AIDS RESEARCH AND HUMAN RETROVIRUSES

Volume 5, Number 2, 1989

Mary Ann Liebert, Inc., Publishers

Inhibitory Effect of Papaverine

HIV Replication In Vitro
ADOLFO

TURANO,1

ROBERTO

on

GEMMA

SCURA,1 ARNALDO CARUSO,1 CARLO BONFANTI,1

LUZZATI,2 DANTE BASSETTI,2 and NINO MANCA1
ABSTRACT

The ability of papaverine to inhibit human immunodeficiency virus (HIV) replication in H9

cell line and in peripheral blood mononuclear cell (PBMC) culture was examined. HIV-infected H9 cells were exposed to different concentrations of papaverine for 20 days. Reverse
transcriptase (RT) activity and the presence of p24 in the supernatant were determined to
assess the level of viral replication in treated and control cultures. The most effective concentration of papaverine in the culture medium was 10 pg/ml, a dose that did not significantly affect cell proliferation. At this drug concentration the treatment resulted in no RT
activity or p24 expression in the supernatant and no virus antigen detection at the cellular
level as demonstrated by Western blot (WB) analysis. The activity of the drug occurred in a
short period of time (60 hours) as shown by radioimmunoprecipitation (RIP) assay and
affected the synthesis of the env precursor protein gpl60. The drug was also effective in
inhibiting HIV replication in PBMC cultures and influenced specific viral markers, namely,
RT and p24. Evidence of the efficacy of papaverine treatment was enforced by the finding
in the treated PBMC cultures, compared with the untreated ones, of a reduced percentage
of cells forming syncitia and of the inhibition of the virus-induced decrease in the number of
cells. When an equal number of virus-infected H9 cells exposed or unexposed to papaverine
was analyzed for HIV-specific proteins, a marked decrease in the expression of the viral
proteins was observed in the treated cultures. At the same time, one cellular protein of
molecular weight 69,000 was not inhibited by papaverine. This may indicate that, at least
for one protein, synthesis may not be affected by the drug. Our data suggest that papaverine merits attention as a possible nontoxic candidate for the treatment of HIV infection.

INTRODUCTION

immune deficiency syndrome

effective preventive
Acquired
be solved

Many problems must

(AIDS) has become a worldwide epidemic.

In the absence of

expansion of the patient population with AIDS is expected.

before an AIDS vaccine can be developed,1 and human immunodeficiency

measures,

continued

istituto di Microbiologa, Universit di Brescia, 25124 Brescia, Italy.

2Istituto di Malattie Infettive, Universit di Verona, 37100 Verona, Italy.

183

TURANO ET AL.
virus (HlV)-infected individuals may not benefit from a vaccine. Thus developing drugs that will be effective against HIV is a major challenge. Several promising agents that inhibit HIV RT are already available.2-5 Other drugs have been identified as noncompetitive inhibitors with anti-HIV activity in vitro.6-9
Molecules that interfere with HIV replication, such as anti-CD4+ monoclonal antibodies10,11 and a CD8 +
cell soluble factor,12 have also been considered for treatment of AIDS virus infection. Papaverine, an
alkaloid having a molecular weight of 375, is an inhibitor of cellular phosphodiesterases,13 the enzyme
responsible for cAMP breakdown, and its addition in cell cultures results in an increased cellular level of
endogenous cAMP. The drug is stable at 20C and may be given orally or intravenously. Its toxicity in
vivo, in clinical treatment, is very low, and its tolerance over a long term is well known since papaverine
has been extensively used for many years as a smooth muscle relaxant. Recently, papaverine was shown to
have antiviral activity with several human viruses14-16 and a murine retrovirus, MSV-Harvey.17 The mechanism of its action is not completely understood, even if interference with cellular DNA synthesis directly,
through a competitive and reversible binding to the DNA molecule,18 or through elevation of endogenous
cAMP19 has been postulated; at least for measles virus, inhibition of viral RNA synthesis and phosphorylation of viral proteins have been demonstrated.16 Because of the wide spectrum of its antiviral activity, it
was interesting to determine whether papaverine could also be effective in inhibiting in vitro HIV replication. We now demonstrate that papaverine, affecting the synthesis of all the viral proteins, interferes with
in vitro replication of HIV both in an immortalized helper T cell line (H9)20 and in PBMC cultures.

