JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2007, p. 477–487
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 45, No. 2
Use of New T-Cell-Based Cell Lines Expressing Two Luciferase
Reporters for Accurately Evaluating Susceptibility to
Anti-Human Immunodeficiency Virus Type 1 Drugs?
Tomoko Chiba-Mizutani,1,2Hideka Miura,1Masakazu Matsuda,1Zene Matsuda,1
Yoshiyuki Yokomaku,1Kosuke Miyauchi,1Masako Nishizawa,1
Naoki Yamamoto,1,2and Wataru Sugiura1*
AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan,1and Department of Molecular Virology and
Microbiology, Tokyo Medical and Dental University, Tokyo, Japan2
Received 17 August 2006/Returned for modification 25 October 2006/Accepted 11 December 2006
Two new T-cell-based reporter cell lines were established to measure human immunodeficiency virus
type 1 (HIV-1) infectivity. One cell line naturally expresses CD4 and CXCR4, making it susceptible to
X4-tropic viruses, and the other cell line, in which a CCR5 expression vector was introduced, is susceptible
to both X4- and R5-tropic viruses. Reporter cells were constructed by transfecting the human T-cell line
HPB-Ma, which demonstrates high susceptibility to HIV-1, with genomes expressing two different lucif-
erase reporters, HIV-1 long terminal repeat-driven firefly luciferase and cytomegalovirus promoter-driven
renilla luciferase. Upon HIV infection, the cells expressed firefly luciferase at levels that were highly
correlated (r2? 0.91 to 0.98) with the production of the capsid antigen p24. The cells also constitutively
expressed renilla luciferase, which was used to monitor cell numbers and viability. The reliability of the
cell lines for two in vitro applications, drug resistance phenotyping and drug screening, was confirmed. As
HIV-1 efficiently replicated in these cells, they could be used for multiple-round replication assays as an
alternative method to a single-cycle replication protocol. Coefficients of variation for drug susceptibility
evaluated with the cell lines ranged from 17 to 41%. The new cell lines were beneficial for evaluating
antiretroviral drug resistance. Firefly luciferase gave a wider dynamic range for evaluating virus infec-
tivity, and the introduction of renilla luciferase improved assay reproducibility. The cell lines were also
beneficial for screening new antiretroviral agents, as false inhibition caused by the cytotoxicity of test
compounds was easily detected by monitoring renilla luciferase activity.
Drug resistance assays have been accepted as standard clin-
ical tests to guide the antiretroviral therapy of human immu-
nodeficiency virus (HIV)-infected patients who have devel-
oped resistance to drug treatment or drug-naı ¨ve patients
infected with drug-resistant virus. These tests have been shown
to improve treatment outcomes by selecting the most effective
drugs and by minimizing the risk of treatment failure (2, 5–7,
9, 34). Drug resistance has been determined by two ap-
proaches. One is drug resistance genotyping, in which drug
resistance is evaluated by sequencing the viral genes targeted
by the drug, such as the HIV-1 protease and reverse transcrip-
tase (RT) genes. The level of drug resistance is estimated by
using observed mutation patterns and interpretation algo-
rithms (23). Several protocols have been used for drug resis-
tance genotyping, including in-house sequencing (10, 13, 38).
Although these protocols differ in some aspects, e.g., the de-
sign of primers, the length of analyses, and amplification pro-
cedures, all are based on the same technical approach, modi-
fied Sanger sequencing.
The other approach to drug resistance assays is phenotyping.
In this method, the levels of drug resistance of patient-derived
viral isolates are evaluated by using in vitro bioassays (17, 26).
Two advantages of the phenotyping assay are its ability to
directly evaluate the drug susceptibilities of patient-derived
viruses and the ease of interpreting its results compared to
those from genotyping. This assay is especially useful in cases
with a high degree of exposure to antiretroviral drugs, there-
fore involving many mutations. In these cases, the evaluation
of resistance levels by genotyping alone may be difficult (35). In
addition, the resistance levels determined by phenotyping pro-
vide important information for updating interpretation algo-
rithms used in genotyping.
Although peripheral blood mononuclear cells (PBMC) are
the natural target of HIV type 1 (HIV-1) and hence are the
best candidates for host cells in phenotyping assays, reporter
cell systems are more commonly used in drug susceptibility
assays (1, 12, 15, 31). Reporter systems are preferred because
their susceptibility to HIV-1 is stable and their output is both
rapidly measured and highly reproducible compared to that of
PBMC assays. Several kinds of reporter cells have been used
with different reporter proteins, such as MAGI cells with ?-ga-
lactosidase (21), GHOST cells with enhanced green fluores-
cent protein (36), MOCHA cells with secreted alkaline phos-
phatase (24), and CEM.NKR-CCR5-Luc cells with luciferase
(31). Although these systems use different cell lines, their basic
strategies for evaluating HIV infectivity are similar (21, 36).
The cell lines carry a reporter protein gene regulated by the
HIV-1 long terminal repeat (LTR) promoter, inducing them to
* Corresponding author. Mailing address: AIDS Research Center,
National Institute of Infectious Diseases, 4-7-1 Gakuenn, Musashimu-
rayama, Tokyo 2080011, Japan. Phone: 81-42-561-0771. Fax: 81-42-
561-7746. E-mail: email@example.com.
?Published ahead of print on 20 December 2006.
produce the reporter protein when they are infected with
HIV-1. Which reporter system is used depends on the prop-
erties of the original cell line and the installed reporter protein.
Reporter systems using MAGI and GHOST cells have the
advantages of high sensitivity and rapidity in determining in-
fectivity. However, MAGI and GHOST cells have been estab-
lished from HeLa cells (21) and human osteosarcoma cells
(36), respectively, which are not naturally susceptible to HIV-1.
Therefore, these cells cannot propagate viruses efficiently. On the
other hand, MOCHA and CEM.NKR-CCR5-Luc cell lines were
established from T-cell lines and secreted alkaline phosphatase
and luciferase, respectively, were installed as reporters. These
reporter systems allow for the evaluation of HIV-1 infectivity by
using enzymatic reactions and demonstrate greater reproducibil-
ity with wider dynamic ranges of reporter proteins. However, for
these cells to produce sufficient reporter protein for accurate
determinations, they must be cultured for 5 to 7 days, longer than
MAGI and GHOST cells. Longer culture periods allow reporter
cells to divide, which may affect the accuracy of the quantification.
