JOURNAL OF VIROLOGY, July 2011, p. 6579–6588
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 13
Chemical Induction of Endogenous Retrovirus Particles from the Vero
Cell Line of African Green Monkeys?
Hailun Ma, Yunkun Ma,† Wenbin Ma, Dhanya K. Williams, Teresa A. Galvin, and Arifa S. Khan*
Laboratory of Retroviruses, Division of Viral Products, Center for Biologics Evaluation and Research,
U.S. Food and Drug Administration, Bethesda, Maryland 20892
Received 21 January 2011/Accepted 22 April 2011
Endogenous retroviral sequences are present in high copy numbers in the genomes of all species and may
be expressed as RNAs; however, the majority are defective for virus production. Although virus has been
isolated from various Old World monkey and New World monkey species, there has been no report of
endogenous retroviruses produced from African green monkey (AGM) tissues or cell lines. We have recently
developed a stepwise approach for evaluating the presence of latent viruses by chemical induction (Khan et al.,
Biologicals 37:196–201, 2009). Based upon this strategy, optimum conditions were determined for investigating
the presence of inducible, endogenous retroviruses in the AGM-derived Vero cell line. Low-level reverse
transcriptase activity was produced with 5-azacytidine (AzaC) and with 5?-iodo-2?-deoxyuridine (IUdR); none
was detected with sodium butyrate. Nucleotide sequence analysis of PCR-amplified fragments from the gag, pol,
and env regions of RNAs, prepared from ultracentrifuged pellets of filtered supernatants, indicated that
endogenous retrovirus particles related to simian endogenous type D betaretrovirus (SERV) sequences and
baboon endogenous virus type C gammaretrovirus (BaEV) sequences were induced by AzaC, whereas SERV
sequences were also induced by IUdR. Additionally, sequence heterogeneity was seen in the RNAs of SERV- and
BaEV-related particles. Infectivity analysis of drug-treated AGM Vero cells showed no virus replication in cell
lines known to be susceptible to type D simian retroviruses (SRVs) and to BaEV. The results indicated that
multiple, inducible endogenous retrovirus loci are present in the AGM genome that can encode noninfectious,
Endogenous retroviral sequences are stably integrated, ge-
netically inherited, and present in multiple copies in the ge-
nomes of all species. The majority of these sequences are
defective; however, some may produce infectious retroviruses
(9, 11, 16, 71, 78). In general, newly acquired endogenous
retroviral sequences are more likely to be associated with an
infectious virus, whereas ancient sequences may be transcrip-
tionally active but defective for virus production (86) or pro-
duce noninfectious particles (38). In rodents, endogenous ret-
roviruses can become activated in animals, as a consequence of
age (4), or in cell lines, either spontaneously by long-term
culture passage (2, 49, 63) or by treatment with a variety of
inducers, including biological, immunological, and chemical
agents (1, 13, 16, 25, 39, 47, 50). In humans or nonhuman
primates (NHPs), spontaneous release of endogenous retrovi-
ruses has been reported from tumor tissues and cell lines, as
well as from normal placenta (9, 12, 14, 15, 19, 20, 27, 28, 34,
43, 44, 51, 52, 58, 62, 64–66, 69, 72, 73). Endogenous retrovi-
ruses have also been isolated from NHP cells by extended
cultivation (6 to 8 months) of normal primary cell cultures and
cell lines (67, 75, 76, 79). The number of endogenous primate
retroviruses isolated thus far is limited, and the virus isolates or
the tissues from which they were recovered are not readily
available for characterization by current state-of-the-art meth-
ods or for the development of reagents for further investiga-
Endogenous retroviruses have been reported from a variety
of NHP species, including Old World primates and New World
primates, but there has been no evidence of endogenous ret-
roviral particles produced from African green monkey (AGM)
tissues or from cell lines derived from this species. The Vero
cell line, derived from the kidney of a normal, adult AGM
(Chlorocebus species, formerly called Cercopithecus aethiops)
(87), is used broadly in research and virus diagnostics as well as
in vaccine development due to its broad susceptibility to infec-
tion by different viruses. This cell line has been shown to be
nontumorigenic at low passage levels (7, 18, 22, 32, 48, 54, 74,
84) and negative for viruses by extensive testing using a variety
of assays, including PCR assays and standard chemical induc-
tion (29, 74). We have recently developed a stepwise strategy
for using chemical inducers to optimize induction conditions
for investigating the presence of latent viruses (37). We have
used this strategy to evaluate activation of endogenous retro-
viral sequences in Vero cells, which, although known to con-
tain numerous copies of endogenous retroviral sequences due
to their AGM species of origin (5, 8, 10, 26, 31, 36, 42, 57, 68,
81), were not expected to contain an inducible virus, based
upon the extensive testing history and broad use of the cell line
(29, 74). Here, we report that treatment of Vero cells with
5-azacytidine (AzaC) and with 5?-iodo-2?-deoxyuridine (IUdR)
induced endogenous retroviral particles related to ancient sim-
* Corresponding author. Mailing address: Laboratory of Retrovi-
ruses, Division of Viral Products, Center for Biologics Evaluation and
Research, U.S. Food and Drug Administration, 8800 Rockville Pike,
HFM-454, Bldg. 29B, Room 4NN10, Bethesda, MD 20892. Phone:
(301) 827-0791. Fax: (301) 496-1810. E-mail: email@example.com.
