(The FASEB Journal. 2000;14:511-515.)
© 2000 FASEB
P-glycoprotein-overexpressing multidrug-resistant cells are resistant to infection by enveloped viruses that enter via the plasma membrane1
YOSSEF RAVIV2,
ANU PURI* and
ROBERT BLUMENTHAL*
Intramural Research Support Program SAIC Frederick, Laboratory of Experimental and Computational Biology and
* Laboratory of Experimental and Computational Biology, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick Maryland 21702, USA
2Correspondence: Intramural Research Support Program SAIC Frederick, Laboratory of Experimental and Computational Biology, National Cancer Institute-Frederick Cancer Research and Development Center, Bldg. 469, Room 211, NCI-FCRDC, Frederick MD 21702, USA. E-mail: yraviv{at}mail.ncifcrf.gov
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ABSTRACT
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The multidrug resistance gene product P-glycoprotein confers drug
resistance to tumor cells by acting as a transporter that blocks the
entry into the cell of a great variety of drugs and hydrophobic
peptides. In this study we find that in drug-resistant cells, the
insertion of the influenza virus fusion protein (hemagglutinin-2) into
the plasma membrane is blocked and that the fusion of the viral
envelope with the plasma membrane of these cells is impaired.
Multidrug-resistant cells display significant resistance to infection
by envelope viruses that invade cells by fusion with the plasma
membrane, but not to infection by pH-dependent viruses that penetrate
cells by fusion with endocytic vesicles. These observations suggest
that multidrug resistance phenomena may protect cells from infection by
a large group of disease-causing viruses that includes human
immunodeficiency virus, herpes simplex virus, and some cancer-inducing
retroviruses. —Raviv, Y., Puri, A., Blumenthal, R.
P-glycoprotein-overexpressing multidrug-resistant cells are
resistant to infection by enveloped viruses that enter via the plasma
membrane.
Key Words: viral invasion • membrane fusion • photosensitized labeling • hydrophobic labeling
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INTRODUCTION
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OVEREXPRESSION OF THE multidrug-resistant (MDR)
gene product P-glycoprotein confers simultaneous resistance of tumor
cells to a great variety of drugs and cytotoxic agents. This phenomenon
is the main obstacle to successful chemotherapeutic treatment of
cancer. The mechanism of P-glycoprotein-mediated multidrug resistance
is not completely understood (for reviews, see refs 1
2
3
).
The difficulty stems from an apparent lack of substrate specificity
manifested by the ability to transport a great variety of drugs and
compounds dissimilar in their molecular structure, size, or biological
action. Since the initial demonstration that P-glycoprotein interacts
with its substrates within the hydrophobic phase of the membrane
(4)
, the information emerging from structural and
functional studies is consistent with the view that substrates first
partition into the lipid phase of the membrane before they access
putative binding sites on the molecule itself. An important feature of
P-glycoprotein-overexpressing multidrug-resistant cells is their
ability to block the entry of a great variety of hydrophobic peptides
(2
, 5)
and become resistant to toxic peptides like
gramicidin D and valinomycin (6)
. Other members of the ATP
binding cassette (ABC) family of transporters function as transporters
of peptides across membranes (7
8
9)
. It was of interest to
test whether P-glycoprotein-overexpressing drug-resistant cells would
block the penetration of hydrophobic fusion peptides of envelope
viruses that facilitate virus invasion into cells (10
, 11)
. We have recently shown that with influenza virus and
vesicular stomatitis virus, fusion of the viral envelope with the
target membrane starts with the insertion of the hydrophobic domain of
the envelope glycoprotein of the virus into the lipid domain of the
cell membrane (12
13
14)
. In this work we show that
1) cells that overexpress P-glycoprotein and phenotypically
display resistance to drugs also display resistance to viral infection,
2) this resistance is exerted at the step of the insertion
of the viral fusion protein into the target cell membrane, and
3) the resistance is limited to viruses that invade the
cells through the plasma membrane.
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MATERIALS AND METHODS
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Materials and chemicals
KB carcinoma cell lines were kindly provided by Dr.
Michael Gottesman, Chief Laboratory of Cell Biology at NCI, National
Institutes of Health. Drug-sensitive (KB-3–1) cells (15)
and drug-resistant (KB-V1) cells (16)
were cultured and
selected as described. Influenza virus (X-31 strain) was purchased from
SPAFAS (Storrs, Conn..) and Sendai virus was propagated in the
allantoic cavity of chicken eggs as described previously
(17)
. Titers for the influenza virus and Sendai virus were
determined by the egg infectious dose (EID) assay (17)
and
found to be 1011 EID50/ml
and 3 x 1010
EID50/ml, respectively.