MATERIALS AND METHODS

Virus strain and cells
H9 cell line and a HIV (HTLV-IIIB)-producing cell line, H9/HTLV-III20 (courtesy of Dr. R.C. Gallo),
used in this study. Both cell lines were maintained in RPMI-1640 medium supplemented with penicillin (250 U/ml), streptomycin (250 pg/ml), L-glutamine (2 mM), HEPES (10 mM), and 20% fetal calf
serum (Flow, UK). PBMC were isolated by Ficoll-Hypaque centrifugation of blood leukocytes and incubated in the same medium containing phytohemagglutinin (PHA, Sigma, St. Louis, MO; 5 pg/ml).
were

Compound
Papaverine hydrochloride
Italy).

Houd

4cg,

in

liquid form,

was

bought

from Lirca

S.p.A. (Limito, Milano,

Liquid competition radioimmunoassay (LICRIA)

major viral core protein, p24, was measured by LICRIA as already described.21 Briefly, lysates of
(0.2 pg), diluted in 50 pi phosphate-buffered saline (PBS) containing calcium and magnesium (pH
7.2), were allowed to dry overnight at 37C on individual wells of 96-well polyvinyl chloride microtiter
plates (detection plates). Volumes of 200 pi PBS containing 5% bovine serum albumin (BSA, w/v) were
added to each well of the detection plates and to each well of a separate set of 96-well microtiter plates
containing no antigen adsorbed to the wells (reaction plates) in order to minimize nonspecific protein
adsorption. After incubation for 1 hour at 37C, the plates were washed once with PBS containing 1% BSA
(w/v). To each well of the reaction plate, 75 pi of the appropriate dilution of an anti-HIV-1 p24 (MB 12) in
PBS containing 1% BSA (w/v) and 0.5% Tween 20 (v/v, assay buffer) and either 75 pi of competitor
antigen (diluted in assay buffer containing 1% Triton X-100) or assay buffer containing 1% Triton X-100
The

HIV

were added. The final detergent concentration in each well was 0.05% Tween 20 and 0.5% Triton X-100.
Reaction plates were incubated overnight at 4C. Aliquots (50 pi) were then transferred from each well of
the reaction plates to duplicate wells of the detection plates. The detection plates were then incubated for 1
hour at 37C and washed twice with assay buffer. A 1:2000 dilution (50 pi in assay buffer) of rabbit
antimouse IgG (H + L chain) (Cappel, Cochranville, MD) was then added to each well. The detection

184

PAPAVERINE AND HIV REPLICATION

plates were incubated for 1 hour at 37C, the wells were then washed twice, and 25 pi of assay buffer
containing 50,000 cpm of [125I]protein A (Amersham, Amersham, UK) was added to each well. Following

an additional 1 hour incubation at 37C, the wells were washed four times with assay buffer, cut from the
detection plates, and counted in a gamma counter. The percentage of bound counts was determined by
dividing the average of cpm bound to the wells of the detection plate in the presence of competitor antigen
by the average of the cpm obtained in the absence of competitor antigen multiplied by 100. Using an
affinity-purified HIV p24 as a standard in the reaction, we could quantitate HIV p24 in the samples tested.

Infection ofH9 cells and papaverine

treatment

HIV was obtained from culture supernatants of H9/HTLV-III as previously described.20 H9 cells were
treated with polybrene (Sigma, St. Louis, MO; 2 pg/ml) for 20 minutes and a number of flasks containing
2 x 106 cells were exposed to 8 x 107 viral particles. Control cells were mock infected with a filtered,
cell-free supernatant from an uninfected H9 cell culture. After 1 hour incubation at 37C, culture medium
with papaverine at a concentration of 3, 6, 8, and 10 |xg/ml, or without the drug, was added to the cell
cultures. After the infection, at intervals of 5 days up to day 20, the supernatants were examined for the
presence of RT22 and p24, the major viral core protein, measured by LICRIA.

