JOURNAL OF VIROLOGY, Feb. 2007, p. 1444–1450
Vol. 81, No. 3
Lysis of Human Immunodeficiency Virus Type 1 by a Specific
Secreted Human Phospholipase A2
Jae-Ouk Kim, Bimal K. Chakrabarti, Anuradha Guha-Niyogi, Mark K. Louder,
John R. Mascola, Lakshmanan Ganesh,† and Gary J. Nabel*
Vaccine Research Center, NIAID, National Institutes of Health, Room 4502, Bldg. 40,
MSC-3005, 40 Convent Drive, Bethesda, Maryland 20892-3005
Received 17 August 2006/Accepted 31 October 2006
Phospholipase A2(PLA2) proteins affect cellular activation, signal transduction, and possibly innate immu-
nity. A specific secretory PLA2, sPLA2-X, is shown here to neutralize human immunodeficiency virus type 1
(HIV-1) through degradation of the viral membrane. Catalytic function was required for antiviral activity, and
the target cells of infection were unaffected. sPLA2-X potently reduced gene transfer of HIV-1 Env-pseudotyped
lentivirus vectors and inhibited the replication of both CCR5- and CXCR4-tropic HIV-1 in human CD4?T
cells. Virions resistant to damage by antibody and complement were sensitive to lysis by sPLA2-X, suggesting
a novel mechanism of antiviral surveillance independent of the acquired immune system.
Mammalian viruses are subjected to a variety of antiviral
host defenses, including recognition by antibody and comple-
ment, lysis of producer cells by cytolytic T cells, interferon
responses, and possibly antibody-dependent cellular toxicity.
However, the mechanisms by which viruses may be subject to
recognition and removal at sites of entry, particularly in the
mucosa, are poorly defined. Among the gene products synthe-
sized in the gut, phospholipases (PLAs) are secreted in high
abundance and play a role in digestion, cellular activation, and
nerve signaling. PLA2s hydrolyze phospholipids at the sn-2
position to release fatty acids and lysophospholipids (3, 18, 24).
They are classified into two major groups, the high-molecular-
weight cytosolic PLA2s and the low-molecular-weight secreted
PLA2s (sPLA2s), which have diverse functions. Human sPLA2
groups (IB, IIA, IID, IIE, IIF, III, V, VII [platelet-activating
factor acetyl hydrolase], X, XIIA, and XIIB) show unique
tissue and cellular localizations and enzymatic properties (20,
23). Of these sPLA2s, group X sPLA2(sPLA2-X) exhibits the
highest capacity to hydrolyze phospholipids in intact mamma-
lian cell membranes when overexpressed (16, 17) or added
exogenously (4, 8). sPLA2-X is converted from a catalytically
inactive zymogen to a mature, catalytically active form by re-
moval of the N-terminal propeptide after the secretion pro-
cess, supporting the extracellular action of this enzyme (14).
Some sPLA2s have bactericidal activity, presumably through
their effect on membrane integrity, raising the possibility that
they may contribute to host antimicrobial defense (7, 11, 12,
26). On the other hand, the role of sPLA2s in viral infections
has not been defined.
In this study, we screened several human sPLA2s for their
potential antiviral effects, and we report that human sPLA2-X
has antiviral activity against lentiviruses due to its catalytic
function and its recognition of the virus envelope. This effect
was observed even when viruses were resistant to antibody-
mediated complement activation. The ability of sPLA2-X to
degrade viruses suggests a novel mechanism of host defense
that may provide a barrier to infection independent of the
adaptive immune response.