† Present address: Division of Product Quality, Center for Biologics
Evaluation and Research, U.S. Food and Drug Administration, Be-
thesda, MD 20892.
?Published ahead of print on 4 May 2011.
ian endogenous type D betaretrovirus (SERV) sequences that
are present in all Old World monkeys (83) and distinct from
pathogenic, type D simian retroviruses (SRVs) (46). Addition-
ally, particles containing baboon endogenous virus (BaEV)-
related type C gammaretrovirus sequences were also induced
from AzaC-treated Vero cells. Infectivity analysis of drug-
treated Vero cells indicated the absence of a replicating virus
using various target cells known to be susceptible to SRVs and
BaEV. The results demonstrate the use of optimized chemical
induction conditions for investigating infectious, endogenous
retrovirus loci in the genomes of primates and other species.
MATERIALS AND METHODS
Cell lines and chemicals. Cell lines were obtained from American Type Cul-
ture Collection (ATCC; Manassas, VA; http://www.atcc.org/). The Vero cell line
(AGM kidney cells; ATCC catalog no. CCL-81, lot no. 3645301; passage 120)
was grown in Eagle minimum essential medium (EMEM) (modified), with Ear-
le’s salts and without L-glutamine (Mediatech, Manassas, VA; catalog no. 15-
010-CV), supplemented with 5% fetal bovine serum (FBS), per ATCC instruc-
tions (heat inactivated at 56°C for 30 min; HyClone, Logan, UT; catalog no.
SH30071.03), 2 mM L-glutamine, 250 U of penicillin per ml, 250 ?g of strepto-
mycin per ml, 1? nonessential amino acids (MEM-NEAA 100?; Quality Bio-
logical Inc., Gaithersburg, MD, catalog no. 116-078-061), and 1 mM sodium
pyruvate (Quality Biological, Inc.), designated complete medium. To maximize
reproducibility of results in the induction assays, a cell bank was established at
passage 123. A new vial was used in each experiment to maintain similar passage
numbers in the induction studies.
For virus-induction studies, Vero cells were treated with different concentra-
tions of IUdR (stock solution, 75 mg per ml in 1 N NH4OH; Sigma, St. Louis,
MO; catalog no. 17125), AzaC (1 mg/ml in complete Vero cell medium; Sigma
catalog no. A1287), and sodium butyrate (NaBut; 0.9 M in sterile H2O; Sigma
catalog no. B5887).
Target cell lines used in infectivity studies were A-204 (human rhabdomyo-
sarcoma; ATCC catalog no. HTB-82), Raji (human B cell lymphoma; ATCC
catalog no. CCL-86), and Cf2Th (dog thymus; ATCC catalog no. CRL-1430).
Cf2Th cells were grown in Dulbecco’s modified Eagle medium (DMEM; Invi-
trogen, Carlsbad, CA; catalog no. 119955) supplemented with 20% FBS; A-204
and Raji cells were grown in RPMI 1640 (Quality Biological catalog no. 112-
024-101) supplemented with 10% FBS and 1? MEM-NEAA. Additionally, both
media contained 2 mM L-glutamine, 250 U of penicillin per ml, and 250 ?g of
streptomycin per ml.
