Effect of betulinic acid and its ionic derivatives on M-MuLV replication

Article abstract of DOI:10.1016/j.bbrc.2018.04.080

Murine leukemia virus (MuLV) is a retrovirus known causing leukemia and neurological disorders in mice, and its viral life cycle and pathogenesis have been investigated extensively over the past decades. As a natural antiviral agent, betulinic acid is a pentacyclic triterpenoid that can be found in the bark of several species of plants (particularly the white birch). One of the hurdles for betulinic acid to release its antiviral potency is its poor water solubility. In this study, we synthesized more water-soluble ionic derivatives of betulinic acid, and examined their activities against Moloney MuLV (M-MuLV). The mouse fibroblast cells stably infected with M-MuLV, 43D cells, were treated with various doses of betulinic acids and its derivatives, and the viral structural protein Gag in cells and media were detected by western blots. Two ionic derivatives containing the benzalkonium cation were found to inhibit the virus production into media and decreased Gag in cells. However, a cell proliferation assay showed that the benzalkonium cation inhibited the growth of 43D cells, suggesting that our ionic derivatives limited virus production through the inhibition of metabolism in 43D cells. Interestingly, all of these betulinic acid compounds exhibited a minimum impact on the processing and release of Gag from 43D cells, which outlines the differences of viral maturation between MuLV and human immunodeficiency virus.

Full text of DOI:10.1016/j.bbrc.2018.04.080

Biochemical and Biophysical Research Communications 500 (2018) 365e369
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Effect of betulinic acid and its ionic derivatives on M-MuLV replication
,
, d, *
Jasmine Phillips a, Iesha Phillips b, Blessing Enya b, Hua Zhao c **, Takayuki Nitta b
a Department of Chemistry and Forensic Science, Savannah State University, 3219 College St., Savannah, GA 31404, USA
b Department of Biology, Savannah State University, 3219 College St., Savannah, GA 31404, USA
c Department of Chemistry and Biochemistry, University of Northern Colorado, Greeley, CO 80639, USA
d Department of Biomedical Sciences, Mercer University School of Medicine, Savannah, 1250 East 66th St., Savannah, GA 31404, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Murine leukemia virus (MuLV) is a retrovirus known causing leukemia and neurological disorders in
mice, and its viral life cycle and pathogenesis have been investigated extensively over the past decades.
As a natural antiviral agent, betulinic acid is a pentacyclic triterpenoid that can be found in the bark of
several species of plants (particularly the white birch). One of the hurdles for betulinic acid to release its
antiviral potency is its poor water solubility. In this study, we synthesized more water-soluble ionic
derivatives of betulinic acid, and examined their activities against Moloney MuLV (M-MuLV). The mouse
fibroblast cells stably infected with M-MuLV, 43D cells, were treated with various doses of betulinic acids
and its derivatives, and the viral structural protein Gag in cells and media were detected by western
blots. Two ionic derivatives containing the benzalkonium cation were found to inhibit the virus pro-
duction into media and decreased Gag in cells. However, a cell proliferation assay showed that the
benzalkonium cation inhibited the growth of 43D cells, suggesting that our ionic derivatives limited virus
production through the inhibition of metabolism in 43D cells. Interestingly, all of these betulinic acid
compounds exhibited a minimum impact on the processing and release of Gag from 43D cells, which
outlines the differences of viral maturation between MuLV and human immunodeficiency virus.
© 2018 Elsevier Inc. All rights reserved.
Received 9 April 2018
Accepted 10 April 2018
Available online 21 April 2018
Keywords:
Murine leukemia virus
Betulinic acid
Viral release
Gag maturation
Human immune deficiency virus
1. Introduction
which has enabled the understandings of general phenomenon of
leukemogenesis; therefore, M-MuLV has been considered as a
Retroviruses comprise a large and diverse family of enveloped
RNA viruses defined by common taxonomic denominators
including the structural, compositional, and replicative properties
[1]. Murine leukemia viruses (MuLVs) are simple retroviruses and
encode only three polyproteins that are used in the assembly of
progeny virus particles; Gag (structural protein), Pol (three retro-
viral enzymes, i.e. protease, reverse transcriptase, and integrase),
and Env [2]. MuLVs are composed of diverse strains in both
endogamous and exogenous viruses, and some of them are caus-
ative agents of leukemia and neurological disorders in mice [3].
