bear in mind that the magnitude of induc-
tion is dependent on the amount of mRNA in the mock con-
trol, as well as the amount in the induced sample. About 3% of
BCBL-1 cells express a high enough level of lytic proteins to be
detected by immunofluorescence in the absence of stimulation
(56, 76). The nut-1 message is a point in case. Its levels are
increased 10- to 100-fold by TPA, depending on the experi-
mental conditions and the method of observation (28, 69, 74).
Since nut-1 mRNA is also the most abundant lytic mRNA,
based on absolute copy number, it is easily detected in mock-
treated PEL cells (63, 75). Yet, in situ hybridization showed
that it is present in only a few, lytically infected, KS and PEL
tumor cells (68). As determined by real-time quantitative RT-
PCR, overall nut-1 mRNA levels were as abundant as GAPDH
mRNA levels and, after TPA induction, nut-1 mRNA was
more abundant than GAPDH mRNA. Similarly, some early
promoters might be leaky in cells even though these cells never
complete the KSHV lytic cycle, resulting in a significant level
of mRNA in mock-treated BSBL-1 cultures. This complicates
any interpretation based solely on a single time point.
To verify that LANA-2’s resistance to lytic reactivation held
true over time, we performed a time course experiment. Again,
BCBL-1 cells were induced by TPA, and mRNA was isolated
at various times after induction and quantified by real-time
RT-PCR. Figure 6 shows the outcome of the experiment. In
this case, dCTvalues (normalized to GAPDH) were sorted
based on the 24-h-mock-treated values and plotted for each
primer pair. In contrast to mock treatment (see Fig. 4), dCT
values (and the underlying mRNA levels) were clearly different
as early as 24 h after TPA induction compared to the mock-
treated control at 24 h. As expected, the real-time RT-PCR
amplification for two cellular genes actin and c-myc (under-
lined in Fig. 6) yielded dCTvalues which were independent of
TPA (c-myc is induced within 30 min of serum stimulation in
3T3 cells  but returns to steady-state levels thereafter). The
primer pairs that measured mRNAs that did not respond to
TPA in a consistent manner were also determined (indicated
by asterisks in Fig. 6). In the extreme, these were the result of
a pipetting or instrument error for that particular well and time
FIG. 6. Plot of dCT(normalized to GAPDH) for each primer pair in the KSHV array for TPA-treated BCBL-1 cells at 24, 48, and 72 h after
induction as well as for mock-treated cells at 24 h. The vertical axis indicates the dCTvalue, and the horizontal axis indicates the percentile of the
total distribution. Note that the ddCTvalues represent a log transformation of the relative abundance of the target mRNA. The arrows indicate
primer pairs for which all three time points, as well as mock-treated cells, exhibit similar dCTvalues, i.e., the mRNA did not change upon TPA
treatment. Primer pairs which measured mRNAs that did not respond to TPA in a consistent manner are indicated with asterisks.
VOL. 76, 2002 CHARTING LATENCY TRANSCRIPTS IN KSHV6219
point. In other cases, they represented mRNAs that were in-
duced with delayed kinetics or which were unusually abundant
in the mock-treated sample. In contrast, the arrows in Fig. 6
denote the results of primer pairs for latency type one mes-
sages, for which all three independent datum points—24, 48,
and 72 h after TPA treatment—were nearly identical. More-
over, their dCTvalues were the same or higher (indicating a
lower mRNA abundance) in TPA-treated BCBL-1 cells as for
mock-treated BCBL-1 cells. This group of mRNAs was qual-
itatively and quantitatively different from the rest of the KSHV
Figure 7 shows a conventional cluster analysis of the data.
All latency type one mRNAs (LANA, LANA-2, v-cyc, and
v-FLIP) group together, and the cellular genes actin, GAPDH,
and myc are also part of this cluster. In contrast, immediate-
early lytic mRNAs (orf50/Rta and 57/Mta), spliced forms for
k-bZIP and gamma-2 mRNAs (orfK8.1 and orf29), are located
elsewhere. The spliced v-cyclin mRNA signal is segregated
from the other latency mRNAs, since its levels are higher in
mock-treated than in TPA-treated samples. This effect is most
pronounced at 72 h, suggesting that spliced v-cyclin mRNA
might be rapidly degraded in lytically replicating cells. Cluster
analysis revealed that orfK1 and orf75 also group with the
latency type one mRNAs, whereas both mRNAs were clearly
induced, as judged by a single 48-h time point comparison. It is
conceivable that, by chance, a summation over the principal
components (time points) for K1 and orf75 gives the same rank
as a latent pattern (K1 is one of the mRNAs for which the
clustering result of the two previous array analyses is discor-
dant [28, 50]). This is one of the theoretical limitations of any
cluster analysis. Since multiple mRNAs are transcribed across
the K1 locus and are regulated differently (62), array analysis
for this ORF is less reliable. By and large every single KSHV
mRNA ordered according to real-time quantitative RT-PCR
analysis follows the transcription pattern that was previously
established by using the KSHV cDNA arrays (28, 50). This
demonstrates that the mRNAs for LANA, v-cyclin, v-FLIP,
and LANA-2 were not induced by TPA at any time after TPA
induction. No other mRNA that can be measured with our
array shared this property. This supports a model in which
latency type one mRNA levels are regulated differently than
the rest of the KSHV genome. They are resistant to the reac-
tivation-induced genome-wide induction.