Given the advantages and limitations of previously con-
structed reporter cell lines, we designed and tested two new
reporter cell lines with dual chemokine receptors for use in
drug resistance phenotypic assays and other HIV infectivity
assays. The cell lines we designed have unique characteristics
in that they originate from the human T-cell line HPB-Ma (16,
29, 40) and were engineered to express the CCR5 receptor and
two different marker proteins, firefly luciferase (FL) and re-
nilla luciferase (RL). FL, which is under HIV-1 LTR promoter
regulation, is produced upon HIV-1 infection. Therefore, fire-
fly luciferase activity can be used as a marker for virus infec-
tivity. RL, which is under cytomegalovirus (CMV) promoter
control, is constitutively expressed in the cells. Therefore, re-
nilla luciferase activity can be used as a marker for cell number
MATERIALS AND METHODS
Construction of luciferase and CCR5 expression vectors. Two different lucif-
erase expression vectors were constructed. The first luciferase construct com-
prised HIV-1 Tat-regulated FL and the red fluorescent protein (DsRed) con-
struct 53LTRN-lucneor#1. The HIV-1 Tat-responsive reporter construct
53LTRN-lucneor#1 was constructed based on the expression vector pGEM-
7Zf(?) (Promega, Madison, WI). Initially, a parent vector was constructed,
53LTRNCNS, which has a rabbit ?-globin unit under the control of the HIV-1
LTR derived from strain HXB2. In this construct, a gene of interest can be
cloned within the second exon of the ?-globin gene and the polyadenylation
signal is provided by the ?-globin unit. A neomycin expression module was
prepared by PCR and cloned upstream of the HIV-1 LTR region to generate
53LTRCNSneo. The reporter gene employed here was a fusion between an FL
gene and a DsRed gene. The FL gene allows HIV-1 replication to be quantita-
tively evaluated by using luciferase activity when the LTR is activated by HIV-
derived Tat, and the DsRed gene allows transfected and HIV-infected cells to be
identified by red fluorescence. The FL portion was derived from pGLuc5 (Pro-
mega), and the DsRed portion was derived from pDsRed1N-1 (Clontech). Both
genes were prepared by PCR, fused, and cloned into the ?-globin unit by using
NcoI and NotI restriction sites.
The second luciferase construct, pRenillaPac, was constructed using the plas-
mid pPUR (Clontech). The PCR-amplified RL gene, derived from phRL-CMV
(Promega), was spliced into the upstream region of the pac gene. This hybrid
gene manifests both RL activity and resistance to puromycin. Expression of the
fusion gene was constitutive under the control of a CMV promoter.
A CCR5 expression vector, pCCR5/CEP4, was constructed based on the
pCEP4 expression vector (Invitrogen), which possesses the Epstein-Barr nuclear
antigen 1 episomal-expression gene. The CCR5 gene was inserted into the vector
by using NotI and SnaB I restriction sites on the vector. Expression of the CCR5
gene was constitutive under the control of a CMV promoter.
Selection of host cell line and establishment of new reporter cell lines. To
design new reporter cell lines for quantifying HIV-1 replication, we selected the
murine leukemia virus-transformed human T-cell line HPB-Ma, established by
Y. K. Shimizu and H. Yoshikura (16, 29, 40), because of its high susceptibility to
HIV-1 and its stable expression of CD4 and CXCR4. HPB-Ma cells were main-
tained at 37°C in 5% CO2in complete RPMI 1640 medium (Sigma, Tokyo,
Japan) supplemented with 10% fetal calf serum (HyClone, Logan, UT) and 1%
penicillin-streptomycin (Invitrogen, Tokyo, Japan). Cells were transfected by
electroporation with the two luciferase expression vectors, 53LTRN-lucneor#1
and pRenillaPac. Plasmid DNA (10 ?g) was mixed with HPB-Ma cells (5 ? 106
cells in 500 ?l phosphate-buffered saline), and the mixture was incubated for 5
min at 4°C and electropulsed with a Gene Pulser II apparatus (Bio-Rad, Her-
cules, CA) at 250 V and 950 ?F. After electroporation, the cells were resus-
pended in complete medium and incubated at 37°C in 5% CO2. Subsequently,
cells with incorporated plasmids were selected with 0.1 ?g/ml puromycin (BD
Biosciences, San Jose, CA) and 250 ?g/ml Geneticin (Invitrogen), maintained in
complete medium for several weeks, and enriched with cell populations express-
ing high levels of CD4 and CXCR4 by fluorescence-activated cell sorting with a
FACSVantage system (BD Biosciences). Finally, clones were generated by lim-
iting dilution and selected if they showed high sensitivity to HIV-1 and low
spontaneous expression of FL and DsRed.
Since the parent HPB-Ma cell line expresses only the CXCR4 receptor, we
extended the spectrum of the reporter cell lines to include R5-tropic viral isolates
by transfecting cells by electroporation with a CCR5 expression plasmid. Clones
were selected by incubating for several weeks with 0.1 ?g/ml puromycin, 250
?g/ml Geneticin, and 150 ?g/ml hygromycin B. Selected cells were recloned, and
the expression of cell surface markers was confirmed by using FACSCaliber
(Becton Dickinson, San Jose, CA). CD4, CXCR4, and CCR5 receptors were
stained with SK-3-Cy5.5, 12G5-phycoerythrin, and 2D7-fluorescein isothio-
cyanate monoclonal antibodies, respectively (all from BD Biosciences, San
Evaluation of introduced reporter gene functions. To confirm the ability of FL
activity to reliably measure virus titer and production, established cell lines were
plated into 96-well plates at 105cells per well and inoculated with 50 to 400 50%
tissue culture infective doses (TCID50) of HXB2 or JRCSF. After 7 days of
culture with the test viruses, cells were harvested and lysed in 75 ?l of luciferase
assay reagent. FL activity was quantified using a Dual-Glo luciferase reporter
assay system (Promega, Madison, WI) and an LMax microplate luminometer
(Molecular Devices, Sunnyvale, CA). Virus production was also quantified by
using the p24 antigen enzyme-linked immunosorbent assay RETROtek kit
(ZeproMetrix Co., Buffalo, NY) and compared with FL activity.
