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Proc Natl Acad Sci U S A. Jun 1, 2010; 107(22): 10178–10183.
Published online May 17, 2010. doi:  10.1073/pnas.0914870107
PMCID: PMC2890444

Prothymosin-α inhibits HIV-1 via Toll-like receptor 4-mediated type I interferon induction


Induction of type I interferons (IFN) is a central feature of innate immune responses to microbial pathogens and is mediated via Toll-like receptor (TLR)-dependent and -independent pathways. Prothymosin-α (ProTα), a small acidic protein produced and released by CD8+ T cells, inhibits HIV-1, although the mechanism for its antiviral activity was not known. We demonstrate that exogenous ProTα acts as a ligand for TLR4 and stimulates type I IFN production to potently suppress HIV-1 after entry into cells. These activities are induced by native and recombinant ProTα, retained by an acidic peptide derived from ProTα, and lost in the absence of TLR4. Furthermore, we demonstrate that ProTα accounts for some of the soluble postintegration HIV-1 inhibitory activity long ascribed to CD8+ cells. Thus, a protein produced by CD8+ T cells of the adaptive immune system can exert potent viral suppressive activity through an innate immune response. Understanding the mechanism of IFN induction by ProTα may provide therapeutic leads for IFN-sensitive viruses.

Keywords: macrophage, TIR-domain-containing adapter-inducing interferon-β

Macrophages are one of the first lines of defense against viruses and infectious microbes. The rapid induction of direct effector and immunomodulatory genes in macrophages is critical for pathogen clearance. In addition, macrophages infected with HIV-1 are a relatively stable reservoir for virus production that presents unique challenges to antiretroviral therapy. In an effort to identify host proteins that protect macrophages against HIV-1 infection, we reported a strategy to screen for postentry HIV-1 inhibitors from CD8+ T cell conditioned medium by using supernatants of herpes virus saimiri-transformed CD8+ T cells (1). Although several molecules with anti-HIV-1 activity, including the β-chemokines RANTES, MIP-1α and β (2), and macrophage-derived chemokine (MDC) (3), have been found in CD8+ T cell supernatants, these do not account for the full spectrum of soluble anti-HIV-1 activity (1, 46). We reported the isolation and identification of prothymosin-α (ProTα) from a fraction derived from the CD8+ T cell culture medium and demonstrated that this protein has potent postentry HIV-1-inhibitory activity in macrophages (7). The mechanism of CD8+ T cell-derived ProTα-mediated inhibition of HIV-1 remains elusive.

ProTα is a small 12.5 kDa, highly acidic protein (pI 3.5) that lacks aromatic and sulfur amino acids and has no intrinsic secondary structure. Posttranslational modifications of ProTα have been reported including unusual phoshorylation at glutamic acid residues and proteolytic cleavage of the N terminus by lysosomal asparaginyl endopeptidase producing a 28 amino acid bioactive peptide called Thymosin alpha1 (Tα1) (8, 9). We have shown that the anti-HIV-1 activity of ProTα is not associated with Tα1 (7) but rather with amino acids 50–89 of ProTα (Fig. 1A; sequence in red) (10, 11).

Fig. 1.
ProTα and the C-terminal peptide ProTα(50-89) induce expression of type I IFN and suppress HIV-1 gene expression. (A) Sequence alignment of human and murine ProTα. (B and C) Time course of IFN-β mRNA induction in human ...

Here, we report that ProTα represents an endogenous TLR4 ligand produced by CD8+ T cells and stimulates type I IFN production to potently suppress HIV-1 after its entry into the cells. Furthermore, ProTα accounts for some of the soluble HIV-1 inhibitory activity ascribed to this cell. Understanding the molecular basis of this activity could provide promising therapeutic compounds for IFN-sensitive viral infections.


ProTα Induces Expression of Type I IFN and Suppresses HIV-1 Gene Expression.

