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Phospholipase A2 (PLA2) proteins affect cellular activation, signal transduction, and possibly innate immunity. 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 complement, 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 synthesized 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 mammalian 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 removal of the N-terminal propeptide after the secretion process, 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.
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 μg of penicillin-streptomycin/ml.
Human sPLA2s (PLA2 groups 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 PLA2 cDNA 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 sPLA2 group 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 (Stratagene) according to the manufacturer's protocol. All plasmids were sequenced to verify the coding regions.
The primer sequences for amplification of the sPLA2 isoforms are as follows: for the IIA isoform, the 5′ sequence is ACCGTTAGCGGCCGCCACCATGAAGACCCTCCTACTGTTGGCAGTGATCATGA and the 3′ sequence is TGCCAGTTCTAGATCAATGATGATGATGATGATGGCAACGAGGGGTGCTCCCTCTGCAGTGTTTATTG; for IID, the 5′ sequence is ACCGTTAGCGGCCGCCACCATGGAACTTGCACTGCTGTGTGGGCTGGTGGTGATGGCTGGTG and the 3′ sequence is TGCCAGTTCTAGATCAATGATGATGATGATGATGGCACCCAGGGGTCTGCCCCCGGCAGTGGGGCC; for III, the 5′ sequence is ACCGTTAGCGGCCGCCACCATGGGGGTTCAGGCAGGGCTGTTTGGGATGCTGGG and the 3′ sequence is TGCCAGTTCTAGATCAATGATGATGATGATGATGCTGGCTCCAGGACTTCTGCTGCCTGT; for V, the 5′ sequence is ACCGTTAGCGGCCGCCACCATGAAAGGCCTCCTCCCACTGGCTTGGTTCCTGGC and the 3′ sequence is TGCCAGTTCTAGATCAATGATGATGATGATGATGGGAGCAGAGGATGTTGGGAAAGTATTGGTAC; for VII, the 5′ sequence is ACCGTTAGCGGCCGCCACCATGGTGCCACCCAAATTGCATGTGCTTTTCTGCC and the 3′ sequence is TGCCAGTTCTAGATCAATGATGATGATGATGATGATTGTATTTCTCTATTCCTGAAGAGTTCTGTAAC; for X, the 5′ sequence is GGTCGACCATGG GGCCGCTACCTGTGTG and the 3′ sequences are GGATCCCCCTCCGCTTCCCCCTCCGCTTCCCCCTCCGCTTCCCCCTCCGTCACACTTGGGCGAGTCCGGCTC (sPLA2-X-linker) and CAGATCTCAATGGTGATGGTGATGATGGGATCCCCCTCCGCTTCCCC (linker-six-His); and for XIIA, the 5′ sequence is ACCGTTAGCGGCCGCCACCATGGCCCTGCTCTCGCGCCCCGCGCTCACCC and the 3′ sequence is TGCCAGTTCTAGATCAATGATGATGATGATGATGAAGATCAGTTTTTTCTTCATAATGACACCTGCA.
The primer sequences used for point mutants are as follows: for the D47K mutant, the 5′ sequence is GACTGGTGCTGCCATGGCCACAAGTGTTGTTACACTCGAGC and the 3′ sequence is GCTCGAGTGTAACAACACTTGTGGCCATGGCAGCACCAGTC; for the H46N, D47E, and Y50F mutants, the 5′ sequence is CTGCCATGGCAACGAGTGTTGTTTCACTCGAGCTGAGGAGGCCGGCTGCAGCC and the 3′ sequence is GGCTGCAGCCGGCCTCCTCAGCTCGAGTGAAACAACACTCGTTGCCATGGCAGC.
293 cells were transfected using calcium 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).
The baculovirus expression vector was made following the standard protocol as described by the company (Invitrogen). 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 polyhedron 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 PLA2 gene and was amplified by reinfecting Sf9 insect cells. This high-titer recombinant virus was later used to make PLA2 protein 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-μm-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 concentrated using 10,000-molecular-weight-cutoff Amicon Ultra filtration devices (Millipore).
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.
To measure sPLA2 enzymatic activity in the cell culture supernatant from the indicated DNA-transfected cells, an sPLA2 assay kit (Cayman Chemical) was used according to the manufacturer's recommendations. 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 sPLA2 was calculated based on the initial slope of the time dependence of absorption at 405 nm, using an extinction coefficient at 405 nm (405) of 12.8 mM− cm−.
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 pCMVΔR8.2 (7 μg), pHR′CMV-Luc (7 μg), and the envelope-expressing vector pSVIII-ADA (10 μg), pRSV-IIIB (10 μg), pVR1012-GP(Z) (50 ng), pNGVL-Env (4070A) (2 μg), 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-μm-pore-size syringe filter, and stored at −80°C.
Adenovirus type 5 (Ad5)-luciferase was made as described previously (2). Wild-type HIV-1BaL and HIV-1MN stocks were prepared in peripheral blood mononuclear cells (PBMCs) as previously described (13).