MATERIALS AND METHODS
Cell lines. The 786-O (human kidney adenocarcinoma) cell line was purchased
from the American Type Culture Collection. The HeLa-derived cell line MAGI-
CCR5 (a subline of HeLa expressing CCR5) was obtained from the NIH AIDS
Research and Reference Reagent Program. The human T-cell leukemia cell line
A3R5 (a subline of A3.01 expressing both CCR5 and CXCR4) was a gift from
Jerome Kim of the Walter Reed Army Institute of Research. Cells were cultured
with Dulbecco’s modified Eagle’s medium or RPMI 1640 (Invitrogen) containing
10% fetal bovine serum (Sigma) and 100 ?g of penicillin-streptomycin/ml.
Construction of expression plasmids. Human sPLA2s (PLA2groups IIA
[GenBank loci NM_000300], IID [NM_012400], III [NM_015715], V [NM_000929],
VII [NM_005084], X [NM_003561], and XIIA [NM_03081]) were first PCR
amplified from corresponding PLA2cDNA clones obtained from Invitrogen or
Openbiosytems and then subcloned into the mammalian expression vector CMV/
R-mcs. A linker (4 repeats of GGGS) and a six-His tag were added to the
carboxy-terminal end of the sPLA2group X gene, and a carboxy-terminal six-His
tag alone was added to the other genes. Point mutants were constructed by using
overlap extension PCR or the QuikChange site-directed mutagenesis kit (Strata-
gene) according to the manufacturer’s protocol. All plasmids were sequenced to
verify the coding regions.
The primer sequences for amplification of the sPLA2isoforms are as follows:
for the IIA isoform, the 5? sequence is ACCGTTAGCGGCCGCCACCATGA
AGACCCTCCTACTGTTGGCAGTGATCATGA and the 3? sequence is TGC
CCCTCTGCAGTGTTTATTG; for IID, the 5? sequence is ACCGTTAGCGG
TGGTG and the 3? sequence is TGCCAGTTCTAGATCAATGATGATGAT
GATGATGGCACCCAGGGGTCTGCCCCCGGCAGTGGGGCC; for III, the
5? sequence is ACCGTTAGCGGCCGCCACCATGGGGGTTCAGGCAGGG
CTGTTTGGGATGCTGGG and the 3? sequence is TGCCAGTTCTAGATCA
V, the 5? sequence is ACCGTTAGCGGCCGCCACCATGAAAGGCCTCCTC
CCACTGGCTTGGTTCCTGGC and the 3? sequence is TGCCAGTTCTAGA
TTGGTAC; for VII, the 5? sequence is ACCGTTAGCGGCCGCCACCATGG
TGCCACCCAAATTGCATGTGCTTTTCTGCC and the 3? sequence is TGC
CCTGAAGAGTTCTGTAAC; for X, the 5? sequence is GGTCGACCATGG
* Corresponding author. Mailing address: Vaccine Research Center,
NIAID, National Institutes of Health, Room 4502, Bldg. 40, MSC-
3005, 40 Convent Dr., Bethesda, MD 20892-3005. Phone: (301) 496-
1852. Fax: (301) 480-0274. E-mail: email@example.com.
?Published ahead of print on 8 November 2006.
GGCCGCTACCTGTGTG and the 3? sequences are GGATCCCCCTCCGCT
AGTCCGGCTC (sPLA2-X–linker) and CAGATCTCAATGGTGATGGTGAT
GATGGGATCCCCCTCCGCTTCCCC (linker–six-His); and for XIIA, the 5?