The validity of using RL activity to monitor MaRBLE cell numbers was
evaluated by measuring RL activity in various numbers of cells and determining
the correlation between RL activity and cell numbers. The correlation between
RL activity and cell viability was also confirmed in cell killing assays with two
anticancer drugs, hygromycin B (Invitrogen, Tokyo, Japan) and blasticidin S
(Funakoshi, Tokyo, Japan). Target cells were plated into 96-well plates at 105
cells per well, and hygromycin B (15.6 to 500 ?g/ml) and blasticidin S (1.25
to 20 ?g/ml) were added. After 7 days of culture, cells were harvested and RL
activity was measured by using the Dual-Glo luciferase reporter assay system
(Promega) and the percentage of cell killing was determined by trypan blue
Preparation of recombinant and patient-derived viruses. Recombinant viruses
with point mutations were constructed as described elsewhere (25). In brief, drug
resistance mutations were introduced into the RT and protease genes of the
HXB2 clone by site-directed mutagenesis (28). MT-2 cells (5 ? 106human
T-lymphoblastoid cells) were then transfected by electroporation with the re-
combinant virus plasmids, and the cells were maintained in 10 ml of complete
medium for 7 to 14 days. Half the culture supernatant was harvested and re-
placed with fresh medium every other day. Viral replication was monitored by
measuring RT activity in the supernatant, and the sample with the highest RT
activity was used in subsequent studies.
Eight clinical samples were selected randomly from patient blood specimens
sent for routine HIV-1 drug resistance testing to the AIDS Research Center,
National Institute of Infectious Diseases, Tokyo, Japan. Patient viruses were
isolated by a standard coculture method described elsewhere (18). In brief, 2 ?
107patient PBMC were mixed with the same number of phytohemagglutinin-
stimulated normal human PBMC and the mixture was cultured for 2 weeks. Half
the culture supernatant was collected and replaced with the same amount of
fresh culture medium every other day. Viral replication was monitored by mea-
478CHIBA-MIZUTANI ET AL.J. CLIN. MICROBIOL.
suring RT activity in the supernatant, and the sample with peak RT activity was
selected and used for infection experiments afterward. RT assays were per-
formed as previously described (37). Viral RNAs in collected supernatants were
sequenced, and drug resistance mutation patterns were determined.
For the reconstructed virus, viral RNA was extracted from 200 ?l of patient
plasma by using a High Pure viral RNA kit (Roche, Basel, Switzerland) accord-
ing to the manufacturer’s instructions. Subsequently, a 1.8-kb gag-pol fragment,
encoding the region from p2gagto whole protease, and the 5? half of the RT gene
fragment were amplified and inserted into the HXB2 backbone. MT-2 cells (5 ?
106) were then transfected by electroporation with the plasmid, and the cells
were maintained in 10 ml of complete medium for 7 to 14 days. Half the culture
supernatant was harvested and replaced with fresh medium every other day.
Viral replication was monitored by measuring RT activity in the supernatant, and
the sample with the highest RT activity was selected for use in subsequent
studies. Viral RNAs in collected supernatants were sequenced, and the drug
resistance mutation patterns were confirmed. For both the patient-derived and
reconstructed viruses, HIV infectivity (TCID50) in the target cell lines was as-
sayed by the Reed-Muench method (27).
Drug resistance genotyping. HIV-1 RNA was extracted from 200 ?l of patient
plasma using a High Pure viral RNA kit according to the manufacturer’s instruc-
tions. For amplification of the 500-bp protease gene fragment, DRPRO5 (5?-
AGA-CAG-GYT-AAT-TTT-TTA-GGG-A) and DRPRO2L (5?-TAT-GGA-TTT-
TCA-GGC-CCA-ATT-TTT-GA) were used for reverse transcription and the
first PCR and DRPRO1M (5?-AGA-GCC-AAC-AGC-CCC-ACC-AG) and
DRPRO6 (5?-ACT-TTT-GGG-CCA-TCC-ATT-CC) were used for the second
PCR. For amplification of the 800-bp RT gene fragment, DRRT1L (5?-ATG-
ATA-GGG-GGA-ATT-GGA-GGT-TT) and DRRT4L (5?-TAC-TTC-TGT-
TAG-TGC-TTT-GGT-TCC) were used for reverse transcription and the first
PCR and DRRT7L (5?-GAC-CTA-CAC-CTG-TCA-ACA-TAA-TTG-G) and
DRRT6L (5?-TAA-TCC-CTG-CAT-AAA-TCT-GAC-TTG-C) were used for
the second PCR. The amplicons were purified by using a MultiScreen PCR filter
plate (Millipore), and sequence reactions were performed by using the BigDye
Terminator v3.1 cycle sequencing kit, followed by electrophoresis using an ABI-
3730 auto sequencer (Applied Biosystems, Foster City, CA).
HIV-1 replication kinetics analyses and drug susceptibility assays. To analyze
the replication kinetics of clinically derived HIV-1 isolates, target cells were
plated into 96-well plates at 105cells per well and infected with 100 TCID50of
test viruses per well. At days 3, 5, and 7, the culture supernatant of each well was
collected and RT activity was measured as previously described (37).
To evaluate anti-HIV-1 drug susceptibility, 107cells were infected with 10,000
TCID50of wild-type control or test viruses in 50-ml tubes and incubated for 2 h
at 37°C. Infected cells were resuspended in culture medium and plated into
96-well plates at 105cells per well. At 2 and 48 h after infection, serial RT
inhibitor dilutions and serial protease inhibitor (PI) dilutions were added, re-
spectively. Each drug was prepared in a fivefold serial dilution and tested over
different dose ranges, as follows. Didanosine, abacavir, and nevirapine were
tested at concentrations from 25.0 ? 101?m to 3.2 ? 10?4?M. Lamivudine and
stavudine were tested at concentrations from 5.0 ? 101?m to 6.4 ? 10?5?M.
Zidovudine, zalcitabine, and the five PIs (saquinavir, indinavir, nelfinavir, lopi-
navir, and amprenavir) were tested at concentrations from 1.0 ? 101?m to
12.8 ? 10?6?M. Efavirenz was tested at concentrations from 0.2 ? 101?m to
25.6 ? 10?7?M. All samples were tested in triplicate. The following manufac-
turers kindly supplied anti-HIV drugs: GlaxoSmithKline, Middlesex, United
Kingdom (zidovudine, lamivudine, and abacavir); Bristol-Myers Squibb, New
York, NY (didanosine, stavudine, and efavirenz); Roche, Basel, Switzerland
(zalcitabine and saquinavir); Boehringer Ingelheim, Ingelheim, Germany (nevi-
rapine); Merck Research Laboratories, Rahway, NJ (indinavir); Japan Tobacco,
Tokyo, Japan (nelfinavir); Vertex Pharmaceuticals, Cambridge, MA (amprena-
vir); and Abbott Laboratories, Abbott Park, IL (lopinavir).