To understand how ProTα mediates its anti-HIV-1 activity, we investigated whether it induced type I IFN production. This question was based on prior work showing that ProTα induces several IFN responsive genes at 24 h after treatment (10, 12). In this investigation, we incubated human macrophages with recombinant human ProTα (rProTα; 200 ng/mL) (sequence shown in Fig. 1A) or a synthetic peptide, spanning amino acids 50–89 of ProTα called ProTα(50-89) at 5 μg/mL (Fig. 1A; sequence in red) or Tα1 (Fig. 1A; sequence in blue) and measured IFN-β mRNA by using reverse transcription quantitative real-time PCR (RT-qPCR). ProTα induced IFN-β mRNA starting at 1–2 h (Fig. 1B) and protein by 4–6 h and returned to baseline by 24 h of treatment (Fig. 1C). This data are consistent with our reported inability to detect type I IFN induction at later time points (10). ProTα(50-89) also induced IFN-β, whereas the N-terminal synthetic peptide, Tα1, reported to have immunomodulatory activity, showed no IFN-β induction (Fig. 1D). The kinetics of induction with ProTα(50-89) differed from that with full-length ProTα, showing a slower and less dramatic peak of induction at 6 h (Fig. 1D). The effects of ProTα were not unique to macrophages because both rProTα and ProTα(50-89) also induced IFN-α1 and IFN-β mRNA in MDCs derived from normal blood donors (Fig. 1E). Interestingly, ProTα also induced TNF-α mRNA in primary human macrophages in a time-dependent manner with a peak at 4 h (Fig. 1F).

Consistent with our previous reports, ProTα suppressed HIV-1 gene expression in human macrophages after viral integration as measured by a decrease in reporter activity of HIV-1 pseudotyped with VSV envelope glycoprotein (HIV-1VSV) and carrying a luciferase reporter gene (Fig. 1G) (7).

ProTα Induction of IFN-β Is TRIF-Dependent, Whereas TNF-α Induction Is MyD88-Dependent.

Because ProTα is an evolutionarily conserved molecule with 98% homology between mouse and human (Fig. 1A), we predicted that human ProTα would induce similar signaling in murine cells, which would allow us to take advantage of genetically engineered mice deficient in expression of individual TLRs or TLR signaling adaptor molecules. Human ProTα induced IFN-β mRNA in macrophages derived from the bone marrow of C57BL/6J mice at the same concentration used for human macrophages and with higher levels of induction that were sustained at 24 h (Fig. 2A). To test whether the effect of ProTα was mediated through TLR activation, we used macrophages derived from mice deficient for the major TLR signaling adaptors, Myeloid Differentiation Primary Response Gene-88 (MyD88) and Toll IL-1 receptor (TIR) domain-containing adaptor inducing IFN-β (TRIF, also known as TICAM-1). MyD88 is an essential adaptor downstream of all TLRs, with the exception of TLR3, whereas TRIF is associated with type I IFN induction by TLR4 and is the only adaptor used by TLR3 (13). Treatment of C57BL/6J wild type (WT) murine macrophages with human ProTα induced IFN-β mRNA, and this induction was dramatically reduced in Trif−/− macrophages (74-fold in WT versus 8.5-fold in Trif−/−) (Fig. 2B Left). As expected, induction of IFN-β mRNA in response to LPS was also reduced dramatically in Trif−/− macrophages (882-fold in WT to 12-fold in Trif−/−). Deficiency in MyD88 had no effect on the induction of IFN-β mRNA by LPS, because this response is known to be mediated via TRIF and independently of MyD88 (Fig. 2B Left). Similarly, IFN-β mRNA induction by ProTα was not abrogated in Myd88−/− murine macrophages (Fig. 2B Left). These data collectively suggested that ProTα induces IFN-β transcription via TRIF and not MyD88.

Fig. 2.
IFN-β induction by ProTα is TRIF-dependent, whereas TNF-α induction is MyD88-dependent and this activity is independent of LPS. (A) Time course of IFN-β mRNA induction in WT murine macrophages by human ProTα. ( ...

We next examined the ability of ProTα to trigger the MyD88-dependent proinflammatory cytokine TNF-α. ProTα induced TNF-α mRNA in human (Fig. 1F) and murine macrophages (Fig. 2B Right), and this response was markedly reduced in macrophages from Myd88−/− mice (524-fold in WT to 7.7-fold in Myd88−/−) but remained similar to WT in Trif−/− macrophages. These results indicate that in contrast to IFN-β induction, ProTα induces TNF-α transcription by using the MyD88 adaptor molecule and not TRIF. As expected, TNF-α mRNA was reduced in Myd88−/− macrophages in response to LPS (955-fold in WT to 6.7-fold in Myd88−/−) (Fig. 2B Right), consistent with the MyD88-dependent induction of TNF-α by LPS in murine macrophages (14).