Genome-wide transcriptional analyses provide valuable in-
sights into the biology of KSHV and human pathogens in
general. The first latent KSHV mRNA (kaposin) and the lytic
mRNA (nut-1) were identified through reverse Northern hy-
bridization of labeled total mRNA from mock- or TPA-treated
BCBL-1 cells with a restriction digest of KSHV genomic frag-
ments as a template (75). A whole-genome Northern blot scan
for KSHV transcripts introduced the current classification into
type one (latent), two (latent or lytic), and three (lytic only)
mRNAs (63). Similar, more detailed data have since been
obtained by using KSHV DNA gene chips, which employed
full-length predicted KSHV cDNAs as a template (28, 50). The
latter experiments classified KSHV transcripts into temporal
classes (alpha, beta, and gamma), analogous to the other her-
FIG. 7. Cluster analysis with dCT(normalized to GAPDH) values
of each primer pair in the KSHV array for TPA-treated BCBL-1 cells
at 24, 48, and 72 h after induction, as well for as mock-treated cells at
24 h. Data were median centered by gene and time point. Hence,
mRNAs that are underrepresented are in shades of green, and those
that are overrepresented are in red.
6220FAKHARI AND DITTMER J. VIROL.
pesviruses. Because DNA arrays are based on solution hybrid-
ization, only substantial differences in the level of each target
mRNA could be discerned, and large amounts of sample [ca.
107cells or ?1 ?g of poly(A) mRNA per routine hybridiza-
tion] were needed (14, 27). A variety of template amplification
methods are currently under development to address this prob-
lem (e.g., linear amplification with T7 polymerase or template
switching [41, 42, 65]). However, these methods may introduce
a bias because of sequence-dependent processivity of the poly-
merase. According to one manufacturer, the concordance of
enzymatically amplified cDNA pools compared to nonampli-
fied mRNA is estimated at no more than 80% (Clontech, Inc.).
In contrast, real-time quantitative RT-PCR allows the quanti-
fication of a smaller set of mRNAs but with, in principal, single
cell sensitivity (22). Since real-time quantitative PCR ampli-
cons are on average 100 bp and all primers fit similar, highly
restrictive performance criteria, enzyme processivity and sec-
ondary structures pose less of an issue. The real-time quanti-
tative RT-PCR array introduced here can be improved to use
as little as 1 ng of poly(A) mRNA or 103cells per experiment
(unpublished observations). Greater sensitivity, i.e., fewer in-
put cells is achievable, but at the limit random target cell
variation becomes a concern. While a Northern blot can dis-
tinguish between differentially spliced transcripts, DNA array
experiments (with PCR-amplified DNA fragments) at present
do not. Since a significant proportion of herpesvirus transcripts
are spliced and many more are 3? coterminal, a genome scan by
using real-time, quantitative RT-PCR and primers designed
for specific splice sites is ideally suited for investigating this
genus of human pathogens.
Automated primer design based on predicted KSHV ORFs
proved surprisingly successful. More than 90% of the primer
pairs, which were designed for real-time, quantitative PCR
under universal cycling conditions, worked without further op-
timization at a single primer concentration (166 nM) and
Mg2?-ion concentration. Genomic DNA contamination was
eliminated through poly(dT) purification of the input RNA,
although limited DNase I digestion suffices for this application
as well (unpublished observations). All primer pairs amplified
a single KSHV-specific product, which in this study was reliably
detected by using a double-strand-specific dye rather than ad-
ditional, more costly fluorescently labeled probes. Whether
this sensitivity and specificity is sufficient for clinical studies is
currently under investigation.
Several groups previously used levels of a single KSHV
mRNAs or protein as a surrogate marker for KSHV infection.
Compared to the whole-genome transcript pattern, we can now
evaluate the appropriateness of these choices. (i) orf29 mRNA
primers were originally developed for the detection of lytically
replicating KSHV by conventional RT-PCR (55). orf29 protein
encodes a capsid component and, based on Northern analysis
in BCBL-1 cells, the orf29 mRNA falls into the gamma-2
temporal class. Since mature orf29 mRNA is spliced by remov-
ing a 3,251-bp intron, primer pairs that are located on opposite
sides of the intron never amplify contaminant DNA. Because
of this property, quantification of orf29 spliced mRNA consti-
tutes the most stringent assay for complete KSHV lytic repli-
cation. (Since a second mRNA encoding orf48 also splices into
orf29 exonB, analysis of intra-exon sequences alone can result
in conflicting data.) Adopting the orf29 assay for real-time
quantitative RT-PCR allowed us to quantify the inhibition of
KSHV de novo lytic replication in SCID-hu Thy/Liv mice by
ganciclovir (16). However, CTvalues for spliced orf29 mRNA
were consistently higher (5 to 10 cycles) than CTvalues of
other spliced gamma-2 amplicons, such as orfK8.1. In other
words, a much higher proportion of late lytically infected cells
must be present in the culture to yield a significant orf29 signal.