After 7 days of culture with test drugs and test viruses, cells were harvested and
lysed in 75 ?l of luciferase assay reagent. Firefly and RL activities were sequen-
tially quantified using a dual-luciferase reporter assay system (Promega) and an
LMax microplate luminometer (Molecular Devices). Data were displayed by
plotting the percentage of luciferase activity versus the log10drug concentration.
The concentration at which 50% of viral replication was inhibited (IC50) was
determined by plotting curves defined by the four-parametric sigmoidal equation
f(x) ? A ? ([B ? A])/(1 ? [C/x]D) using XLfit4 software (CTC Laboratory
Systems Corporation, Tokyo, Japan). To determine susceptibility or resistance,
results for test viruses were compared to those for wild-type HIV-1 and evaluated
by Student’s t test.
Establishment of new T-cell-based cell lines with two lucif-
erase reporter proteins. Two luciferase expression vectors
were successfully constructed and used for transfection of the
HPB-Ma cell line. These vectors were 53LTRN-lucneor#1,
with FL under HIV-1 LTR regulation, and pRenillaPac, with
RL under CMV promoter regulation. HPB-Ma cells with these
vectors were subjected to several rounds of selection for cells
resistant to Geneticin and puromycin and were enriched by
flow cytometry with populations expressing high levels of CD4
and CXCR4 to establish the new cell line HPB-Ma/LTR-FL/
Since the parent HPB-Ma cell line expresses only CXCR4,
the spectrum of the X4-MaRBLE cell line was extended to
include R5-tropic viruses by transfection with a CCR5 expres-
sion plasmid, thus establishing the R5-MaRBLE cell line. Ex-
pression levels of CD4 were comparable among the parent
HPB-Ma, X4-MaRBLE, and R5-MaRBLE cell lines (Fig. 1a
to c), whereas the proportion of CXCR4-positive cell popula-
tions and CXCR4 expression levels were slightly higher in X4-
and R5-MaRBLE cells than in the parent HPB-Ma cell line
(Fig. 1d to f). This difference is due to the cell sorter’s selecting
for populations expressing high levels of CXCR4. As for CCR5
expression, HPB-Ma and X4-MaRBLE cells did not signifi-
cantly express the receptor (Fig. 1g and h). On the other hand,
more than 76% of the R5-MaRBLE cell population expressed
CCR5 (Fig. 1i).
To confirm the susceptibility of cell lines to X4- and R5-
tropic viruses, each cell line was inoculated with HXB2 (X4-
tropic) and JRCSF (R5-tropic) viruses. X4-MaRBLE cells in-
oculated with HXB2 expressed FL activity in a dose-dependent
manner but did not show any FL activity after inoculation with
JRCSF (Fig. 2a). On the other hand, R5-MaRBLE cells were
susceptible to both HXB2 and JRCSF, which induced FL ac-
tivity in a dose-dependent manner (Fig. 2b). To validate the
use of FL activity to evaluate viral production, FL levels were
compared to amounts of the viral capsid antigen, p24, in both
X4- and R5-MaRBLE cell lines, and the correlation between
FL levels and the amounts of p24 was determined. As shown in
Fig. 2c, FL activity in cell lysates and the amount of p24 antigen
in the culture supernatant were positively and linearly corre-
lated in X4-MaRBLE cells infected with HXB2 (r2? 0.98), in
R5-MaRBLE cells infected with HXB2 (r2? 0.91), and in
R5-MaRBLE cells infected with JRCSF (r2? 0.94). These
results verify that FL activity expressed by both X4- and R5-
MaRBLE cell lines accurately represents the levels of viral
replication and production. These good correlations also indi-
cate a small likelihood of interference between the two LTRs
in infected cells, the one driving luciferase and the other con-
tained in the infecting virus.
The second type of luciferase, RL, was inserted into
MaRBLE cells to monitor and evaluate their number and
viability. As shown in Fig. 3a, RL activity demonstrated a
positive, linear correlation with cell number (r2? 0.99). Thus,
RL activity can be used to assess cell number in culture. An-
other useful parameter evaluated by RL activity was the cyto-
toxicity of test compounds added to cultures. To confirm the
relationship between RL activity and cell viability, cell killing
assays were performed with two anticancer drugs, hygromycin
VOL. 45, 2007NEW T-CELL-BASED CELL LINES EXPRESSING HIV-1 REPORTERS479
B (Fig. 3b) and blasticidin S (Fig. 3c). As the percentages of
cells killed by both test chemicals increased, RL activity de-
clined (Fig. 3b and c). The concentrations of hygromycin B and
blasticidin S needed to kill 50% of the cells were 100 ?g/ml and
2 ?g/ml, respectively, in agreement with data from previous
reports (3, 32). Thus, RL activity can be used to measure
In addition, the replication of two patient-derived viral iso-
lates, 8 and 9, in R5-MaRBLE cells was compared to that in
PBMC. As shown in Fig. 4, the two clinical isolates efficiently
replicated in R5-MaRBLE cells. Isolate 8 replicated more ef-
ficiently in R5-MaRBLE cells than in PBMC, as indicated by
the 10-fold-higher RT activity at day 7 in R5-MaRBLE cells
(Fig. 4a). Isolate 9 had comparable day 7 RT activities in
R5-MaRBLE cells and PBMC (Fig. 4b). These data clearly
show that R5-MaRBLE cells can efficiently propagate clinical
Evaluation of HIV-1 drug susceptibility using X4- and R5-
MaRBLE cells is highly reproducible. Having confirmed the
quantitative reliability of FL expressed by HIV-infected X4-
and R5-MaRBLE cell lines, we next used the cell lines to
evaluate HIV-1 susceptibility to antiretroviral drugs. First, we
evaluated the precision of phenotyping using the X4- and R5-
MaRBLE cell lines. By using the wild-type HXB2 strain as a
target virus for both X4- and R5-MaRBLE cells and JRCSF as
a target virus for R5-MaRBLE cells, the IC50s of four repre-
sentative drugs from three classes of antiretroviral agents
(zidovudine, lamivudine, efavirenz, and lopinavir) were deter-
mined by measuring FL activity. As shown in Fig. 5, well-
characterized dose-response curves were obtained for the four
drugs. The mean IC50s, standard deviations (SD), and coeffi-
cients of variation (CV) for each cell line are summarized in
Table 1. The CV ranged from 17 to 41%, demonstrating high
reproducibility for drug susceptibility assays using both HIV-
FIG. 1. Levels of expression of CD4, CXCR4, and CCR5 in parent HPB-Ma cells and X4-MaRBLE and R5-MaRBLE cells. Parent HPB-Ma
(a, d, g), X4-MaRBLE (b, e, h), and R5-MaRBLE (c, f, i) cells were stained with monoclonal antibodies to CD4 (a, b, c), CXCR4 (d, e, f), and
CCR5 (g, h, i). To calculate the percentage of each population positive for the expression of cytokine receptors (bars), 2,000 to 5,000 cells were
analyzed by fluorescence-activated cell sorting. To calculate the percentage of each population positive for expression of CD4 and cytokine
receptors (bar), 2,000 to 5,000 cells were analyzed by FACSCalibur and compared with fluorescence-negative control cells. Histograms with gray
shading indicate cell populations stained with each monoclonal antibody; histograms without shading indicate negative control populations.