Because of the similarities in responses mediated by ProTα and LPS, we were careful to exclude potential contamination of our ProTα with residual levels of endotoxin. Endotoxin removing gel was used to clean each batch of rProTα protein we purchased. The measured amount of endotoxin in the proteins was well below the threshold level of <1 EU (endotoxin units) per 2 μg of protein, making it unlikely that the anti-HIV-1 activity (Fig. 1G), induction of IFN-β (Fig. 1B), and TNF-α (Fig. 1F) by ProTα were due to contaminating LPS. Furthermore, although as expected the anti-HIV-1 activity of LPS was blocked by treatment with polymyxin B (PMB), there was no loss of anti-HIV-1 activity mediated by ProTα after treatment with two different concentrations of PMB, (1 μg/mL and 10 μg/mL) (Fig. 2C). More importantly, the anti-HIV-1 and IFN-β mRNA-inducing activities of ProTα were completely abrogated with proteinase treatment (Fig. 2 D and E, respectively), whereas proteinase treatment had no effect on corresponding LPS-mediated activities. The efficiency of proteinase treatment was confirmed by gel electrophoresis of digested and undigested rProTα (Fig. 2F), demonstrating loss of the protein with digestion. These results collectively exclude LPS contamination from our rProTα preparations and argue that the responses in macrophages we observed were primarily mediated through ProTα.

Induction of IFN-β and the Anti-HIV-1 Activity of ProTα Are TLR4 Dependent.

Utilization of both TRIF and MyD88 TLR signaling-adaptors by ProTα is reminiscent of the signaling pathways downstream of TLR4. We thus determined whether TLR4 might be mediating the cellular responses to ProTα. Tlr4−/− macrophages showed marked reduction in IFN-β expression in response to ProTα as compared with WT macrophages (6.8-fold in WT to 1.2-fold in Tlr4−/−) (Fig. 3A). The blunted response compared with Fig. 1 is due to 10-fold less ProTα. ProTα-induced TNF-α was also abrogated in the absence of TLR4 (48-fold in WT to 0 Tlr4−/−) (Fig. 3A). To determine whether this effect was specific to ProTα recognition by TLR4 but not other TLRs, which might cooperate with TLR4, we tested IFN-β induction by ProTα in TLR3-deficient macrophages. As expected, induction of IFN-β by the TLR3 ligand polyinosinic:polycytidylic acid (polyI:C; a synthetic double-stranded RNA) (15) was markedly reduced in Tlr3−/− macrophages as compared with WT (47-fold in WT to 12-fold Tlr3−/−), whereas LPS-induced IFN-β expression was not impaired (148-fold in WT to 217-fold Tlr3−/−) (Fig. 3B). Importantly, ProTα-induced IFN-β was not altered in the absence of TLR3 (115-fold in WT to 181-fold Tlr3−/−), strongly suggesting specific activation of TLR4 but not TLR3 by ProTα (Fig. 3B).

Fig. 3.
Induction of IFN-β and TNF-α mRNA by ProTα is TLR4-dependent, whereas the anti-HIV-1 activity is both TLR4 and IFN-β-dependent. (A) WT or Tlr4−/− macrophages were treated with 20 ng/mL ProTα (*, ...

We next determined whether ProTα-mediated anti-HIV-1 activity in murine macrophages also required TLR4. WT and Tlr4−/− murine macrophages were infected with HIV-1VSV carrying a luciferase reporter gene, followed by treatment with ProTα, LPS or PolyI:C. Consistent with the effects on cytokine induction, ProTα also suppressed HIV-1 gene expression in WT mouse macrophages. In contrast, this suppression was abolished in Tlr4−/− macrophages (Fig. 3C), whereas the anti-HIV-1 activity of poly I:C remained intact. As expected, the anti-HIV-1 activity of LPS was abrogated in Tlr4−/− murine macrophages (Fig. 3C).

To determine whether the anti-HIV-1 activity of ProTα is fully accounted for by its ability to induce type I IFN, macrophages derived from WT and type I IFN receptor 1 deficient (Ifnar1−/−) mice were infected with HIV-1VSV, followed by treatment with ProTα or LPS. Suppression of HIV-1 gene expression by ProTα was completely abolished in Ifnar1−/− macrophages (Fig. 3D), suggesting that type I IFN induction by ProTα is essential for HIV-1 suppression in this model. Not only was suppression by LPS lost in the Ifnar1−/− macrophages, but stimulation of HIV-1 gene expression was above background (from 15% in WT to 164% in Ifnar1−/− compared with macrophages treated with control medium 100%), which may be due to the stronger unopposed proinflammatory response (Fig. 3D).