Growth curve and population doubling time (PDT). Cells were counted using
an automated Guava PCA flow cytometer according to the manufacturer’s pro-
tocol (Guava ViaCount assay; Hayward, CA). Cells were diluted, and the num-
bers of viable cells, dead cells, and apoptotic cells were counted in triplicate. The
average count of the viable cell numbers was used in the experiments. For cell
cycle analysis, 0.5 ? 106cells were processed and stained according to the
manufacturer’s protocol (Guava cell cycle assay).
To determine the optimum number of cells for obtaining a sigmoidal growth
curve, Vero cells (0.5 ? 106, 0.75 ? 106, and 1.0 ? 106) were planted in 5 ml
complete medium in 25-cm2flasks, and viable cells were counted at various times
using a Guava PCA cytometer. Cell cycle analysis was done to determine the cell
phases. PDT was calculated as 1/k ? T (where T ? PDT) from the linear curve
in the log phase from the formula N ? N02kt(where N ? total viable cell number
at end time t; N0? total viable cell number at initial time t0, t ? hours from N0
to N, and k ? regression constant) (60). Results were confirmed in three inde-
Drug dose evaluation. Vero cells (1 ? 106; passages 125 to 131) were planted
for 16 h in 25-cm2flasks before replacing medium with fresh medium containing
different concentrations of AzaC (0.3125 to 40 ?g/ml), IUdR (50 to 3,200 ?g/ml),
or NaBut (1 to 6 mM). After 48 h of drug treatment, cells were washed with
medium three times (designated day 0), trypsinized, and counted by using a
Guava PCA cytometer. Another set of flasks were further incubated after the
medium change, and the cells were trypsinized and counted at day 3. Untreated
cells, at confluence, were used as a control to evaluate cell toxicity and cell
recovery based upon the cell-confluence ratio at day 0 and at day 3, which was
calculated by dividing the number of viable cells in the drug-treated flask by the
number of viable cells at confluence in the untreated control flask (2.3 ? 106
cells) and expressed as a percentage. Furthermore, in the case of IUdR-treated
cells, additional controls were included to evaluate toxicity due to NH4OH used
for dissolving the drug. In case the number of cells in the NH4OH-treated flask
was less than that of the untreated control flask, the number of viable cells in the
IUdR-treated flask was divided by the number of viable cells in the NH4OH-
treated flask before determining the cell-confluence ratio.
Chemical treatment and evaluation for induced retroviruses by the STF-
PERT assay. Vero cells were drug treated under optimized induction conditions:
cells (1 ? 106) were planted for 16 h and then treated with drug for 48 h (AzaC,
1.25 ?g/ml; IUdR, 200 ?g/ml; and NaBut, 3 mM); untreated cells were included
as a control. For kinetics of virus induction, medium was replaced daily and
filtered supernatant collected for detection of reverse transcriptase (RT) activity
by the single-tube fluorescent PCR-enhanced reverse transcriptase (STF-PERT)
assay (53). Supernatants were collected and filtered (Costar Spin-X centrifuge
tube filters, 0.45-?m-pore-size CA membrane; Corning, Corning, NY, catalog
no. 8162) on the day of drug removal (day 0), prior to washing the cells, and then
daily, at each medium change. Filtered supernatants were stored at ?80°C in
single-use, 10-?l aliquots for STF-PERT analysis and in 0.5-ml aliquots for
additional use. Each sample was tested at a 1:10 dilution (per the assay protocol),
and results were obtained from triplicate samples. The PERT assays for testing
supernatant from drug-treated cells met the acceptability criteria (53): IUdR,
slope ? ?3.96, y intercept ? 48.06, r2? 0.999; AzaC, slope ? ?3.14, y inter-
cept ? 42.59, r2? 0.996; NaBut, slope ? ?3.97, y intercept ? 48.05, r2? 0.996.
Negative controls were cells without drug (or with NH4OH in the case of IUdR
cell toxicity studies) and were set up in parallel.
RT-PCR. Total cellular RNAs were extracted by using the RNeasy Plus minikit
(Qiagen, Valencia, CA; catalog no. 74134) in combination with the RNase-free
DNase set (Qiagen; catalog no. 79254) according to the manufacturer’s instruc-
tions. Concentration and purity were determined by using UV absorbance.