3,3'-Dioctadecyloxacarbocyanine (DiO), octadecylrhodamine (R-18), and
calcein were from Molecular Probes (Eugene, Oreg.).
1,5-Iodonaphthylazide (125INA) was prepared as
described (14)
and purchased from Lofstrand Labs
(Gaithersburg, Md.). Goat anti-influenza hemagglutinin antibody-FITC
conjugate was from ViroStat (Portland, Maine). All other chemicals were
from Sigma Chemical Co. (St. Louis, Mo.).
Measurements of the insertion of influenza virus fusion peptide
into the target cell membrane
Insertion of the viral fusion protein into KB-V1 and
KB-3–1 cells was measured by site-directed photosensitized
radiolabeling as described previously for red blood cells
(12)
and chromaffin cells (14)
. DiO and
[125I]-INA were inserted in the target cell
membranes. The virus was bound to the cell, and lowering the pH to 5.0
at room temperature triggered fusion. Radiolabeling of cell membrane
proteins was initiated by simultaneous irradiation of the cells with
visible light (
460 nm) for 5 min. Hemagglutinin-2 (HA-2) was
isolated by immunoprecipitation, followed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Extent of labeling was
evaluated by scintillation counting of excised bands in a gamma
counter.
Measurement of fusion of the viral envelope with the target cell
membrane
Influenza virus (X-31 strain) was labeled with (R-18) as
described previously (18
, 19)
. The labeled virus was bound
to the cells at 4°C. Unbound virus was removed and an aliquot was
placed in a spectrofluorimeter cuvette at room temperature at pH 7.4
and stirred. Fusion was triggered on lowering the pH to 5.0 by addition
of an appropriate volume of 0.5 M citric acid. The time-dependent
increase in fluorescence intensity was monitored for 400 s. Triton
X-100 0.1% was added to obtain maximum fluorescence.
Measurement of the extent of infection of cells by
influenza virus and Sendai virus
Cells (KB-3–1 and KB-V1) were plated on 6-well plates at a
density of 106 cells/well. One microliter of
either influenza (108
EID=5x107 viral particles) or Sendai virus
(3x107 EID=1.6x107 viral
particles) was added to each well in 1 ml Dulbecco’s modified Eagle
medium (DMEM) +2% fetal bovine serum (FBS). This amount was the lowest
plateau concentration as determined by dose response curves (Fig. 1
). The cells were incubated for 90 min at 37°C, washed with DMEM+10%
FBS, and further incubated overnight with fresh medium for expression
of viral proteins. Binding of fluorescent antibody and/or fluorescent
erythrocytes was evaluated by flow cytometry. Binding of fluorescent
erythrocytes was also evaluated by measuring total fluorescence
obtained after lysis of cells with 1% Triton X-100, as described
previously (20)
. The high capacity of erythrocytes for
R-18 provided the opportunity to use the same sample to evaluate the
extent of infection by two independent assays: by fluorescence analysis
in a flow cytometer and by the more quantitative assay of measuring
total fluorescence in an SLM Aminco fluorimeter. The fluorescent signal
generated by the bound antibodies was below the detection limits of the
fluorimeter. For Sendai virus only R-18-labeled erythrocytes were used.
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Figure 1. The effect of influenza viral dose on the infection of drug-resistant
(KB-V1) and drug-sensitive (KB-3–1) cells.
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Induction of viral infection by penetration through the
plasma membrane
Cells were incubated with medium (DMEM +10% FBS) containing 50
mM NH4Cl for 30 min at 37°C. The medium was
aspirated and cells were incubated with 1 µl purified virus (X31
strain) in DMEM + 2% FBS + 50 mM NH4Cl at 4°C
for 1 h (21)
. The virus was washed away and 1 ml of
warm medium pH 5.0 was added to each well for 1 min. The acidic medium
was quickly removed and replaced with medium + 50 mM
NH4Cl for 2 h at 37°C. The medium was then
replaced with fresh medium without NH4Cl and the
cells were incubated for additional 12 h to allow protein
synthesis.
Determination of susceptibility of membrane probes to
extrusion by the multidrug-resistant cells
KB-V1 and KB-3–1 cells were labeled in suspension with
either DiO or R-18 for fluorescence measurements and with
125INA for radioactive measurement as in the
respective experiments described above. The extent of dye uptake in
drug-resistant and drug-sensitive cells was measured by fluorescence in
an SLM Aminco fluorimeter. For DiO, settings were Ex=480 nm, Em=520 nm;
for R-18, Ex=560, Em=600 nm. For 125INA, total
radioactivity associated with the cells was measured with a gamma
counter. We used calcein as a positive control for a dye extruded by
KB-V1 cells. Excitation emission settings were as for DiO. The extent
of calcein fluorescence retained in drug-resistant cells was less than
2% of that observed in drug-sensitive cells.