sequence is ACCGTTAGCGGCCGCCACCATGGCCCTGCTCTCGCGCCC
CGCGCTCACCC and the 3? sequence is TGCCAGTTCTAGATCAATGATG
The primer sequences used for point mutants are as follows: for the D47K
mutant, the 5? sequence is GACTGGTGCTGCCATGGCCACAAGTGTTGTT
ACACTCGAGC and the 3? sequence is GCTCGAGTGTAACAACACTTGT
GGCCATGGCAGCACCAGTC; for the H46N, D47E, and Y50F mutants, the
5? sequence is CTGCCATGGCAACGAGTGTTGTTTCACTCGAGCTGAGG
AGGCCGGCTGCAGCC and the 3? sequence is GGCTGCAGCCGGCCTCC
Transfection and Western blot analysis. 293 cells were transfected using cal-
cium phosphate (Promega), and cell culture supernatants were harvested 2 days
after transfection and kept at ?80°C. Cell culture supernatants were resolved by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane
was incubated with a rabbit polyclonal anti-His antibody (1:1,000; Santa Cruz
Biotechnology) for 1 h at room temperature in blocking buffer (Tris-buffered
saline, 3% skim milk, 0.5% Triton X-100), followed by washing. The blot was
further incubated in blocking buffer with horseradish peroxidase-conjugated goat
anti-rabbit immunoglobulin G (IgG; 1:5,000; Santa Cruz) for 30 min and then
washed. Detection was performed with the ECL reagent (Amersham).
Recombinant sPLA2protein purification. The baculovirus expression vector
was made following the standard protocol as described by the company (Invitro-
gen). Briefly, sPLA2-X cDNA and three sPLA2-X amino acid mutants (with an
H46N, D47E, or Y50F mutation) were cloned into pVL1393 (transfer vector),
which has an Autographa californica multiple nucleopolyhedrosis virus polyhe-
dron enhancer-promoter sequence to drive high expression. The recombinant
DNA was verified by sequencing. This plasmid was cotransfected with linearized
BD baculoGold baculovirus DNA (BD Biosciences) in Sf9 insect cells to make a
recombinant baculovirus. The plaque-purified virus was checked for the presence
of the PLA2gene and was amplified by reinfecting Sf9 insect cells. This high-titer
recombinant virus was later used to make PLA2protein in High Five (Hi5) cells.
Culture supernatant from 1 liter of Hi5 cells infected with the baculovirus
described above was harvested after a 48-h incubation at 27°C. The sample was
adjusted to 1? phosphate-buffered saline and 10 mM imidazole with a 1 M stock,
filtered (through a 0.45-?m-pore-size polyethersulfone membrane), applied to a
5-ml HisTrap column (GE Healthcare), and eluted with a 20-column-volume
linear imidazole gradient to 400 mM. The fractions were analyzed by SDS-
PAGE. Final samples were dialyzed to 1? phosphate-buffered saline and con-
centrated using 10,000-molecular-weight-cutoff Amicon Ultra filtration devices
Wild-type sPLA2-X and mutant sPLA2-X (D47K) were also expressed in 293
cells, and the culture supernatants were applied to a 5-ml HisTrap column and
eluted as described above.
sPLA2enzymatic assay. To measure sPLA2enzymatic activity in the cell
culture supernatant from the indicated DNA-transfected cells, an sPLA2assay
kit (Cayman Chemical) was used according to the manufacturer’s recommenda-
tions. This assay uses the 1, 2-dithio analog of diheptanoyl phosphatidylcholine
as a substrate for sPLA2s. Upon hydrolysis of the thio ester bond at the sn-2
position by sPLA2, free thiols are detected using 5, 5-dithio-bis-(2-nitrobenzoic
acid) (DTNB) at 405 nm. The specific activity of sPLA2was calculated based on
the initial slope of the time dependence of absorption at 405 nm, using an
extinction coefficient at 405 nm (ε405) of 12.8 mM?cm?.
Viruses. Luciferase-expressing lentiviral vectors pseudotyped with envelopes
from HIV-1ADA, HIV-1IIIB, Ebola virus, and Moloney murine leukemia virus
(MoMuLV) were prepared by transient cotransfection of 293T cells with calcium
phosphate (Promega) (28). Briefly, the packaging vector pCMV?R8.2 (7 ?g),
pHR?CMV-Luc (7 ?g), and the envelope-expressing vector pSVIII-ADA (10
?g), pRSV-IIIB (10 ?g), pVR1012-GP(Z) (50 ng), pNGVL-Env (4070A) (2 ?g),
or CMV/R-8kb influenza virus H5 [A/Thailand/1(KAN-1)/2004] HA-wt/h (50 ng)
were cotransfected. Supernatants were harvested 72 h after transfection, filtered
with a 0.45-?m-pore-size syringe filter, and stored at ?80°C.