CD8+ T Cell-Derived ProTα Induces IFN-β mRNA and Suppresses HIV-1 Gene Expression via TLR4.

Finally, to determine whether ProTα directly contributes to the soluble anti-HIV-1 activity associated with primary CD8+ T cells, fractions were collected from human CD8+ T cell conditioned medium by using a similar fractionation scheme that had originally identified ProTα in transformed cell supernatants (7). Fractions derived from supernatants of normal CD8+ T cell-conditioned medium were separated by anion exchange chromatography using concentrations of NaCl that increased in increments of 100 mM. Fractions were then tested for anti-HIV-1 activity and induction of IFN-β mRNA in macrophages. The fraction that eluted at 300 mM NaCl contained ProTα as assessed by polyacrylamide gel electrophoresis (PAGE). This result was in agreement with our earlier work where we had identified ProTα within the 300 mM NaCl eluate of anion exchange chromatography of supernatants of herpesvirus saimiri (HVS)-transformed CD8+ T cells (7). The ProTα-containing fraction derived from the supernatant of normal CD8+ T cells stimulated IFN-β mRNA to similar levels in human and WT mouse macrophages, but this induction was impaired in Tlr4−/− macrophages (Fig. 4A). In contrast, the total unfractionated supernatant from CD8+ T cells induced IFN-β mRNA only in human macrophages (Fig. 4A), suggesting that human macrophages are more sensitive than mouse macrophages to the lower concentrations of native human ProTα in the unfractionated sample. Moreover, depletion of ProTα from fraction 3 by using an affinity column conjugated with anti-ProTα antibodies abolished not only IFN-β mRNA induction (Fig. 4B), but also the anti-HIV-1 activity associated with this fraction (Fig. 4C). Depletion of ProTα was confirmed by gel electrophoresis (Fig. 4D). We compared the anti-HIV-1 activity of unfractionated supernatant from the HVS-transformed CD8+ T cell-line K#1 50k cells (1), from which we had previously isolated ProTα, with an aliquot of the same supernatant from which ProTα had been removed by affinity chromatography. Total unfractionated supernatant of K#1 50K suppressed HIV-1 gene expression to 24% of control. Affinity removal (depletion) of ProTα reduced suppressive activity to 44% of control (Fig. 4E). Therefore, depletion of ProTα reduced HIV-1 gene expression by 56% and did not completely abrogate suppression of HIV-1 gene expression after viral integration, suggesting the presence of other inhibitors of HIV-1 gene transcription (Fig. 4E). Depletion was confirmed by determining ProTα concentration by sandwich ELISA [30 ng/mL in the total supernatant and below level of detection (<0.3 ng/m) after depletion]. The more potent suppression of HIV-1 gene expression by affinity-purified ProTα in Fig. 4C compared with Fig. 4E is accounted for by different activity of the protein after viral integration (Fig. 4E) compared with after viral entry (Fig. 4C). Therefore, ProTα accounts for some of the postentry HIV-1-inhibitory activity long ascribed to supernatants derived from CD8+ T cells (1).

Fig. 4.
ProTα isolated from human CD8+ T cells induces IFN-β mRNA and suppresses HIV-1 gene expression in primary macrophages via TLR4. (A) Human or mouse (WT or Tlr4−/−) macrophages were treated with 4 ng/mL fraction 3 derived ...


These data strongly suggest that full-length ProTα can act as an endogenous signaling ligand for TLR4, triggering both the TRIF-dependent pathway for IFN-β induction and the MyD88 pathway for induction of proinflammatory cytokines such as TNF-α. The net effect is potent inhibition of HIV-1 in macrophages. The postentry inhibition of viral expression is mediated via type 1 interferons as indicated by the complete loss of inhibition of HIV-1 gene expression in murine macrophages lacking type I IFN receptor 1(Ifnar1−/−). The demonstration of a direct role for TLR4 signaling and IFN in this antiviral pathway is consistent with reports demonstrating up-regulation of the signaling protein IRAK4 and IFN responsive genes in peripheral blood mononuclear cells (PBMCs) or MDCs exposed to ProTα (7, 12). Other host proteins including the heat shock antigens Hsp60 and Hsp70, or HMGB1 and Tenascin-C expressed during inflammation, stress, and/or necrosis (1618), have also been reported to serve as endogenous ligands for TLR4, but binding of these ligands to TLR4 is not associated with the induction of type I IFN.