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RESULTS
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The experimental system used in this study included the
parental drug-sensitive cell line (KB-3–1) (15)
, from
which P-glycoprotein-overexpressing drug-resistant cells were selected
by vinblastine containing selection medium (KB-V1) (16)
.
The viruses used were influenza virus (X-31 strain), which enters by
fusion with the acidic endosomes, and Sendai virus, a neutral pH virus.
Viral invasion into cells was evaluated using three different
parameters: 1) insertion of the viral fusion protein into
the target cell membrane, 2) lipid mixing induced by fusion
between the viral envelope and the target cell membrane, and
3) expression of viral envelope proteins on the surface of
the target cell after infection with virus.
We recently used site-directed hydrophobic labeling to monitor the
insertion of the fusion protein of the virus into the lipid domain of
the cell plasma membrane (12
13
14)
. Using the same
approach, the insertion of the influenza HA into the plasma membrane of
drug-resistant vs. drug-sensitive cells was monitored. The virus was
allowed to bind to the cell at neutral pH (7.4). The pH was lowered to
5.0 and radiolabeling was performed under conditions that confine
labeling exclusively to integral membrane proteins of the target cell
(12
, 14)
. The viral fusion protein, HA-2, was isolated by
immunoprecipitation and the extent of labeling was measured by
scintillation counting. Figure 2
shows that the extent of penetration of the viral fusion protein (HA-2)
into drug-resistant cell membranes is significantly reduced as compared
with that observed in drug-sensitive cells. In control experiments we
established that DiO and 125INA are not extruded
as drugs in KB-V1 cells (data not shown).
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Figure 2. Insertion of the influenza virus fusion peptide (HA-2) into
drug-resistant (R) vs. drug-sensitive (S) cells. Left panel shows the
extent of radioactive labeling of HA-2 obtained with drug-resistant (R)
and drug-sensitive (S) cells, either before (pH 7.0) or after (pH-5.0)
fusion of the viral envelope with the target cell membrane. The right
panel represents the counts monitored for the excised gel bands by a
gamma counter. The experiment was repeated four times with highly
reproducible results. Radiolabeling was carried out by selective
photosensitized activation of [125I]-1-
Iodonaphthyl-5-azide ([125I]-INA) in the membrane of the
target cells.
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Lipid mixing as a result of fusion between the viral envelope and
the membrane of KB carcinoma cells was monitored by following the
time-dependent dequenching of R-18 introduced earlier into the viral
envelope (18
, 19)
. Figure 3
demonstrates that fusion of the viral envelope with the target cell
membrane is significantly reduced in drug-resistant cells, consistent
with the data in Fig. 2
. Control experiments showed that KB-V1 cells do
not extrude R-18 and that the extent of virus binding was similar in
both cell types (data not shown). Virus binding was determined by
measuring the total fluorescence obtained after incubation of
R-18-labeled virus with cells at neutral pH at 4°C. Some of the lipid
mixing events observed might not result in successful infection of the
cell (10)
; therefore, it was essential to measure the
extent of viral infection. The virus was allowed to infect the cells
and the expression of viral envelope proteins on the cell membrane was
measured by the extent of binding of fluorescent antibodies and/or
fluorescent human erythrocytes to the cell surface. The surprising
result was that no significant difference between the drug-sensitive
and drug-resistant cells could be detected in the extent of influenza
virus infection (Fig. 4A
) (see also Fig. 1
). In the hydrophobic labeling and lipid
mixing experiments, data were collected after triggering the fusion
between the viral envelope and the cell membrane by low pH at the
surface of the cell after a short (20 min) incubation with the virus
for binding. In viral infection experiments, viral invasion and
expression of viral proteins were allowed to proceed through their
natural course, the endocytic pathway. Influenza virus, like other
pH-dependent viruses, penetrates the cell by fusing with the membranes
of acidic endosomes (for reviews, see refs 10
, 11
). It has
been shown that P-glycoprotein is located in the plasma membrane and
cannot be detected in endocytic vesicles of multidrug-resistant KB
cells (22)
. Exclusion of P-glycoprotein from endocytic
vesicles could account for the results obtained in Fig. 4A
.
To test this hypothesis, we measured the extent of viral protein
expression in cells where the endocytic pathway was blocked by
NH4Cl (50 mM), and penetration of the virus into
the cell was induced at the surface of the cell by lowering the pH
(20)
. Under these conditions, a significant reduction of
influenza virus infection in drug-resistant cells could be observed
(Fig. 4B
), in agreement with the data in Figs. 2
and 3
.