Adenovirus type 5 (Ad5)–luciferase was made as described previously (2).
Wild-type HIV-1BaLand HIV-1MNstocks were prepared in peripheral blood
mononuclear cells (PBMCs) as previously described (13).
Infection of cells with pseudoviruses and luciferase assay. A total of 30,000
cells were plated into each well of a 48-well dish the day before infection:
MAGI-CCR5 cells for HIV-1ADA, HIV-1IIIB, and MoMuLV, and 786-O cells for
Ebola virus and Ad5. The pseudovirus supernatant (50 to 100 ?l) or 1.5 ? 107
viral particles of Ad5 (500/cell) were incubated with sPLA2or its mutant-trans-
fected cell culture supernatant for 1 h at 37°C and added to the target cells. Cells
were replenished with fresh medium at 16 to 18 h postinfection. After 48 h, cells
were lysed in 80 ?l of cell lysis buffer (Promega) in the plate, and 20 ?l of the cell
lysate was used in a luciferase assay with luciferase assay reagent (Promega)
according to the manufacturer’s recommendations. Luciferase assay results were
measured using Top Count (Packard).
HIV single-round replication assay. To assess the effect of sPLA2-X on live
wild-type HIV-1BaLand HIV-1MN, the virus (100 ng of p24) was incubated with
53 ng of purified sPLA2-X (activity, 400 nmol/min) or its H46N, D47E, or Y50F
mutant for 60 min at 37°C. A3R5 cells (1 ? 106cells) were added to the
above-described mixtures for 2 h, allowing infection. Cells were washed and
incubated with fresh medium. After 64 h, cells were stained with a fluorescein
isothiocyanate (FITC)-conjugated anti-p24Gag antibody (KC-57–FITC; Beck-
man Coulter) and analyzed (13).
Analysis of p24 release from virions by use of a density gradient. Density
gradient-purified Ebola pseudovirus (50 ?l) or an HIV-1BaL(2.5 ?g of p24)–
sPLA2mixture was added to the same volume of OptiPrep (Axis-Shield PoC). A
FIG. 1. The antiviral effect of the sPLA2-X isoform is specific. (a)
(Top) The enzymatic activity of each indicated sPLA2gene product in
culture supernatant was assessed by a colorimetric assay using an
sPLA2assay kit. Symbols indicate significant differences from the con-
trol: †, P ? 0.05;*; P ? 0.01. (Bottom) Expression in supernatants was
determined by Western blot analysis with an anti-His antibody. (b) An
HIV-1IIIBenvelope-pseudotyped lentivirus vector encoding luciferase
(100 ?l each) was incubated with 1 ml of cell culture supernatant,
made from a control or the indicated sPLA2isoform from transfected
293 cells, for 60 min at 37°C. The virus-cell culture supernatant mixture
was added to MAGI-CCR5 cells and incubated for 16 h. The mixture
was removed, and luciferase reporter activity was evaluated 48 h after
replacement with fresh medium. Data are averages ? the standard
deviations from triplicates and each value is representative of two
VOL. 81, 2007 SPECIFIC INHIBITION Of HIV-1 BY HUMAN GROUP X sPLA2
density gradient was formed by centrifugation at 421,000 ? g for 3.5 h with an
NVT100 rotor (Beckman). The collected fractions were weighed, and density
was calculated. An equal amount of each fraction (20 ?l) was separated on a
4-to-15% SDS-PAGE gel (Bio-Rad), transferred to a polyvinylidene difluoride
membrane, and blotted with human anti-HIV-1 IgG or rabbit anti-p24Gag serum
(Advanced Biotechnologies). Each lane of the Western blot represents one
fraction of the density gradient.