This suggests that correctly spliced orf29 mRNA is present in
low quantities, late in the infectious cycle, with either a short
half-life or unstable and that this assay therefore underesti-
mates the number of lytically infected cells. (ii) Earlier on, we
also developed conventional primers for spliced v-cyclin latent
mRNA, as well as corresponding real-time quantitative PCR
primers (16). Like the primers for orf29, the primers for v-
cyclin mRNA are located on opposite sides of a large (4,037-
nucleotide) intron. Hence, primers for spliced v-cyclin mRNA
provide a specific probe for latently infected cells. (iii) Other
real-time quantitative PCR primers for KSHV have been pub-
lished, but the corresponding amplicons do not cross splice
sites (3, 6, 18, 34). This has the advantage that the same
primer-probe combination can be used to quantify KSHV viral
load, but it relies on careful and more extensive provisions to
remove contaminating viral DNA if these primers are used for
transcript analysis or transmission studies. Based on these in-
sights we compiled a set of primer pairs and corresponding
TaqMan probes for high-throughput, high-sensitivity, high-
specificity staging of KSHV-infected cells (see Table 1). These
encompass well-characterized spliced latent, alpha, beta, and
gamma mRNAs, most of which have been independently val-
idated by protein expression studies and/or in situ analysis in
The principal question of this inquiry was the assignment of
LANA-2/vIRF-3 as a class one latency mRNA, similar to
LANA, v-cyclin and v-FLIP. LANA-2/vIRF-3 is comprised of
the predicted K10.5 and K10.6 orfs and was initially identified
based on its sequence similarity to viral interferon regulatory
factors (vIRFs) (37, 58). Indeed, like KSHV vIRF-1/K9 and
vIRF-2/K11.1, LANA-2/vIRF-3 inhibits interferon signaling in
experimental systems. LANA-2/vIRF-3 stands out, however,
because of its uniform, widespread expression in PEL and
multifocal Castleman’s disease. Overall, the transcriptional or-
ganization of the K10 region is quite complicated. LANA-2/
vIRF-3 is encoded by a 1,701-bp ORF, which is readily de-
tected in untreated PEL cell lines (37, 58). All three primer
pairs, which were specific for either exon I or exon II or which
spanned the splice junction in between, yielded concordant
results, and these results paralleled the pattern of LANA/v-
cyc/v-FLIP transcription. Hence, LANA-2/vIRF-3 is encoded
by a latency type one mRNA. In contrast, vIRF-1/K9 and
vIRF-2/K11.1 mRNAs are induced by TPA with early kinetics
in PEL cells, as evidenced by the simultaneous comparison
Of major concern in comparing transcription patterns be-
tween various studies are the myriad technical differences such
as timing, drug concentration, and single gene probe charac-
teristics. In the case of KSHV-infected PEL cell lines, culture
conditions and time in culture exert a dramatic influence on
the timing and efficiency of viral reactivation. Relating the
transcription profiles for all KSHV mRNAs provides a com-
VOL. 76, 2002CHARTING LATENCY TRANSCRIPTS IN KSHV 6221
mon frame of reference within in a single experiment, but this
is still a reflection of the particular experimental setup.
While KSHV lytic transcription appears to be an all-or-
nothing response, which proceeds to completion once orf50 is
expressed, KSHV can enter very distinct latency programs in
terms of temporal regulation (latency type one or two) and
tissue specificity (KS tumor versus B-cell lineage). All class one
latency mRNAs are resistant to the effects of other chemical
inducers such as ionomycin or butyrate, as well as TPA (data
not shown), whereas no other KSHV transcripts share this
feature. This suggests that LANAp, the promoter for LANA,
v-FLIP, and v-cyclin (15, 29), and the LANA-2/v-IRF-3 pro-
moter share common regulatory features. The fact that latency
type one mRNAs do not increase upon viral reactivation in
BCBL-1 cells suggests that their expression might be latency
specific and that latency type one mRNAs might be expressed
in latently infected cells to the exclusion of lytic mRNAs. Al-
ternatively, latency type one mRNAs might be constitutively
transcribed, but their promoters are isolated from the orf50-
mediated upregulation of neighboring lytic transcription units.
We thank Mike Lagunoff and Jeffry Martin for critical reading of the
manuscript, Rebecca Hines-Boykin for invaluable technical help, and
David Dyer for oligonucleotide synthesis.
This work was supported by a grant from the Oklahoma Center for
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VOL. 76, 2002CHARTING LATENCY TRANSCRIPTS IN KSHV6223