480CHIBA-MIZUTANI ET AL.J. CLIN. MICROBIOL.
FIG. 2. FL reporter activity in MaRBLE cells accurately represents viral production\chatn. (a) X4-MaRBLE cells are susceptible to HXB2 (X4-tropic) but not JRCSF (R5-tropic) viruses.
FL activity was confirmed as a reliable measure of X4-tropic HIV-1 in X4-MaRBLE cells by inoculating the cells with various amounts of HXB2 or JRCSF and reading FL activity 7 dayslater. Solid and dashed lines indicate HXB2 and JRCSF, respectively. (b) R5-MaRBLE cells are susceptible to both HXB2 (X4-tropic) and JRCSF (R5-tropic) viruses. FL activity wasconfirmed as a reliable measure of X4- and R5-tropic HIV-1 in R5-MaRBLE cells by inoculating the cells with various amounts of HXB2 or JRCSF and reading FL activity 7 days later. Solid and dashed lines indicate HXB2 and JRCSF, respectively. (c) FL activity and the amount of capsid antigen p24 are correlated in HIV-infected MaRBLE cells. The reliability of using FL activityinstead of the amount of p24 to quantify HIV-1 production was evaluated by measuring intracellular FL activity and the amount of p24 antigen in the supernatant from the same culture. Solid,dashed, and dotted lines indicate HXB2-infected X4-MaRBLE cells, HXB2-infected R5-MaRBLE cells, and JRCSF-infected R5-MaRBLE cells, respectively. Percentages of FL activity and of p24 production were calculated from the following formula: percentage ? (observed value with the drug ? background value)/(observed value without the drug ? background value) ?
100. RU, relative units.
VOL. 45, 2007NEW T-CELL-BASED CELL LINES EXPRESSING HIV-1 REPORTERS 481
1-inoculated X4- and R5-MaRBLE cells. Interestingly, while
the efavirenz and lopinavir susceptibilities of wild-type HXB2
were identical in evaluations with both X4- and R5-MaRBLE
cells, the IC50s for HXB2 and JRCSF were significantly differ-
ent (P ? 0.001) in the R5-MaRBLE cell line. Thus, in our
assay, JRCSF appeared to be more susceptible than the HXB2
HIV-1 strain to efavirenz and slightly less resistant to lopinavir.
Drug susceptibility of drug-resistant HIV-1 can be evaluated
using X4- and R5-MaRBLE cell lines. Given the accuracy and
reproducibility of assays using MaRBLE cells to determine the
drug susceptibilities of wild-type HXB2 and JRCSF, we then
evaluated the reliability of using the cell lines for drug resis-
tance phenotyping. Recombinant viruses with representative
drug resistance mutations were constructed, and the drug re-
sistance levels of the viruses were determined using X4-
MaRBLE cells. The drug resistance levels associated with five
patterns of nucleoside RT inhibitor (NRTI) resistance muta-
tions are summarized in Table 2. Of the five mutant viral
clones tested, four showed significant resistance to zidovudine,
with resistance levels in the following order from lowest to high-
est: M41L/M184V/T215Y ? D67N/K70R ? M41L/T215Y ?
M41L/D67N/K70R/T215Y. Thus, zidovudine resistance in-
creased with the accumulation of thymidine analogue muta-
tions (TAMs), and the M184V mutation caused reversion to
the zidovudine resistance phenotype in the M41L/T215Y mu-
tant, with a change in the susceptibility level of 12.5- to 3.5-fold
relative to that of the wild-type virus, similar to results in
previous reports (11, 19). Two clones with the M184V muta-
tion demonstrated over 500-fold (?533.7- and ?1,339.3-fold)-
greater resistance to lamivudine but no significant resistance to
didanosine and zalcitabine, although M184V has been re-
FIG. 3. Constitutively expressed RL in MaRBLE cells provides a reliable measure of cell number and viability. (a) RL activity accurately indicates MaRBLE cell numbers. The validity of using RL
activity to monitor MaRBLE cell numbers was evaluated by measuring RL activity in various numbers of cells and plotting the corresponding values. RL activity and cell number were positively and
linearly correlated (r2? 0.99). (b and c) RL activity reliably indicates hygromycin B and blasticidin S cytotoxicity in MaRBLE cells. The reliability of RL activity as a marker of cytotoxicity was evaluated
for hygromycin B (b) and blasticidin S HCl (c). Cells were cultured for 1 week with serial dilutions of each drug and lysed, and their RL activities were determined. In graphs in both panels b and c,
solid lines represent the RL activities of cell lysates and dashed lines indicate percentages of dead cells as determined by trypan blue staining. RU, relative units.
FIG. 4. Clinically derived isolates replicate in R5-MaRBLE cells as
efficiently as in PBMC. The replication kinetics of two clinical isolates,
8 and 9, were compared after inoculation into both R5-MaRBLE cells
and human PBMC. (a) Replication kinetics of isolate 8. Open and
closed triangles indicate kinetics in R5-MaRBLE cells and PBMC,
respectively. (b) Replication kinetics of isolate 9. Open and closed
circles indicate kinetics in R5-MaRBLE cells and PBMC, respectively.
cpm, counts per minute.
482CHIBA-MIZUTANI ET AL.J. CLIN. MICROBIOL.
ported to confer a three- to fivefold increase in the level of
resistance (14). Our data confirm the recent revalidation of
using didanosine for cases involving the M184V mutation (39).