To define the potential of mammalian sPLA2to confer pro-
tection against viral infection, plasmid expression vectors en-
coding the human group IIA, IID, III, V, VII, X, and XIIA
isoforms were prepared and tagged with a COOH-terminal
polyhistidine epitope to facilitate detection. When tested for
enzymatic activity, groups IIA, III, VII, and X displayed sig-
nificant sPLA2enzymatic activity compared to control super-
natants (vector) (P ? 0.05 for IIA; P ? 0.01 for III, VII, and
X), and sPLA2-X was the most active (Fig. 1a, top). Expression
of each sPLA2was also confirmed by Western blotting with an
anti-His antibody (Fig. 1a, bottom). The antiviral effects of
recombinant human sPLA2cell culture supernatants were
tested first by measuring the luciferase reporter gene activity of
HIV-1 pseudoviruses on MAGI-CCR5 target cells, a human
cervical carcinoma (HeLa) cell line expressing CD4 and core-
ceptors CXCR4 and CCR5. Among the different sPLA2s, the
group X isoform showed marked inhibition of the HIV-1IIIB
pseudotype reporter (Fig. 1b). Though sPLA2-X displayed the
highest enzymatic activity on this substrate, other isoforms
were readily detectable by protein expression. There is evi-
dence that different sPLA2s have different substrate affinities
that may determine their biologic effects (20), suggesting that
there is specificity for this effect among the isoforms. In addi-
tion, the substrate, a 1,2-dithio analog of diheptanoyl phos-
phatidylcholine, may be useful in predicting antiviral efficacy,
possibly because it may be related to viral envelope lipids.
FIG. 2. Antiviral effect of sPLA2-X: dependence of enzymatic activity on the virus and not on the target cells, and specificity of inhibition. (a)
sPLA2-X acts on the virus rather than the target cells. (Top left) The enzymatic activity of purified sPLA2-X (WT) or the inactive ?sPLA2-X
(D47K) mutant (?) made from 293 cells was assessed by a sPLA2assay kit. (Bottom left) Protein amounts in 10 ?l are shown by Western blot
analysis using an anti-His antibody. (Center) HIV-1ADApseudovirions were incubated with sPLA2-X or inactive ?sPLA2-X (0.3 ml) for 60 min at
37°C and ultracentrifuged at 48,400 ? g for 1 h to pellet the virus. Viral pellets were resuspended with fresh medium and incubated with
MAGI-CCR5 target cells for 17 h. Infectivity was assessed with a luciferase reporter 48 h after replacement with fresh medium. (Right)
MAGI-CCR5 target cells were incubated with sPLA2-X or the catalytically inactive ?sPLA2-X (D47K) (0.3 ml) for 2 h at 37°C, washed, and
transduced with pseudotyped HIV-1ADAvirions. Cells were again washed at the indicated times to remove the virions and were cultured with fresh
medium. Infectivity was assessed by luciferase reporter activity 3 days later. (b) sPLA2-X exerts specific antiviral activity. Cell culture supernatants
(1 ml) made from 293 cells transfected with sPLA2-X (activity, 33 to 78 nmol/min/ml) or ?sPLA2-X (D47K; catalytically inactive mutant) were
incubated for 60 min at 37°C with the indicated pseudovirions. The virus-supernatant mixture was added to MAGI-CCR5 cells (for HIV-1ADA,
HIV-1IIIB, and MoMuLV) or 786-O cells (for Ebola virus and Ad5), incubated for 16 h, and replaced with fresh medium, and luciferase-reporter
activity was assayed 48 h later. Data are averages ? standard deviations from triplicates.