Moloney murine leukemia virus (M-MuLV) is a well-studied repli-
cation-competent oncogenic retrovirus of gammaretrovirus genus
model retrovirus [4]. The viruses bind to the host cell via in-
teractions between the envelope protein and its receptor on target
cells. Subsequently, the penetrated viral cores are endocytosed and
the uncoating occurs. The single stranded viral RNA genome is
reverse transcribed and cDNA is integrated into the host genome by
integrase. In the late phase, viral transcripts make the viral proteins.
At the same time, the unspliced viral RNA works as the viral
genome. These viral materials are trafficked to plasma membranes
and assembled, and new viruses bud from the membranes at the
end.
Betulinic acid (3b-hydroxyl-lup-20(29)-en-28-oic acid) is a
natural pentacyclic lupine type triterpene that can be extracted
from plants (e.g. birch tree bark) [5]. This natural compound has a
number of medically relevant biological properties such as anti-
inflammatory, anti-cancer, and anti-viral activities [5e8]. A major
obstacle with the releasing of the antiviral potency of betulinic acid
is due to its poor solubility in aqueous solutions and common
organic solutions (such as esters, alcohols, and ethers). As a matter
of fact, the solubility of betulinic acid in water is only approximately
* Corresponding author. Department of Biology, Savannah State University, 3219
College St., Savannah, GA 31404, USA.
** Corresponding author. Department of Chemistry and Biochemistry, University
of Northern Colorado, Greeley, CO 80639, USA.
E-mail addresses: hua.zhao@unco.edu (H. Zhao), nittat@savannahstate.edu
(T. Nitta).
https://doi.org/10.1016/j.bbrc.2018.04.080
0006-291X/© 2018 Elsevier Inc. All rights reserved.

366
J. Phillips et al. / Biochemical and Biophysical Research Communications 500 (2018) 365e369
0.02
m
g/ml at room temperature [9]. In common organic solvents,
duration of 48 h. After the reaction completed, the precipitate
(dicyclohexylurea as byproduct) was filtered off and the filtrate was
further evaporated under vacuum to remove THF. The crude
product was then dissolved in 100 ml of ether/ethyl acetate (2:1, v/
v) and extracted with 100 ml of water to remove the excess water-
soluble carbodiimide reagent and any remaining dicyclohexylurea.
The organic layer was further extracted with 1.0 N HCl (2 ꢁ 100 ml)
to remove DMAP [17], saturated NaHCO3 solution (1 ꢁ 100 ml), and
lastly with water (1 ꢁ 100 ml) [18,19]. The purified organic layer
was then dried over Na2SO4. After filtration, ethyl acetate was
evaporated under vacuum to yield the glycine methyl ester con-
jugate of betulinic acid. The methyl ester (298 mg) was dissolved in
100 ml of THF/H2O (4:1, v/v) solution, followed by the addition of
LiOH (67.5 mg). The reaction mixture was then stirred at room
temperature for 4 h under argon. Once the reaction completed, THF
was evaporated under vacuum and the product obtained was dis-
solved in 200 ml of ethyl acetate, followed by sequential washing
with water, 0.1 M HCl, and water once again. The organic layer was
then dried with Na2SO4, and after filtration the solvent was
removed under vacuum to yield glycine conjugate of betulinic acid
(231.4 mg). The choline hydroxide was prepared from choline
chloride following an anion-exchange column approach used pre-
viously [15]. The glycinylated betulinic acid, BA4, was dissolved in
100 ml of THF/H2O (4:1, v/v), followed by the addition of a five-fold
molar excess of choline hydroxide (0.55 g). The reaction mixture
the solubility of betulinic acid is also fairly low, e.g. 1% (w/v) in
ethanol and 5% (w/v) in DMSO at 25 ꢀC [10]. Some derivatives of
betulinic acid had shown improved water solubility, as well as
enhanced biological activities when compared with betulinic acid
itself [6,11,12]. Recently, we developed new ionic derivatives of
betulinic acid with a higher water solubility and thus a stronger
antiviral activity [10,13,14]. In the present study, we further
examined the effect of betulinic acid and its derivatives (see Fig. 1)
on the retroviral replication of M-MuLV.
2. Materials and methods
Synthesis of ionic derivatives of betulinic acid. Betulinic acid,
glycine methyl ester hydrochloride, N, N0-dicyclohexylcarbodiimide
(DCC), 4-(dimethylamino)pyridine (DMAP), choline chloride, ben-
zalkonium chloride, and Amberlyst A26 hydroxide form (Sigma-
Aldrich, St. Louis, MO) were used to prepare ionic salts of betulinic
acid conjugated with glycine (Fig. 1). The procedures for preparing
ionic derivatives of betulinic acid are described previously [15,16].