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 μl) or 1.5 × 107 viral particles of Ad5 (500/cell) were incubated with sPLA2 or its mutant-transfected 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 μl of cell lysis buffer (Promega) in the plate, and 20 μl 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).
To assess the effect of sPLA2-X on live wild-type HIV-1BaL and 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 × 106 cells) 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; Beckman Coulter) and analyzed (13).
Density gradient-purified Ebola pseudovirus (50 μl) or an HIV-1BaL (2.5 μg of p24)-sPLA2 mixture was added to the same volume of OptiPrep (Axis-Shield PoC). A 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 μl) 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 sPLA2 to confer protection against viral infection, plasmid expression vectors encoding 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 significant sPLA2 enzymatic activity compared to control supernatants (vector) (P < 0.05 for IIA; P < 0.01 for III, VII, and X), and sPLA2-X was the most active (Fig. (Fig.1a,1a, top). Expression of each sPLA2 was also confirmed by Western blotting with an anti-His antibody (Fig. (Fig.1a,1a, bottom). The antiviral effects of recombinant human sPLA2 cell 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 coreceptors CXCR4 and CCR5. Among the different sPLA2s, the group X isoform showed marked inhibition of the HIV-1IIIB pseudotype reporter (Fig. (Fig.1b).1b). Though sPLA2-X displayed the highest enzymatic activity on this substrate, other isoforms were readily detectable by protein expression. There is evidence 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 addition, the substrate, a 1,2-dithio analog of diheptanoyl phosphatidylcholine, may be useful in predicting antiviral efficacy, possibly because it may be related to viral envelope lipids.
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 ΔsPLA2-X, were generated. Though equivalent amounts of proteins were detected, ΔsPLA2-X showed no catalytic activity (Fig. (Fig.2a,2a, left). While enzymatically active sPLA2-X markedly inhibited reporter gene expression, similar protein concentrations of inactive ΔsPLA2-X exerted no effect (Fig. (Fig.2a,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. (Fig.2a,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 ΔsPLA2-X but did not show significant inhibition of reporter gene expression by the nonenveloped virus recombinant Ad5 (Fig. (Fig.2b),2b), suggesting that the antiviral activity required the presence of a lipid-containing viral membrane.
The antiviral effect of sPLA2-X was assessed against HIV-1BaL (CCR5-tropic) and HIV-1MN (CXCR4-tropic) stocks produced in PBMCs. Virus preparations were incubated with purified sPLA2-X or a different catalytically inactive mutant, Δ3sPLA2-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. (Fig.3a,3a, right) compared to the catalytically inactive Δ3sPLA2-X (Fig. (Fig.3a,3a, left). A similar reduction in viral replication was seen when sPLA2-X was incubated with replication-competent CXCR4-tropic HIV-1MN (Fig. (Fig.3b),3b), suggesting that this antiviral mechanism is effective against diverse lentiviruses with alternative chemokine receptor specificity.
To understand the mechanism of the sPLA2-X antiviral effect, the ability of sPLA2-X to lyse virus was examined both for pseudotyped lentivirus vectors and for replication-competent HIV-1BaL derived from PBMCs. For the pseudotyped lentivirus vector, Ebola virus GP pseudotypes were analyzed first, using gradient-purified virions. The presence of p24Gag in different gradient fractions was first confirmed by immunoprecipitation followed by Western blotting, with peak activity at a density of 1.10 (Fig. (Fig.4a,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. (Fig.4a,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 gradient (Fig. (Fig.4a,4a, right panel, lanes 5 to 7). In contrast, treatment with sPLA2-X, but not ΔsPLA2-X (D47K), caused Gag release when these virions were refractionated through a density gradient (Fig. (Fig.4a,4a, lower right panel, sPLA2-X versus ΔsPLA2-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. (Fig.4b),4b), confirming its effect on native virus.
In this study, the ability of sPLA2s to inhibit HIV-1 replication has been evaluated. We find that sPLA2-X displays antiviral 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, suggesting that this mechanism acts in cases where the acquired immune 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 enzymatic 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 specificity (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 viruses.
sPLA2-X efficiently hydrolyzes cell membranes primarily because 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 zwitterionic phospholipids (phosphatidylcholine and sphingomyelin) 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, glycosylation of envelopes, curvature of the viral particle, and cellular 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, derived 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 recombinant 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 report here.
The disruptive action of sPLA2-X on the viral membrane was strongly confirmed by p24Gag protein redistribution in a high-density gradient fraction (Fig. (Fig.4).4). Antibody and complement did not show p24 release from virions. Although some reports have suggested that HIV-1 is susceptible to complement-mediated lysis (1, 22, 25), these studies have utilized poorly defined sera from HIV-infected subjects or nonphysiological 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 inactivators such as CD46, CD55, and CD59 on their viral membranes (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 primary infection.
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, NIAID.
Published ahead of print on 8 November 2006.