Low-level but significant stavudine resistance in M41L/T215Y
(change, 2.2-fold), M41L/M184V/T215Y (change, 2.9-fold),
and M41L/D67N/K70R/T215Y (change, 3.8-fold) was found,
consistent with data from previous reports (22, 33). All five
clones demonstrated significant resistance to abacavir. The
highest resistance was observed in the M41L/M184V/T215Y
mutant, consistent with findings in previous reports that TAMs
with M184V reduce susceptibility to abacavir 10-fold (1).
The drug resistance levels associated with the two most
common nonnucleoside RT inhibitor (NNRTI) resistance mu-
tations (K103N and Y181C) are summarized in Table 3. The
K103N mutant virus demonstrated reduced susceptibility to
both nevirapine (change, 78.6-fold) and efavirenz (change,
54.7-fold), whereas the Y181C virus was resistant only to ne-
virapine (change, 47.5-fold) but remained susceptible to efa-
virenz (change, 1.5-fold).
The drug resistance levels of three PI-resistant mutant
clones (M46I, V82T, and L90M) are summarized in Table 4.
Clones with M46I and L90M mutations did not demonstrate
significant resistance to any PI tested, except for nelfinavir, to
which the L90M clone demonstrated low-level (change, 3.3-
fold) resistance. The clone with the V82T mutation demon-
strated low-level resistance to indinavir (change, 3.8-fold),
nelfinavir (change, 5.4-fold), amprenavir (change, 2.9-fold),
and lopinavir (change, 5.0-fold), consistent with results in pre-
vious reports (8). Thus, the drug susceptibilities of viruses with
resistance mutations evaluated with the MaRBLE cell lines
matched those from previous reports of drug resistance, indi-
cating the reliability of using the new cell lines to evaluate drug
To assess the reliability of using R5-MaRBLE cells to eval-
uate the levels of drug resistance of viral isolates from patients
for whom treatment failed, seven cases were selected and vi-
ruses were isolated by coculture with normal human PBMC.
Among the isolates from these seven cases, isolate 7 did not
yield measurable virus by coculture. Therefore, a protease-RT
gene fragment was amplified by RT-PCR and inserted into the
HXB2 backbone. As shown in Table 5, isolate 1 had three
minor mutations in the protease region and the virus was
susceptible to all four inhibitors tested. The increase in resis-
tance, calculated by comparison to the drug resistance of
JRCSF, was ?1.0-fold for zidovudine, lamivudine, and lopina-
FIG. 5. Results of assays for HIV-1 drug susceptibility with MaRBLE reporter cell lines are highly reproducible. Dose-response curves for four
representative agents against wild-type HXB2 and JRCSF are shown. Solid and dotted lines indicate HXB2 and JRCSF, respectively. (a to d)
Results of assays for susceptibility to zidovudine, lamivudine, efavirenz, and lopinavir, respectively, using X4-MaRBLE cells. (e to h) Results of
assays for susceptibility to zidovudine, lamivudine, efavirenz, and lopinavir, respectively, using R5-MaRBLE cells. The percentage of inhibition was
calculated as follows: percentage ? (observed FL activity with the drug ? background FL activity)/(FL activity without the drug ? background
FL activity) ? 100.
TABLE 1. Susceptibility of wild-type HXB2 and JRCSF to
representative antiretrovirals as determined using
X4- and R5-MaRBLE cells
Mean IC50(nM) ? SD (CV ?%?) fora:
(n ? 18)
(n ? 21)
(n ? 24)
0.9 ? 0.4 (41)
12.3 ? 3.9 (32)
2.7 ? 1.0 (37)
6.0 ? 1.0 (17)
1.3 ? 0.4 (31)
13.6 ? 4.8 (35)
2.1 ? 0.5b(24)
4.2 ? 1.4b(33) 17.6 ? 6.9b(39)
1.5 ? 0.6 (40)
13.4 ? 3.6 (27)
1.0 ? 0.3b(30)
an, number of isolates of the indicated virus strain.
bIC50s of efavirenz and lopinavir were significantly different for HXB2 and
JRCSF in R5-MaRBLE cells (P ? 0.001).
VOL. 45, 2007NEW T-CELL-BASED CELL LINES EXPRESSING HIV-1 REPORTERS483
vir. The patient in case 2 had been heavily treated with anti-
retroviral agents, and isolate 2 had a high accumulation of
NRTI and PI resistance mutations but no NNRTI resistance
mutation. This isolate had six TAMs and an M184V mutation
in the RT region and demonstrated resistance to zidovudine
(change, 32.9-fold) and lamivudine (change, ?380.7-fold) but
not to efavirenz. As for the protease region, 11 mutations were
detected, including three major mutations (M46I, V82F, and
L90M). Of these 11 mutations, 10 matched known lopinavir
resistance mutations (International AIDS Society—USA drug
resistance chart) (20). Indeed, high resistance to lopinavir
(change, ?76.5-fold) was observed.
Isolate 3 had 6 TAMs (M41L, E44D, D67N, V118I, L210W,
and T215Y), and a high level of resistance to zidovudine
(change, 114.5-fold) was observed. As the virus in this isolate
had all four known NRTI resistance mutations responsible for
hypersusceptibility to NNRTIs, it was hypersusceptible to efa-
virenz (change, 0.2-fold). Efavirenz hypersusceptibility was de-
fined as a change in resistance of ?0.4-fold compared to that
of the wild type by statistical analysis (mean value minus 2 SD)
and by data from a previous report (4). Isolate 4 had accumu-
lated five TAMs and demonstrated 191.7-fold-higher resis-
tance to zidovudine. No lamivudine resistance and NNRTI
resistance mutations were observed, and the virus was suscep-
tible to lamivudine. Isolate 4 also had M41L, V118I, L210W,
and T215Y mutations, and the virus demonstrated hypersus-
ceptibility to efavirenz (change, 0.2-fold). Two major muta-
tions, D30N and L90M, and eight secondary mutations in the
protease region were observed. Of these eight secondary mu-
tations, five matched lopinavir resistance mutations, with our
assay indicating a 9.4-fold increase in resistance to lopinavir.