1446 KIM ET AL.J. VIROL.
To examine whether catalytic activity was required for the
inhibitory effect of sPLA2-X, wild-type, enzymatically active
protein and a catalytically inactive point mutant (with the
D47K mutation), termed ?sPLA2-X, were generated. Though
equivalent amounts of proteins were detected, ?sPLA2-X
showed no catalytic activity (Fig. 2a, left). While enzymatically
active sPLA2-X markedly inhibited reporter gene expression,
similar protein concentrations of inactive ?sPLA2-X exerted
no effect (Fig. 2a, center). sPLA2-X acted primarily through
damage to virions, as evidenced by the fact that treatment of
the target cells of infection did not significantly reduce viral
gene transfer (Fig. 2a, right).
The specificity of the sPLA2-X antiviral effect was assessed
on different viral envelopes expressed on lentivirus vectors,
including CXCR4-tropic HIV-1IIIB, CCR5-tropic HIV-1ADA,
amphotropic MoMuLV, Ebola virus glycoprotein (GP), or a
nonenveloped viral vector, recombinant Ad5. Wild-type
sPLA2-X showed significant antiviral activity against CCR-5-
or CXCR-4-tropic HIV Env, amphotropic MoMuLV, and
Ebola virus compared to ?sPLA2-X but did not show signifi-
cant inhibition of reporter gene expression by the nonenvel-
oped virus recombinant Ad5 (Fig. 2b), suggesting that the
antiviral activity required the presence of a lipid-containing
The antiviral effect of sPLA2-X was assessed against HIV-1BaL
(CCR5-tropic) and HIV-1MN(CXCR4-tropic) stocks pro-
duced in PBMCs. Virus preparations were incubated with pu-
rified sPLA2-X or a different catalytically inactive mutant,
?3sPLA2-X (H46N, D47E, and Y50F mutations) (9, 19), prior
to infection of the human T-cell leukemia cell line A3R5, a
subline of A3.01 cells (10) expressing both CCR5 and CXCR4.
Flow cytometric analysis of intracellular Gag protein was used
to assess viral replication. sPLA2-X treatment substantially
reduced T-cell infection by CCR5-tropic HIV-1BaL(Fig. 3a,
FIG. 3. sPLA2-X inhibits productive replication of CCR5- or CXCR4-tropic HIV-1 strains in T cells. HIV-1BaL(a) or (b) HIV-1MN(100 ng of
p24) stocks were incubated with 53 ng of purified sPLA2-X (activity, 400 nmol/min) or ?3sPLA2-X (H46N, D47E, and Y50F mutations) for 60 min
at 37°C. The virus-sPLA2mixture was incubated with the human T-cell leukemia cell line A3R5 (a subline of CEM expressing both CCR5 and
CXCR4; 1 ? 106) for 2 h. Cells were then washed and replaced with fresh medium. HIV-1 replication was analyzed 64 h after infection by flow
cytometry, staining for intracellular p24 with an FITC-conjugated anti-p24 antibody. The percentage of p24-positive cells was subtracted from that
of mock-infected cells.
VOL. 81, 2007SPECIFIC INHIBITION Of HIV-1 BY HUMAN GROUP X sPLA2
right) compared to the catalytically inactive ?3sPLA2-X (Fig.
3a, left). A similar reduction in viral replication was seen when
sPLA2-X was incubated with replication-competent CXCR4-
tropic HIV-1MN(Fig. 3b), suggesting that this antiviral mech-
anism is effective against diverse lentiviruses with alternative
chemokine receptor specificity.