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Figure 3. Inhibition of fusion of the influenza viral envelope with the membrane
of drug-resistant (R) and drug-sensitive (S) cells. Time-dependent
increase in fluorescence (dequenching) of R-18 represents the extent of
dilution of the viral envelope lipids with the target cell membrane
lipids, which results from fusion between the two. Fusion was initiated
by lowering the pH to 5.0 at room temperature. 100% is the total
fluorescence obtained after maximal dilution of the dye by lysis of
cells with Triton X-100 (0.1%).
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Figure 4. Infection of drug-resistant (R) and drug-sensitive (S) cells by
influenza virus. Cells were plated in 6-well culture plates as
described in Materials and Methods. Viral protein expression is
represented by the extent of binding of R-18-labeled human erythrocytes
to the infected cells. Comparison between infection by fusion with
endosomes (A) and infection by fusion with the plasma
membrane (B). Left panel: Fluorescence analysis of cells
by flow cytometry. Gates were selected to include cells larger than
erythrocytes. R, solid line; S, shaded area. Right panel: Fluorimetric
readings obtained independently with an SLM Aminco fluorimeter after
lysis of the bound erythrocytes with 0.1% Triton X-100. Experiments
were carried out in triplicate and repeated three times. The flow
cytometry experiments were repeated using fluorescein-tagged anti-HA
antibody as the tracer of viral protein expression, and results were
similar to that obtained by erythrocyte binding. Uninfected cells were
used as background (5% of signal).
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This result indicates that drug-resistant cells may not be
protected from infection by pH-dependent viruses that penetrate into
the cell via the endocytic pathway. However, these results also suggest
that P-glycoprotein-expressing multidrug-resistant cells could be
protected from infection by pH independent viruses that presumably
infect cells by fusion with the plasma membrane. To test this
possibility, we examined Sendai virus, a neutral pH fusing virus, for
its ability to infect drug-resistant cells. The results presented in
Fig. 5
show that, as expected, Sendai virus infection is significantly reduced
in drug-resistant cells.
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Figure 5. Infection of drug-resistant (solid line) and drug-sensitive (shaded
area) cells with Sendai virus. Analysis by flow cytometry of cells
tagged with R-18-labeled erythrocytes as in Fig. 3
. Infection with
Sendai virus was performed as described with influenza virus.
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DISCUSSION
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In this study we present for the first time evidence that
P-glycoprotein-overexpressing multidrug-resistant cells are also
resistant to infection by viruses that penetrate the cell through the
plasma membrane. Blocking the insertion of the viral fusion protein
into the target membrane may be the mechanism for this protection. The
observation that viral protein expression is not affected in
drug-resistant cells when infection is allowed to proceed through the
endocytic pathway strongly suggests that viral infection is blocked by
a mechanism associated with P-glycoprotein overexpression in the plasma
membrane. Exclusion of the protein from the endocytic vesicle clears
the obstacle for fusion between the viral envelope and the endosomal
membrane. An indirect effect of P-glycoprotein overexpression on the
structure of the membrane cannot be excluded, as is the possible
involvement of other factors. Additional studies are required in order
to understand the role of P-glycoprotein in blocking viral infection.
Yet the link established in this study between the phenomenon of
P-glycoprotein-associated multidrug resistance and the process of
envelope virus invasion may provide new insight into the mechanisms
underlying both phenomena. The finding that
P-glycoprotein-overexpressing cells can block invasion and pathogenesis
of disease causing viruses may have some obvious practical
implications. The MDR-1 gene can be introduced and overexpressed in
target cells by a variety of vectors and used as a marker for selection
of the transfected cells in vivo, in the presence of drugs
(for review, see refs 23
, 24
). The results reported in
this study suggest that the selective transfection of the MDR-1 gene
coupled with an in vivo selection process in the presence of
drugs may also generate conditions that will prevent infection by
disease-causing viruses that penetrate the cell through the plasma
membrane.
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ACKNOWLEDGMENTS
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We thank Carol Cardarelli and Dr. Michael Gottesman from the
Laboratory of Cell Biology at the National Cancer Institute for their
generous supply of cell lines, as well as Dr. Caroline Lee from the
same laboratory for useful discussions and sharing of data. We thank
Dr. Thomas Korte and Andrew Hendifar from our laboratory for their help
in the preparation of this manuscript. The content of this publication
does not necessarily reflect the views or policies of the Department of
Health and Human Services, nor does mention of trade names, commercial
products, or organizations imply endorsement by the U.S. government.
This project has been funded in whole or in part with federal funds
from the National Cancer Institute, National Institutes of Health under
contract no. N01–56000.
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FOOTNOTES
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1 The publisher or recipient acknowledges the right of the U.S. government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. 
Received for publication June 23, 1999. Revised for publication November 1, 1999.
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ABSTRACT
INTRODUCTION