In brief, betulinic acid (BA1, 204.4 mg), glycine methyl ester
(104.0 mg), and triethylamine (8 mg) were dissolved in 50 ml of
anhydrous tetrahydrofuran (THF) at room temperature. Into the
reaction mixture, DCC (107.9 mg) and DMAP (30.3 mg) were added
and the mixture was stirred at room temperature under argon for a
Fig. 1. Structures of betulinic acid (BA1) and ionic derivatives (BA2‒BA5).

J. Phillips et al. / Biochemical and Biophysical Research Communications 500 (2018) 365e369
367
was stirred at room temperature for a duration of 24 h. THF was
removed by rotary evaporation. The crude product was extracted
into 100 ml of ethyl acetate, and washed with 100 ml of distilled
water to remove the excess choline hydroxide. The organic layer
was dried and ethyl acetate was removed under vacuum to give the
cholinium salt of betulinic acid-glycine([Choline][BA-Gly], (BA2,
171 mg) (Fig. 1). Using the above reaction strategies, we prepared
the benzalkonium salt of glycinylated betulinic acid ([Bzk][BA-Gly],
BA3, and cholinium betulinate ([Choline][BA], BA5) starting with
betulinic acid. These compounds were used to treat mouse fibro-
blast cells.
of 3% SDS. Absorbance of 570 nm/600 nm was measured by
NanoDrop 2000 UVeVis Spectrophotometer (Thermo Scientific) to
determine reduction of resazurin dye. The DMSO treated cells were
set to 100% and then the viability readings from the treated wells
were calculated as a percentage of the control.
Statistical analysis. Students T-test was used to evaluate viral
release efficiency from 43D cells treated with betulinic acid or its
derivatives.
3. Results
Treatment of 43D cells with betulinic acid and its derivatives.
Establishment of 43D mouse fibroblast cell line stably infected with
the wild-type M-MuLV was described previously [20]. The 43D cells
were cultured with DMEM media containing 10% Hyclone calf
serum (GE Healthcare Life Sciences, PA), 100 IU/ml of Penicillin and
100 ug/ml of Streptomycin (Corning, VA) at 37 ꢀC with 5% CO2. Cells
were cultivated in 60 mm and 100 mm BioLite cell culture treated
dishes (Thermo Scientific, MA, USA). The 43D cells were treated
with betulinic acid, its derivatives, or benzalkonium chloride (BKC)
for 24 h, and then the media were replaced. The cells and viruses
were gathered 24 h further incubation after the second treatment
of respective compounds.
Detection of MuLV and beta-Tubulin. The sodium dodecyl
sulfate (SDS) samples were prepared from the cells and the viruses
released into media. The proteins were extracted from the cells
with radioimmunoprecipitation assay (RIPA) buffer which is a
common buffer used to lyse cultured mammalian cells. This buffer
was composed of 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40,
1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, and 1 x
AMRESCO Protease Inhibitors (AMRESCO LLC, Solon OH, USA). In
To determine the inhibition of betulinic acid and its ionic de-
rivatives against M-MuLV release, 43D cells were treated with
30 mM of these compounds for 48 h. Viral Gag protein is translated
as poly-Gag, Pr65Gag which is further cleaved into some smaller Gag
proteins such as p30CA through the maturation of viruses [2]. Since
the cleavage of Gag proteins to yield p30CA occurs during budding/
viral release, the majority of Gag proteins in secreted viruses is
p30CA (Fig. 2A). Poly-Gag proteins Pr65Gag in cells and p30CA in cells
and viruses (media) were detected by SDS-PAGE and western blots
with rabbit polyclonal anti-p30CA antibodies [22]. Compounds BA1,
BA2, and BA5 did not show any significant impact on the intracel-
lular Gag production and its processing, but compounds BA3 and
BA4 decreased the intracellular Gag proteins. The signals for beta-
Tubulin in the cells treated with compounds BA3 and BA4 exhibi-
ted a small decrease. Compounds BA3 and BA4 decreased the
amount of p30CA in media, but the virus release efficiency was not
affected by any of the compounds BA1-BA5 (Fig. 2B). Treatment of
10 mM of betulinic acids and its derivatives did not change Gag in
order to obtain SDS samples, 43D cells were incubated with 120 ml
of RIPA on ice. The cell debris was span down by centrifugation at
13,000 rpm for 15 min at 4 ꢀC. The supernatant was transferred into
new micro-centrifuge tubes, and 4 x SDS buffer was added and
boiled for 3 min. Separately, the viruses in media were concen-
trated by ultracentrifugation at 25,000 rpm with a Beckman SW28
ultracentrifuge rotor for 1 h at 4 ꢀC [21]. All media were removed,
and the viruses were suspended in 160
(62.5 mM Tris-HCl (pH 6.8), 2.5% SDS, 0.002% bromophenol blue,
0.7135 M -mercaptoethanol, 10% glycerol). The viral proteins were
ml of 1 x SDS sample buffer
b
detected by SDS-PAGE and western blots with the rabbit serum for
M-MuLV p30CA [22], and anti-rabbit IgG conjugated with horse-
radish peroxidase. The images were acquired by the SuperSignal
West Femto Maximum Sensitivity Substrate (Thermo Scientific)
and Gel Doc™ XR þ Gel Documentation System (Bio-Rad, CA, USA).