A low-concentrated (10?) supernatant sample was prepared from normal and
drug-treated cells by ultracentrifugation of filtered supernatant (1.5 ml) at 45,000
rpm (Beckman TLA 45 rotor) for 90 min at 4°C. RNA was prepared from the
pellet by resuspending it in 130 ?l of Promega DNase buffer and adding 10 ?l
DNase (1 U per ?l; RNase-free DNase; Promega, Madison, WI, catalog no.
M6101) for incubation at 37°C for 30 min. RNA was extracted from the entire
sample by using the QIAamp viral RNA minikit (Qiagen catalog no. 52904).
A high-concentrated (1,000?) supernatant sample was prepared from normal
and from AzaC-treated Vero cells (1.25 ?g/ml for 48 h) on day 4 after drug
treatment (medium was changed on day 1) by ultracentrifugation of pooled (180
ml), filtered supernatant (tube top vacuum filters, 0.45-?m-pore-size CA mem-
brane, Corning catalog no 430314) on a 20% sucrose cushion (25,000 rpm in a
Beckman SW-28 rotor for 4 h at 4°C). Pellets were pooled, resuspended in 4 ml
phosphate-buffered saline (PBS) (pH 7.4), and ultracentrifuged immediately at
35,000 rpm (Beckman SW-41 rotor) for 90 min at 4°C. The pellet was resus-
pended in 180 ?l in PBS (pH 7.4) and stored in aliquots at ?80°C to minimize
freeze-thaw of test samples. RNA was extracted from 50 ?l using the QIAamp
viral RNA minikit after DNase I digestion (1 U per ?l), as described above.
One-half of the RNA sample was used for cDNA synthesis using the iScript
cDNA synthesis kit (Bio-Rad, Hercules, CA; catalog no. 170-8890) according to
the manufacturer’s instructions. The other half of the RNA was used for control
without RT. Additionally, PCR amplification using human ?-actin primers was
performed to demonstrate absence of cellular DNA according to the manufac-
turer’s protocol (Clontech, Mountain View, CA; catalog no. 639008).
Consensus PCR primers (SRV/SERV) were designed based upon GenBank
sequences of SRV-1 type D retrovirus (M11841), SRV-2 complete genome
(AF126467), simian Mason-Pfizer D-type retrovirus or SRV-3 (M12349), and
simian type D virus 1, complete proviral genome (U85505; designated SERVbab
in this paper). The location of the SRV/SERV primers is given in Table 1: a long
terminal repeat (LTR) gag fragment (553 bp) was amplified using forward primer
F04, 5?-CTGTCTTGTCTCCATTTCT-3?, and reverse primer R10, 5?-ACSGC
AGCCATKACTTGYGG-3?; a pol fragment (610 bp) was amplified using for-
ward primer F41, 5?-TACAAGAYCCMTAYACCTA-3?, and reverse primer
R46, 5?-TTDGGTGGRTAATGGTTRTC-3?; and an env fragment (548 bp) was
amplified using forward primer F65, 5?-CAYATNTCYGATGGAGGAGG-3?,
and reverse primer R70, 5?-CCYGTCCARTTTGTRGGTA-3?. PCR conditions
were 95°C for 3 min, followed by 35 amplification cycles of 95°C for 30 s, 56°C for
1 min, and 72°C for 1 min, with a final extension at 72°C for 10 min.
Primers for amplification of BaEV sequences and PCR cycle conditions were
as described previously (82): RT1 and RT2 in the pol region, and ENV1/ENV4
with nested primers ENV2/ENV3 in the env region. Additional primers were
made for PCR amplification in the gag region: outer primer pairs were GAG1
(5?-GAGTGGCCCACCCTTCATGT-3?) and GAG2 (5?-CAGTACTGGATCG
TGCGGTT-3?), at nucleotide positions 1108 to 1127 and 1697 to 1678, respec-
tively, and inner primer pairs were GAG3 (5?-CCCCGGGACGGAACTTTTG
A-3?) and GAG4 (5?-GATGAGGTAGAGGGTCTTGGAAG-3?) at nucleotide
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