There have been a number of other immunomodulatory activities attributed to extracellular ProTα or peptides derived from it, including up-regulation of HLA-DR, IL- 2 receptor, dendritic cell maturation, chemotaxis, anti-viral, anti-cancer, and anti-fungal activities (1921). In fact, the Tα1 peptide, which did not display anti-HIV-1 activity, has been associated with direct stimulation of TLR9, activation of the MyD88 pathway, and production of IL-10 in dendritic cells in which Tα1 peptide has immunomodulatory activity and stimulates an anti-fungal response in a murine model of candida (20). Interestingly, all of these diverse functions exerted by ProTα or peptides derived thereof could involve TLR activation.

TLR4 triggering by LPS is inextricably linked to several other extracellular proteins capable of recognizing and binding LPS. The most important of these ancillary proteins is MD-2, because MD-2 knockout mice do not respond to LPS (22) nor do cultured cells expressing certain mutant forms of MD-2 (23). MD-2 is associated with the extracellular domain of TLR4 (22, 24) and has been shown to directly interact with LPS (25). The LPS binding site of MD-2 is comprised of several clusters of basic residues and a hydrophobic region, which presumably exist as a pocket (26, 27). Thus, MD-2 can establish complementary interactions with the negatively charged phosphate groups and hydrophobic acyl chains of the lipid A portion of LPS. The phosphate groups of LPS also form ionic interactions with positively charged residues in TLR4 that contribute to multimerization of TLR4 and MD-2 (28). ProTα is highly negatively charged, even at neutral pH values, and exists in a random coil conformation under physiological conditions (29, 30). Based on these features, we hypothesize that ProTα has complementary interactions with the cationic clusters of MD-2 and, possibly, TLR4, and its highly flexible nature allows it to adopt a conformation matching that of the LPS binding site. However, more experiments are necessary to test this hypothesis.

A number of soluble factors released by CD8+ cells have non-MHC-dependent anti-HIV-1 activity including the β-chemokines that block entry. These factors have not accounted for the strong CD8+-antiviral activity (CAF) that acts at the level of viral transcription. Our work showed that removal of ProTα from a potent inhibitory fraction of primary CD8+ cell supernatants was associated with both a decrease in IFN induction and a loss of HIV-1 transcriptional inhibition. However, depletion of ProTα from total supernatant demonstrated that this protein contributes to, but does not completely account for, the activity in unfractionated supernatants. In addition to its ability to inhibit HIV-1 transcription, ProTα has some of the characteristics attributed to CAF including size (12 kDa), sensitive to V8 proteinase digestion (5). ProTα can be added to the list of endogenous soluble anti-HIV-1 factors released from CD8+ cells. Understanding the molecular basis of this activity could provide promising therapeutic compounds for IFN-sensitive viral infections.

Materials and Methods

Animal Strains and Reagents.

MyD88−/−, TRIF−/−, and TLR4−/− mice have been described (14, 31, 32) and were originally provided by S. Akira (Osaka University, Osaka). Type I IFN receptor knockout (Ifnar1−/−) mice (Sv129 background) were obtained from B & K Universal, and murine macrophages from these animals were prepared as described below.

Recombinant human ProTα and antibodies to ProTα (2F11 and 4F4) for sandwich ELISA were purchased from Alexis Biochemicals. Endotoxin removal columns from Pierce were used to remove LPS from recombinant preparations of ProTα protein. Endotoxin levels were measured by CTS Analyst (Associates of Cape Cod) by using the turbidimetric method. Endotoxin levels were below 1 EU per 2 μg of recombinant protein in all preparations used.

General Procedure for the Solid-Phase Synthesis of Human and Murine ProTα Peptides.

The human ProTα(50-89) peptide was prepared by solid-phase synthesis on an automated PS3 synthesizer (Proteins Technologies) using standard fluorenylmethoxycarbonyl (Fmoc) methods. The peptides were characterized by electrospray ionization mass spectrometry on a Micromass Q-ToF micro mass spectrometer. Tα1 was purchased from Bachem.

Mouse Macrophage Isolation.

Murine macrophages were derived from the bone marrow of mice as described by using adhesion (31) and grown in medium supplemented with 30% M-CSF. Macrophages were serum-starved in RPMI 1640 for 2–4 h before treatment with ProTα or different peptides.