Protein extract

ofH9 cells and Western blot analysis

of HIV antigens in the H9 cells was determined by WB analysis. H9/HTLV-III cells and
control H9 cells were cultured for 15 days in the presence or absence of papaverine at a concentration of 10
pg/ml. At days 5, 10, and 15, an equal number of cells (5 x 106) from each culture were washed twice in
serum-free RPMI-1640 and resuspended in phosphate-buffered saline (PBS) containing 1% Triton X-100
(w/v), pH 7.2, and then frozen and thawed three times. Nuclei and debris were pelleted at 1200 x g for 10
minutes, and the clarified supernatants were boiled for 2 minutes. Following a centrifugation at 3500 x g
for 10 minutes, the supernatants were assayed for the presence of HIV proteins. Markwell determination23
gave a similar protein concentration in all the samples. An equal amount of protein from each sample (100
pg) was subjected to electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel, and the proteins
were then transferred to a nitrocellulose sheet by electroblotting24 for 5 hours at 200 mA. The blot was
incubated for 2 hours at 25C in PBS containing 5% BSA (w/v). Serum positive for antibodies to HIV was
then added to a final dilution of 1:400 in PBS containing 1% BSA (w/v) and allowed to react for 16 hours at
4C. The blot was then washed and 125I-labeled goat antihuman Ig (Amersham, Amersham, UK) at a final
dilution of 1 X 106 cpm/ml was added. After incubation for 1 hour at 37CC, the label was removed and the
nitrocellulose blot was washed, dried, and exposed to Amersham x-ray MP-Hyperfilm.

Expression

Radioimmunoprecipitation assay
H9/HTLV-III cells were exposed or not to a single dose of papaverine at a concentration of 10 pg/ml,
cultured for 48 hours, and then radioactively labeled by incubation for 12 hours at 37C in the presence of
[3H]leucine (Amersham, Amersham, UK; 100 pCi/ml). Labeled cells were washed twice in serum-free
RPMI-1640 medium and disrupted at 4C by repeated aspiration through a 25-gauge needle in 10 mM
sodium phosphate (pH 7.6) containing 0.5% NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and
0.1% SDS (SDS/TDS). The lysates were absorbed for 3 hours at 25C with protein A-Sepharose and equal
portions of normal serum. Immunoprecipitation analysis was performed as already described.25 Briefly, 10
ml of a HIV immune serum and 0.2 ml of a 10% suspension of protein A-Sepharose were added to 1 ml of
labeled and clarified extract. The samples were then incubated for 18 hours at 4C. Immunoprecipitates
were collected by centrifugation at 2000 x g for 10 minutes, washed repeatedly in SDS/TDS, suspended in
50 pi of 0.65 M Tris-hydrochloride (pH 6.7), 1% SDS, 10% glycerol, 2.5% 2-mercaptoethanol, and 0.1%
bromophenol blue, heated for 2 minutes at 90C, and analyzed by SDS-PAGE.

185

TURANO ET AL.

Infection of PBMC cultures and antiviral agent treatment

PBMC were grown in culture medium containing PHA (5 pg/ml) for 48 hours at 37C. They were then
refed with complete RPMI-1640 medium containing 10% purified T cell growth factor (TCGF; Sigma, St.
Louis, MO). After 12 hours of exposure to TCGF, PBMC were treated with polybrene (2 pg/ml) for 20
minutes and 1 x 107 cells were distributed in each of 12 flasks: 3 flasks of PBMC were maintained in
complete RPMI-1640 medium containing papaverine at a concentration of 10 pg/ml; 3 flasks of PBMC
were infected with 8 x 107 HIV particles and maintained in complete RPMI-1640 medium containing
papaverine at the same concentration as earlier; 3 flasks of PBMC were infected with 8 x 107 viral
particles and maintained in complete RPMI-1640 medium; and 3 flasks of PBMC were maintained in
complete RPMI-1640 medium and cultured as a growth control. After 5 days of culture, the presence of
HIV was detected in the supernatant by RT and LICRIA. Expression of p24 was also quantified on an equal
number of PBMC harvested from each culture by LICRIA. Number of living PBMC and percentage of
cells forming syncitia26 were also evaluated.