Isolate 5 had an RT inhibitor resistance pattern similar to that
of isolate 2, having accumulated six TAMs and the M184V
lamivudine resistance mutation in RT and demonstrating
104.1-fold-higher resistance to zidovudine and ?380.7-fold-
higher resistance to lamivudine. This virus also had M41L,
V118I, L210W, and T215Y mutations. Though this virus ap-
peared to be slightly more susceptible to efavirenz (change,
0.6-fold), this effect was not statistically significant. Isolate 5
had accumulated 11 lopinavir resistance mutations in the pro-
tease region and demonstrated 75.5-fold-higher resistance to
lopinavir in our assay.
Isolate 6 had accumulated six TAMs in RT and showed
224.0-fold-higher resistance to zidovudine. This isolate had
E44D and V118I mutations (low-level-lamivudine-resistance
mutations) and showed 6.7-fold-higher resistance to lamivu-
dine. Similar to those in cases 3 to 5, the virus in case 6 had
M41L, V118I, L210W, and T215Y mutations and demon-
strated slight hypersusceptibility to efavirenz (change, 0.4-
fold). As for the protease region, isolate 6 had three major
mutations, M46I, I84V, and L90M, and six minor resistance
mutations. Of these nine mutations, eight were listed as lopi-
navir resistance mutations, and intermediate-level resistance to
lopinavir (change, 14.6-fold) was observed. Isolate 7R had
K103N and showed high-level resistance to efavirenz (change,
66.8-fold). No other drug resistance mutations in the RT re-
gion were found, and thus the isolate was susceptible to zidovu-
dine and lamivudine. The protease region did show two minor
mutations, L63C and V77I, and the isolate was susceptible to
TABLE 2. Drug resistance levels associated with NRTI resistance mutations as determined using X4-MaRBLE cells infected with HIV-1 clones
IC50(nM) ? SD (change, n-fold)afor:
0.8 ? 0.4
10.6 ? 6.9c(12.5 ? 1.9)
0.4 ? 0.1 (0.5 ? 0.01)
1.9 ? 1.0b(3.5 ? 0.5)
4.6 ? 1.0c(4.6 ? 1.2)
54.0 ? 32.3c,d,e(48.1 ? 16.0)
2,097.7 ? 1,101.4
2,899.8 ? 1,627.9 (1.4 ? 0.4)
2,164.6 ? 1,019.8 (1.3 ? 0.2)
2,893.7 ? 1519.0 (2.1 ? 0.8)
3,754.2 ? 1,468.3 (1.4 ? 0.3)
5,082.9 ? 2,397.5b(1.8 ? 0.5)
2.5 ? 1.1
3.8 ? 3.3 (1.5 ? 0.8)
3.4 ? 0.6 (1.3 ? 0.4)
3.9 ? 2.0 (2.2 ? 0.8)
3.0 ? 0.8 (1.2 ? 0.3)
3.5 ? 2.3 (1.2 ? 0.6)
8.7 ? 4.5
20.9 ? 14.5b(3.8 ? 3.2)
17.8 ? 6.9b(2.4 ? 1.3)
44.3 ? 27.7c(6.8 ? 7.4)
14.4 ? 5.2
29.9 ? 14.0b(2.2 ? 0.4)
17.5 ? 7.6 (0.9 ? 0.3)
40.7 ? 23.8b(2.9 ? 1.1)
31.3 ? 24.9 (2.3 ? 1.7)
50.3 ? 24.2c(3.8 ? 1.6)
348.7 ? 122.8
985.5 ? 306.3c(3.2 ? 1.1)
1,497.5 ? 589.4c(3.9 ? 1.2)
3,300.0 ? 1,986.9c,d(10.6 ? 3.1)
836.2 ? 522.5b(2.1 ? 0.7)
2,015.1 ? 842.8c,d(5.2 ? 0.9)
aChange (n-fold) ? (observed IC50for strain)/(IC50for wild type).
bP, ?0.05 for comparison with wild-type virus.
cP, ?0.005 for comparison with wild-type virus.
dP, ?0.05 for comparison with M41L/T215Y virus.
eP, ?0.05 for comparison with D67N/K70R virus.
484CHIBA-MIZUTANI ET AL. J. CLIN. MICROBIOL.
Thus, the increases observed in levels of resistance to
zidovudine and lopinavir were associated with the accumula-
tion of known resistance mutations associated with those
drugs. Similarly, hypersusceptibility to efavirenz was associated
with the accumulation of M41L, V118I, L210W, and T215Y
mutations in four out of six clinical isolates (30). Taken to-
gether, these results confirm the reliability of using X4- and
R5-MaRBLE cells in drug resistance phenotyping.
The development of reliable methodologies to evaluate drug
susceptibility in vitro has been a major thrust of drug resistance
research. Although several phenotypic assays are commercially
available for clinical usage, they are expensive and may not be
readily available either in developing or developed countries.
As for use in research laboratories, these commercial assays
target only the protease and reverse transcriptase gene regions
of the HIV-1 genome, limiting their flexibility as a tool for
basic research. Thus, there is still a need for easy-to-use assay
systems with high reproducibility, for both clinical and research
usage. To fill this gap, we drew a blueprint for a new type of
reporter cell line by considering the advantages and drawbacks
of several previously reported cell lines (12, 15, 17, 21, 24, 26,
31, 36). Based on this blueprint, we chose the T-cell-based cell
line HPB-Ma (16, 29, 40) as the parent cell line to introduce
reporter genes and to establish new reporter cell lines.
HPB-Ma is a murine leukemia virus-transformed human T-cell
line demonstrating high susceptibility to HIV-1, which can
replicate efficiently in these host cells. As HPB-Ma cells natu-
rally express CD4 and CXCR4, but not CCR5, we introduced
the CCR5 expression gene to widen the susceptibility spectrum
of the cell line to include R5-tropic viruses. The reporter pro-
tein chosen to measure HIV infectivity was FL, as it has a
wider dynamic range than other known reporter candidates.
Another type of luciferase, RL, was introduced as a marker of
cell number and viability.
Finally, two types of new reporter cell lines were established,
X4-MaRBLE and R5-MaRBLE. As expected, these new cell
lines had several advantages over previously described cell
lines. First, viruses efficiently propagated in these cell lines,
making multiple-round replication assays possible. In addition,
viruses could be isolated from patient plasma and PBMC by
using the cell lines. Since other reporter cells may not allow
replication-competent viruses to be efficiently produced, their
use is largely limited to single-cycle replication assays. These
assays are currently accepted as the major method for measur-
ing viral infectivity due to their rapid readout of results. How-
ever, single-cycle replication assays cannot evaluate the
postintegration late phase of the viral life cycle. To evaluate
late-phase inhibitors, such as PIs, by using single-cycle replica-
tion assays, an additional step is required prior to the assay to
produce viruses under test drug pressure. On the other hand,
multiple-round replication assays allow late-phase inhibitors to
be directly evaluated, just as early-phase inhibitors, without
additional culture. Furthermore, multiple-round replication
assays allow for a clearer readout of drug susceptibility, as the
differences in drug susceptibilities between the reference and
test viruses may be amplified by each round of replication.