Isolation of Human Monocyte-Derived Macrophages and CD8+ Cells.

CD14+ and CD8+ cells were isolated from Ficoll-Hypaque (Sigma Aldrich)-purified PBMCs from healthy donorsby using magnetic beads from Miltenyi Biotec. Monocytes were differentiated into macrophages by culture for 10–15 days in Dulbecco's modified Eagle medium containing 10% FBS. CD8+ cells were cultured for 24 h in serum-free RPMI supplemented with IL-2 (50 U/mL), insulin (50 ng/mL), and transferrin (Sigma) before conditioned supernatant was collected for fractionation.

Pseudotyped-Virus Production and Infection.

The envelope-deficient, luciferase expressing NL4-3. Luc.RE- virus, obtained from National Institutes of Health AIDS Research and Reference Reagent Program, was pseudotyped with VSV envelope and viral stocks generated as described (7). Virus stocks were analyzed for HIV-1 p24 antigen concentration by an ELISA.

RNA Extraction and RT-PCR.

Total RNA was extracted by using RNeasy spin columns (Qiagen). After treatment with RNase-free DNase (Qiagen), RT was performed on 1μg of RNA by using Omniscript RT kit (Qiagen) and oligo dT primers according to the manufacturer's protocol. RT-qPCR was performed on an ABI Prism 7700. Reactions were carried out in 10 μL by using SYBR Green PCR master mix according to the manufacturer's protocol (Qiagen) with 5–10 ng of cDNA. The concentration of primer pairs was 300 nM. All samples and controls were performed in triplicate. To make comparisons between samples and controls, the CT (cycle threshold, defined as the cycle number at which the fluorescence is above the fixed threshold) values were normalized to the CT of ribosomal protein cDNA in each sample.

Human IFN-β ELISA.

Supernatants from primary human macrophages treated with rProTα were collected, spun down, and used in an ELISA (PBL) to determine IFN-β protein concentrations.

Protein Fractionations.

CD8+ cell supernatants were loaded onto a MonoQ (GE Healthcare) column by using an AKTA explorer 100 (GE Healthcare). The column was washed with 50 mM phosphate buffer (pH 7.4) containing 100 mM NaCl, and bound molecules were eluted in 100 mM NaCl steps each for 5 column volumes from 200 to 900 mM. The fractions were dialyzed across 0.5 kDa MWCO dialysis membrane against 50 mM phosphate buffer at pH 7.4. Dialyzed samples were concentrated by lyophylization. Protein concentrations were determined by BCA assay (Pierce).

Affinity Chromatography.

Monoclonal antibody to ProTα (Alexis Biochemicals) was immobilized on 4% beaded agarose by using an AminoLink Immobilization kit (Pierce) following the manufacturer's instructions. ProTα columns were equilibrated with 50 mM phosphate buffer, pH 7.4. Samples were applied to the ProTα column, allowed to bind for 15 min at room temperature with shaking, then drained by gravity flow. Nonbound solutes were removed by washing with 50 mM phosphate buffer, pH 7.4. Bound solutes were eluted with 200-μL aliquots of 0.2 M glycine (pH 3) (until absorbance at 214 nm was at baseline) and equilibrated to pH 7 with 10 μL sodium phosphate (pH 9). Aliqouts with elevated 214 nm values were pooled and dialyzed across 0.5 kDa MWCO dialysis membranes against 50 mM phosphate buffer, pH 7.4. Dialyzed samples were concentrated by lyophylization. Protein concentrations were determined by BCA assay.

Proteinase Digestion.

Aliquots of endotoxin-free ProTα and LPS were digested by using immobilized Staphylococcus aureus V8 proteinase from Pierce according to the manufacturer's suggestions. Digestion was confirmed by PAGE.


We thank Carolina Lopez and Ana Fernandez of Mount Sinai School of Medicine for reagents; Vladimer Uversky for guidance on TLR4 work; Goar Mosoyan for technical assistance; and Miriam Torchinsky, Ben Chen, and Sergio Lira for critical reading of the manuscript. This research was supported by North Carolina Biotechnology Center Grant 2008 BRG-12139 (to C.S.B.) and National Institute of Allergy and Infectious Diseases Grant AI76092-01A1 (to M.E.K.).


The authors declare no conflict of interest.

This article is a PNAS Direct Submission.


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