To understand the mechanism of the sPLA2-X antiviral ef-
fect, the ability of sPLA2-X to lyse virus was examined both for
pseudotyped lentivirus vectors and for replication-competent
HIV-1BaLderived from PBMCs. For the pseudotyped lentivi-
rus vector, Ebola virus GP pseudotypes were analyzed first,
using gradient-purified virions. The presence of p24Gag in
different gradient fractions was first confirmed by immunopre-
cipitation followed by Western blotting, with peak activity at a
density of 1.10 (Fig. 4a, right panel, lane 3). Analysis of virions
from this purified fraction revealed reactivity with monoclonal
antibody 13C6, known to bind Ebola virus GP on virions (27)
(Fig. 4a, left panel). This subtype IgG2a antibody has been
shown to fix complement (27). Gradient-purified pseudotyped
virions were treated with control mouse IgG or 13C6 plus
mouse complement. Though virions reacted with this antibody
and are able to fix complement, no release of p24Gag was
detected, as shown by refractionation through the density gra-
dient (Fig. 4a, right panel, lanes 5 to 7). In contrast, treatment
with sPLA2-X, but not ?sPLA2-X (D47K), caused Gag release
when these virions were refractionated through a density gra-
dient (Fig. 4a, lower right panel, sPLA2-X versus ?sPLA2-X,
lanes 12 to 14). A similar effect was observed with 2F5, a
broadly neutralizing human monoclonal antibody of subtype
IgG1 that binds HIV-1BaL(Fig. 4b), confirming its effect on
In this study, the ability of sPLA2s to inhibit HIV-1 replica-
tion has been evaluated. We find that sPLA2-X displays anti-
viral activity against diverse lentiviruses by degradation of the
viral membrane. sPLA2-X inhibits replication of both CXCR4-
and CCR5-tropic HIV-1 in primary human CD4?cells. This
effect was observed despite the resistance of virus preparations
to lysis by antibody-mediated complement activation, suggest-
ing that this mechanism acts in cases where the acquired im-
mune response is ineffective.
The antibacterial effects of sPLA2s against gram-positive
bacteria (ranked in order of strength, from highest to lowest, as
follows: IIA, X, V, XII, IIE, IB and IIF) and the gram-negative
bacterium Escherichia coli have been reported previously; only
human group XII displays detectable bactericidal rather than
bacteriostatic activity (7, 11, 12, 26). In this study, group X
alone exerted an antiviral effect on enveloped lentiviruses,
documenting the specificity of this effect. While it may be
suggested that the efficacy of sPLA2-X is due to its high enzy-
matic activity, it should be recognized that this activity relates
to the specificity of the enzyme for the substrate used in this
assay and that various sPLA2s have divergent substrate speci-
ficity (20). For example, sPLA2-XII is not active in this assay
yet mediates a significant antimicrobial effect. Taken together,
this study raises the possibility of a novel and specific role for
this gene product in innate immunity to a specific class of
sPLA2-X efficiently hydrolyzes cell membranes primarily be-
cause of its high binding affinity for phosphatidylcholine, a
phospholipid that is enriched in the outer leaflet of the plasma
membrane. The viral membrane of HIV-1 (5) is rich in zwit-
terionic phospholipids (phosphatidylcholine and sphingomye-
lin) and may be more susceptible to sPLA2-X. The sensitivity
of lentiviruses to sPLA2-X despite their resistance to antibody
and complement should be noted. This difference suggests that
other factors, including the spike density of the virion, glyco-
FIG. 4. sPLA2-X potently damages viral membranes compared to
antibody-mediated complement fixation. (a) 13C6, a complement-fix-
ing antibody, binds to Ebola virus pseudovirions but, unlike sPLA2-X,
does not damage the viral membrane. Gradient-purified Ebola virus
pseudovirions were incubated with a control mouse IgG or 13C6 for 30
min at 4°C and then immunoprecipitated with protein G-Sepharose.
The immunoprecipitate was analyzed for p24 by Western blot analysis
using human anti-HIV-1 IgG (left panel). Gradient-purified Ebola
virus-pseudotyped virions were incubated with mouse IgG (67 ?g/ml)
or 13C6 (333 ?g/ml) plus mouse complement (10%) for 90 min at 37°C
(top right panels) or with 1 ml of sPLA2-X or ?sPLA2-X (D47K) from
transfected 293 cell culture supernatants for 60 min at 37°C (bottom
right panels). A density gradient was formed by centrifugation using
OptiPrep, and the fractions were collected. p24Gag in each fraction is
shown by Western blot analysis with anti-HIV-1 IgG. Gag released
from damaged virus forms aggregates found in higher-density frac-
tions. (b) 2F5, an antibody known to fix complement, binds to HIV-
1BaLbut, unlike sPLA2-X, does not damage the viral membrane. Pu-
rified live HIV-1BaLwas incubated with KZ52 (IgG1) or 2F5 (IgG1)
for 30 min at 4°C and immunoprecipitated with protein G-Sepharose.