Cytotoxicity assay
The effects on cell proliferation were studied by the addition of papaverine at a concentration of 3, 6, 8,
10, 20, 30, and 40 pg/ml to actively replicating H9 cells. Cell numbers were measured after incubation for
72 hours by the trypan blue exclusion staining method.

RESULTS
H9 cell treatment

Figure 1 shows a high level of RT activity at day 10 in the

cultures maintained in RPMI-1640 containing papaverine at

supernatant of HIV-infected H9 cells. Parallel

a

concentration of 3

pg/ml

showed

positive

DAYS AFTER INFECTION

FIG. 1. Detection of HIV replication in H9 cell cultures. RT activity on clarified supernatants from HIV-infected
cells untreated (O) or treated with papaverine at a concentration of 3 (), 6 (A), 8 (T), and 10 (xg/ml ( ).

186

PAPAVERINE AND HIV REPLICATION

activity only at day 15. No RT activity at higher drug concentrations (namely, 6,8, and 10 pg/ml) was
detected at day 20. The drug had no effect on our RT assay system when it was added at the highest
concentration used (10 pg/ml) to the reaction mixture or to purified RT (data not shown). In the supernatant of the same cultures treated with papaverine at concentrations of 3, 6, and 8 pg/ml, HIV p24 was still
detectable at day 20. However, the levels were 3-, 60-, and 80-fold lower than in the supernatant of the
HIV-infected and untreated cultures, respectively. In the supernatant of H9 cells treated with papaverine at
a concentration of 10 |xg/ml, no p24 was detectable (Table 1). The different effect of papaverine shows that
lower doses can completely inhibit RT while p24 is still present and may be explained by de facto higher
expression of p24 in the viral particle or by the difference in sensitivity between RT and p24 detection
assays. The drug, at each concentration used, did not significantly affect H9 cell proliferation (data not
shown), and the cells were viable and actively replicating. Papaverine was unequivocally effective in
inhibiting HIV replication at a concentration of 10 jjug/ml, so that in further experiments we used the drug at
this concentration.
RT

HIV protein

expression in H9 cells

Owing to the decreased release of virus observed in H9 cell cultures when exposed to papaverine, we
decided to investigate the rate of HIV protein synthesis at the cellular level. An equal number of H9/
HTLV-III cells (5 x 106) exposed or unexposed for 15 days to papaverine at a concentration of 10 pg/ml
were extracted and analyzed by WB. As shown in Figure 2, the extracts from infected and untreated H9
cells showed a complete typical HIV protein pattern at days 5, 10, and 15. A partial reduction of all the
HIV proteins was detected in the extract of cells grown for 5 days in complete RPMI-1640 medium containing papaverine, and an almost complete disappearance of all the viral bands, with only a residual trace
of p24, was observed in the extract of cells grown for 15 days in complete RPMI-1640 medium containing
papaverine. The amount of an unrelated cellular protein, recognized by the antiviral immune serum used in
the reaction and present in all the samples analyzed, including the negative control, was not affected by

papaverine treatment (Fig. 2).