To quantify the viral release efficiency, each Pr65Gag and p30CA (a
precursor and a cleaved MuLV Gag proteins) band in the cells and
media was quantified with the densitometry software AlphaEaseFC
(Alpha Innotech, CA, USA), and the percentage of released p30CA
divided by the total Gag proteins (Pr65Gag and p30CA) in cells and
media was calculated [20]. Different exposures of the blots were
analyzed to ensure that densitometry was in the linear range. Beta-
Tubulin was detected by SDS-PAGE and western blots with anti-
beta-Tubulin (Cell Signaling Technology, MA) and goat anti-
mouse IgG conjugated with horseradish peroxidase (ImmunoRe-
agents, NC).
AlamarBlue Cell Viability Assay. Cell viability was assessed
using the AlamarBlue Cell Viability Assay [23] from Bio-Rad
following the manufacturer's protocols. Briefly, 5 ꢁ 103 43D cells
Fig. 2. Effect of betulinic acid and its ionic derivatives on MuLV replication. (A) 43D
were placed on 96-well plates (100
m
l of media/well). After over-
M, 30
M of betulinic acid, its derivatives, or BKC for 48 h
respectively, then 10 l of AlamarBlue solution was added to the
wells. Following 2-h incubation, the reaction was stopped by 50
cells were treated with 30 mM of the compounds for 48 h, and Gag proteins in cells and
night incubation, the cells were treated with DMSO, 10
and 100
m
mM
media were detected by western blots using the rabbit serum for anti-p30CA. (B) The
intensity of Gag signals detected by western blots was quantified by immunodensi-
tometry and the viral release efficiency was calculated. The figure shows the relative
virus release efficiency (% Gag release) with standard deviation.
m
m
ml

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J. Phillips et al. / Biochemical and Biophysical Research Communications 500 (2018) 365e369
cells and in viruses as detected by western blots (data not shown).
It was reported that betulinic acid suppressed some signaling
pathways involved in the cellular metabolism and could induce cell
death [24]. To assess the relationship between the inhibition of M-
MuLV Gag production and the general cellular metabolism in 43D
cells treated with these compounds, cell growth was measured by
AlmarBlue cell proliferation assay which monitored the reducing
environment of living cells [23]. The cells were treated with
different concentrations of betulinic acids and its derivatives for
48 h. Compounds BA3 and BA4 reduced the cellular activities by
site determined the viral sensitivity to DSB [8,25,26]. We confirmed
that betulinic acid inhibited the cleavage of p25CAeSP1
and
,
increased the amount of p25CAꢂSP1 by western blots (data not
shown), but did not observe the inhibition of Gag cleavage in MuLV
with betulinic acid and its ionic derivatives (Fig. 2A) although our
ionic derivatives of betulinic acid displayed improved water solu-
bilities, and exhibited a stronger antiviral activity against herpes
simplex virus type-2 (HSV-2) than betulinic acid [14]. MuLV Gag is
cleaved during virus maturation into MA, p12, p30CA, and NC [2],
while amino acids flanking the MA-p12 are Leucine and Tyrosine,
amino acids flanking the p12-p30CA are Alanine and Phenylalanine,
and amino acids flanking the p30CA-NC are Leucine and Leucine.
Since DSB failed to show any significant inhibition of HIV-1 PR
in vitro using a synthetic peptide and recombinant Gag [25,27], this
compound seems to target unique sequences in Gag to prevent viral
maturation through unknown mechanisms. Our results are in
agreement with the previous data indicating that DSB targeted the
specific amino acid sequence flanking to the cleavage site, and
suggest that amino acid sequence found in MuLV Gag and MuLV
protease are not targeted by betulinic acid and its derivatives.