The immunoprecipitate was analyzed for p24 by Western blot analysis
using anti-p24 rabbit serum for the presence of 2F5 bound to HIV-1BaL
(left panel). HIV-1BaLwas incubated with 100 ?g/ml of monoclonal
antibody 2F5 or KZ52 with human complement (10%) (top right
panel) or 1 ml of culture supernatants from 293 cells transfected with
sPLA2-X or ?sPLA2-X (D47K) (bottom right panel) for 3 h at 37°C. A
density gradient was formed by centrifugation using OptiPrep, and the
fractions were collected. p24Gag in each fraction is shown by Western
blot analysis using rabbit anti-p24Gag serum. Gag released from dam-
aged virus forms aggregates found in higher-density fractions.
1448 KIM ET AL.J. VIROL.
sylation of envelopes, curvature of the viral particle, and cel-
lular proteins on the viral envelope, may affect susceptibility to
different antiviral immune and inflammatory responses.
The enzymatic activity of sPLA2-X is necessary and sufficient
for the antiviral effect. This finding contrasts with a previous
report showing that addition of a nonmammalian sPLA2, de-
rived from bee venom, blocked the entry of HIV-1 by steric
inhibition of the chemokine receptor on target cells, in which
case catalytic activity was not required (6). Another recombi-
nant sPLA2-X derived from bacteria inhibited adenovirus
plaque formation through its ability to hydrolyze phospholipids
on host cell membranes (15), implying a different mechanism
of action with a nonphysiological gene product, unlike the
The disruptive action of sPLA2-X on the viral membrane
was strongly confirmed by p24Gag protein redistribution in a
high-density gradient fraction (Fig. 4). Antibody and comple-
ment did not show p24 release from virions. Although some
reports have suggested that HIV-1 is susceptible to comple-
ment-mediated lysis (1, 22, 25), these studies have utilized
poorly defined sera from HIV-infected subjects or nonphysi-
ological concentrations of polyclonal antibodies and can be
explained by factors other than complement that might cause
viral lysis, possibly even including sPLA2, which is found in the
circulation as well. Here, purified monoclonal antibodies
known to fix the complement on the HIV-1 envelope do not
mediate lysis. It is also well known that HIV-1 virions escape
complement-mediated lysis in vivo through complement inac-
tivators such as CD46, CD55, and CD59 on their viral mem-
branes (21). Since sPLA2-X readily degrades such viruses, we
suggest that sPLA2-X may overcome resistance to antibody
and complement virolysis. Further, because it is expressed at
the highest levels in the intestinal mucosa, a primary site of
HIV replication in natural infection, we suggest that
sPLA2-X be studied further to explore its potential role in
innate immunity against HIV in the gut. Expression of this
recombinant protein, as well as the stimulation of increased
endogenous sPLA2-X, may help to limit viral replication and
reduce the incidence of productive replication at sites of
We dedicate this paper to the memory of Lakshmanan Ganesh, a
most promising scientist whose career was tragically cut short by illness
while this paper was in preparation.
We thank Ati Tislerics and Tina Suhana for help with manuscript
preparation, Toni Miller and Brenda Hartman for figure preparation,
and members of the Nabel lab for advice and discussions.
J.-O.K. was supported by the Postdoctoral Fellowship Program of
the Korea Research Foundation. This research was supported by the
Intramural Research Program of the NIH, Vaccine Research Center,
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