Second, the cell lines were successfully transfected with RL
to broaden their application. The constitutive expression of
this second luciferase in the cell line has made it possible to
easily and accurately evaluate cell number and the cytotox-
icities of test compounds. As we planned to conduct multi-
ple rounds of assays, the cells were cultured for at least a
week, long enough for them to propagate and increase the
background level of FL. The extent of this increase de-
pended on the amount of viral inocula or the level of inhi-
bition by antiretroviral agents. By monitoring RL activity,
TABLE 3. Drug resistance levels associated with NNRTI resistance mutations as determined using X4-MaRBLE cells
infected with HIV-1 clones
IC50(nM) ? SD (change, n-fold)afor:
206.3 ? 68.2
1.4 ? 0.4
16,110.7 ? 6,445.7b(78.6 ? 19.7)
79.3 ? 33.3b(54.7 ? 15.6)
9,586.3 ? 6,396.4c(47.5 ? 23.8)
2.3 ? 0.6c(1.5 ? 0.1)
aChange (n-fold) ? (observed IC50for strain)/(IC50for wild type).
bP, ?0.005 for comparison with wild-type virus.
cP, ?0.05 for comparison with wild-type virus.
TABLE 4. Drug resistance levels of PI-resistant mutants analyzed using HIV-1-infected X4-MaRBLE cells
IC50(nM) ? SD (change, n-fold)afor:
Wild typeM46I V82T L90M
11.3 ? 4.3
7.4 ? 3.4
4.9 ? 2.3
6.7 ? 3.1
6.6 ? 3.8
19.5 ? 17.0 (2.2 ? 2.7)
5.0 ? 2.4 (0.7 ? 0.3)
7.4 ? 5.3 (1.9 ? 1.5)
9.7 ? 5.4 (2.0 ? 2.0)
6.3 ? 2.9 (1.1 ? 0.7)
38.0 ? 6.8b(3.8 ? 1.6)
6.5 ? 1.3 (1.0 ? 0.4)
20.0 ? 5.5b(5.4 ? 3.7)
17.2 ? 5.2c(2.9 ? 1.0)
29.1 ? 9.0c(5.0 ? 1.8)
11.5 ? 1.5 (1.1 ? 0.3)
6.9 ? 1.1 (1.0 ? 0.4)
11.5 ? 0.7d(3.3 ? 2.2)
10.7 ? 2.1 (2.0 ? 1.3)
5.6 ? 0.9 (1.1 ? 0.7)
aChange (n-fold) ? (observed IC50for strain)/(IC50for wild type).
bP, ?0.005 for comparison with wild-type virus.
cP, ?0.05 for comparison with wild-type virus.
dP, ?0.01 for comparison with wild-type virus.
VOL. 45, 2007NEW T-CELL-BASED CELL LINES EXPRESSING HIV-1 REPORTERS485
we could easily evaluate culture conditions and their effects
on assay results.
These two characteristics confer a great advantage to using
the MaRBLE cell lines for screening new antiretroviral agents.
They allow both early- and late-phase inhibitor candidates to
be evaluated under the same protocol, as the assay permits
multiple viral replications. Moreover, monitoring of RL activ-
ity allows false-positive results (inhibition by test drugs due to
cytotoxicity) to be detected and eliminated. Finally, the use of
RL activity greatly improved the efficacy of screening.
The MaRBLE cell lines stably expressed the transfected
genes, as confirmed by the stable expression of CD4, CXCR4,
and CCR5 on the surfaces of cells maintained in culture for up
to 6 months with continuous passage. We also confirmed that
the two reporter genes were stably expressed and that IC50s
were identical for both newly plated and 6-month-old cultures
(data not shown).
In conclusion, we successfully established two unique cell
lines, X4-MaRBLE and R5-MaRBLE, which are useful for
assaying viral drug resistance and for screening new antiret-
roviral compounds. Although the cost of phenotypic assays
using our cell lines may be less than that of commercial
systems, the assays require a biosafety level 3 laboratory,
general culture equipment, and a luminometer for readout.
Since these are all expensive items, the assay price should be
reduced and the assay protocol should be simplified for
wider usage of the assay.
We thank Hiroshi Yoshikura, Mari Takizawa, and Mitsuo Honda
for their help and discussions. We also thank Claire Baldwin for her
help in preparing the manuscript.
This study was supported by a grant from the Human Sciences
Foundation and the Program for Promotion of Fundamental Studies
in Health Sciences of the National Institute of Biomedical Innovation
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RT mutation(s)Protease mutation(s)
IC50(nM) ? SD (change, n-fold) of:
M41L, D67N, K70R,
M41L, E44E/D, D67N,
M41L, E44A, D67N,
L63P/T, A71A/V, V77I
L10I, L33F, M46I, F53L,
I54V, L63P, A71V,
K20T, D30N, M36I,
M46M/L, L63P, A71V,
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A71V, I84V, L90M
1.4 ? 0.6 (1)
13.6 ? 4.6 (1)
0.9 ? 0.1 (1)
19.3 ? 8.4 (1)
3 (F)X4, R5 195.1 (114.5)34.5 (2.6)0.1 (0.2) NDc(ND)
4 (B)X4, R5326.5 (191.7) 13.5 (1.0)0.2 (0.2) 123.4 (9.4)
5 (B) X4, R5M41L, E44D, D67N,
?5,000 (?380.7) 0.5 (0.6)986.8 (75.5)
6 (F)R5M41L, E44D, D67N,
381.7 (224.0)88.0 (6.7) 0.3 (0.4) 190.6 (14.6)
X42.1 (1.5)22.3 (1.6)60.2 (66.8)7.6 (0.4)
aRecombinant HXB2 with patient-derived protease and RT sequences.
bThe tropism of each virus was determined by using X4-GHOST and R5-GHOST cells.
cND, not determined.
486CHIBA-MIZUTANI ET AL.J. CLIN. MICROBIOL.
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