As shown in Fig. 3, compounds BA3 and BA4 reduced the cellular
metabolism, and inhibited the growth of 43D cells, which was
largely due to the amount of beta-Tubulin in cells treated with the
compounds (Fig. 2). Compounds BA3 and BA4 are quaternary
ammonium salts that contain the benzalkonium cation, and our
experiments using BKC suggested that the benzalkonium cation in
BA3 and BA4 showed cytotoxic effects (Fig. 4). Therefore, these
35% at 30
show a strong inhibitory effect at 30
correlated with the beta-Tubulin in the 43D cells treated with
30 M and 100 M of compounds BA3 and BA4 for 48 h (Fig. 2A).
mM and by 95% at 100
mM, but other compounds did not
mM (Fig. 3), which was
m
m
These data suggest that compounds BA3 and BA4 could decrease
the M-MuLV Gag production by inhibiting the cellular metabolisms.
Since both compounds contain the benzalkonium cation, we tested
the inhibitory effect of benzalkonium cation on cell growth. The
43D cells treated with BKC impaired metabolism in a dose-
dependent manner, and BA2 did not show synergistic effects with
BKC (Fig. 4A). BKC decreased the signals for Gag and beta-Tubulin in
cells and p30CA in media (Fig. 4B), which suggested that the ben-
zalkonium cation inhibited viral production by decreasing the
metabolism in 43D cells, and compounds BA3, BA4 and BKC seem
not to affect intracellular Gag production and its processing.
4. Discussion
Our results demonstrated that two ionic derivatives of betulinic
acid (compounds BA3 and BA4) reduced the cellular metabolisms,
and decreased the MuLV production from 43D cells without alter-
ation of the virus release efficiency or the maturation of viral par-
ticles. It was reported that both betulinic acid and platanic acid
isolated from the leaves of Syzigium claviforum inhibited human
immunodeficiency virus (HIV) replication in H9 lymphocyte cells
[7]. A subsequent study [8] demonstrated that the major inhibitory
steps in HIV-1 replication by betulinic acid and its derivatives are
membrane fusion, viral assembly, and maturation. One betulinic
acid derivative with alteration at C3, 3-O-(3,3-dimethylsuccinyl)
betulinic acid/PA-457/DSB blocks a late step of virus replication
through inhibiting the conversion of p25CAꢂSP1 to p24 [25]. Cleav-
ages of the precursor form of Gag including p24 and SP1 Gag spacer
peptide is crucial to the structural alterations necessary for the
formation of mature HIV-1 particles. The study using lentiviruses
and their mutants responsible for DSB resistance indicated that
Valine and Leucine immediately flanking the p25CAeSP1 cleavage
Fig. 4. Effects of benzalkonium chloride on 43D growth and MuLV replication. (A) 43D
cells were treated with different doses of benzalkonium chloride (BKC) and the
cholinium salt of betulinic acid-glycine, BA2 for 48 h. The effect of the compounds on
cell growth was measured by the AlmarBlue cell proliferation assay. The figure illus-
trates the relative cell proliferation to the control. The averages of two experiments are
Fig. 3. Cytotoxicity of betulinic acid and its ionic derivatives in 43D cells. 43D cells
were treated with different doses of betulinic acid and its ionic derivatives for 48 h. The
effect of these compounds on cell growth was measured by the AlmarBlue cell pro-
liferation assay. The figure illustrates the relative cell proliferation to the control. The
averages of two experiments are shown.
shown. (B) 43D cells were treated with 30
proteins in cells and media were detected by western blots using the rabbit serum for
anti-p30CA
mM of the compounds for 48 h, and Gag
.

J. Phillips et al. / Biochemical and Biophysical Research Communications 500 (2018) 365e369
369
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The authors appreciate Dr. Hung Fan (University of California,
Irvine) for his rabbit serum (anti-p30CA) and helpful comments. We
acknowledge the assistance of the undergraduate program coor-
dinator, Ms. Devi Chellu, and research associate, Mr. Harshavardhan
Kenche, and faculty members at Savannah State University. The
authors also thank Drs. Melissa Visalli and Robert Visalli at Mercer
University, School of Medicine, and Yoko Nitta for their technical
assistance and helpful discussion. The research was supported in
part by the NIH RISE program (1R25GM096956) and the Savannah
State University mini-grant to TN.
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