Radioimmunoprecipitation assay
To define whether

influences the de

of HIV gene

products, H9/HTLV-III
analyzed by RIP assay.
The proteins immunoprecipitated from the HIV-infected H9 cells exposed to papaverine showed a marked
reduction in all the immunoprecipitated viral proteins and a complete disappearance of the HIV envelope
papaverine

novo

synthesis

cells, exposed or not for 48 hours to a pulse dose of 10 pg/ml of papaverine,

Table 1. Effect of Papaverine on the HIV

Infectivity of H9 Cells3
Concentration

of papaverine
(\[Link])
10
8
6
3
0

aData
ments.

are

p24b

(lig/mg)
0
0.08
0.12
2.27
7

averages of three independent experi-

bp24 is detected and quantified by LICRIA in

the supernatant of the HIV-infected cell cultures.
187

were

TURANO ET AL.

gpl60
gp120
p51
gp41-45
p31

p24
p17
Identification of HIV antigens in infected cells by Western blot. Analysis of extracts from normal H9 cells
(lane 7), H9/HTLV-III cells grown in complete RPMI-1640 medium for 5 days (lane 1), 10 days (lane 3), and 15 days
(lane 5), and H9/HTLV-III cells grown in complete RPMI-1640 medium containing 10 |xg/ml of papaverine for 5 days
(lane 2), 10 days (lane 4), and 15 days (lane 6).

FIG. 2.

protein gpl60 (Fig. 3). The presence in the same sample of small amounts of the env proteins
gpl20
gp41 can be explained by the processing of all the de novo synthesized gpl60.
The core precursor protein p53 only was reduced. Whether the difference in the inhibition of the expression of the HIV env and core proteins is due to a preferential action of papaverine on the env gene or
whether it is the most visible effect on a less expressed protein needs further investigation.
precursor

and

Cytotoxicity
were observed in uninfected H9 cells treated with papaverine. Howreplication was adversely affected by papaverine, H9 cells were grown in
T25 flasks for 72 hours with different concentrations of drug. At 10 |xg/ml papaverine did not significantly
affect cell proliferation. At 20, 30, and 40 pg/ml the number of papaverine-treated cells was significantly
different from the control (p < 0.05), and 40 jig/ml was more inhibitory than 20 or 30 pg/ml (p < 0.05).
The number of cells in cultures treated with 40 u.g/ml increased 1.5-fold daily compared with the untreated
control cultures, which increased 2.17-fold daily (30% difference in cell growth).

No

morphological

abnormalities

ever, to determine whether cell

PBMC treatment
The data obtained in the HIV-H9 cell system are similar to those in the HIV-PBMC (Table 2). The
number of cells decreased by approximately 26% 5 days after infection with HIV. The presence of HIV
was confirmed by high levels of RT activity in the supernatant, by the expression in the supernatant and in
cells of p24 measured by LICRIA, and by the number of cells forming syncitia. Papaverine was able to
inhibit the virus-induced decrease in PBMC at a concentration (10 |xg/ml), which had little effect on cell
proliferation. The other specific viral markers, RT and p24, were greatly influenced by the drug treatment.
RT decreased by approximately 40%, and the level of p24 expression in the supernatant was sevenfold less
than in those unexposed to the drug. Similarly, on equal numbers of cells, a decrease of sevenfold in p24

188

PAPAVERINE AND HIV REPLICATION

MW

2 3

200K-

14.3K-

FIG. 3.

Immunoprecipitation of metabolically labeled HIV proteins by a HIV-seropositive serum. Proteins were

immunoprecipitated from H9 cells (lane l), H9/HTLV-III cells grown in complete RPMI-1640 medium containing
papaverine (lane 2), and H9/HTLV-III cells grown in complete RPMI-1640 medium without papaverine (lane 3).

expression in the PBMC exposed to papaverine with respect to the unexposed cells was observed. A
significant reduction in the percentage of cells forming syncitia, with a statistical significance of/? < 0.01,
was

also observed.

Compound removal
In order to

papaverine

at

the presence of replicating virus in the PBMC cultured for 5 days in the presence of
concentration of 10 pg/ml, 2 x 106 lymphocytes infected with HIV and exposed or not to

study
a

Table 2. Effect of Papaverine on the HIV Infectivity of Human Peripheral Blood Lymphocytes8

Control
Condition

Uninfected
Uninfected

plus
papaverine (10 (ig/ml)

Infected
Infected

plus
papaverine (10 |xg/ml)

106 Cells
25.0

RT activity

Syncitia formation

p24b

p24c

100

100

1.53

20.0 1.62
18.5 1.20
20.0 1.43

100
37.9

54.4

14.7

13.6

"All data presented are means ( standard deviation) from at least three independent experiments and from three
five replicates per assay.
bp24 is detected and quantified in the HIV-infected cells by LICRIA.
cp24 is detected and quantified in the supernatant of the HIV-infected cell cultures by LICRIA.

189

to

TURANO ET AL.
the drug were cocultivated, in the absence of papaverine, with 2 x 106 lymphocytes from a healthy donor.
After 10 days an RT activity of 3.7 x 104 cpm per reaction was detected in the supernatant of infected and
untreated lymphocytes cocultivated with lymphocytes from a normal donor. A reduction of almost 92% in
the RT activity (3.3 x 103) was detected in the supernatant from lymphocytes cocultivated with the HIVinfected and papaverine-treated cells.

DISCUSSION
The results presented here demonstrate that papaverine interferes with HIV replication in both H9 and
PBMC cultures. The effect of papaverine on HIV replication was dose dependent, with an optimal inhibition of the virus replication achieved at a drug concentration of 10 pg/ml (Fig. 1). In fact, when higher
papaverine concentrations were used, namely 20, 30, and 40 pg/ml, a complete inhibition of HIV replication was achieved, but the drug at these concentrations had a dose-dependent inhibitory effect on cell
proliferation. Papaverine activity occurred in a short period of time (60 hours) affecting the synthesis of the
HIV proteins, especially the env precursor protein gpl60, as demonstrated by RIP data. On the other hand,
the expression of an unidentified cellular protein of molecular weight 69,000 was not affected during 20
days of papaverine treatment, showing that the synthesis of this cellular protein at least is not inhibited.
Besides, when an equal number of HIV-infected cells cultured in the presence or absence of papaverine
was examined by WB or LICRIA, a reduction in the expression of the specific viral proteins in the treated
cells, compared with the untreated cells, was observed.
It is known that papaverine is able to interfere with DNA synthesis through a competitive and reversible
binding to the DNA molecule18 and to increase endogenous cAMP.19 However, the mechanism of the
antiviral action of papaverine is not fully understood. From our data, papaverine seems to affect late steps
of HIV replication. In fact, the selective effects on different proteins suggests that papaverine acts after
reverse transcription. On the other hand, a concomitant interaction of papaverine with the viral DNA when
it is still in an unintegrated form, or an interference with its integration in the DNA of the host cell, cannot
be ruled out.
Many compounds are currently being explored for their efficacy in the treatment of AIDS. However,
considering the severity of AIDS and the necessity of long-term chemotherapy for this disease, more
effective or less toxic drugs are required. The toxicity of papaverine is low, and its tolerance over a
long-term therapy is well known, since the drug has been extensively used clinically for many years.
Nevertheless, papaverine has been shown to reach a peak plasma concentration as high as 1 pg/ml following the administration of 300 mg orally every 6 hours to humans,27 which is only 1/10 the amount
required in vitro to produce a complete inhibition of HIV replication. Since there is no correlation between
the levels of antiviral drugs that produce a significant inhibitory effect in vitro and the level required to
produce a desired effect in vivo, it is possible that only 1 pg/ml of papaverine in serum might be required
to interfere in vivo with HIV replication. Only appropriate studies in animal models and in patients may
answer this question.

ACKNOWLEDGMENTS
We wish to thank Dr. R.C. Gallo for the generous

supply

of H9 cells and HTLV-IIIB. His continuous

support and critical interest is deeply acknowledged. Supported in part by CNR of Italy and World Labora-

tories.

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J Pharm

Address

reprint requests to;

Adolfo Turano
Istituto di Microbiologa
Universit di Brescia
Piazza Spedali Civili
1-25124 Brescia, Italy

192

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