Highly conserved serine residue 40 in HIV-1 p6 regulates capsid processing and virus core assembly
The HIV-1 p6 Gag protein regulates the final abscission step of nascent virions from the cell membrane by the action of two late assembly (L-) domains. Although p6 is located within one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds at least to two cellular budding factors (Tsg101 and ALIX), is a substrate for phosphorylation, ubiquitination, and sumoylation, and mediates the incorporation of the HIV-1 accessory protein Vpr into viral particles. As expected, known functional domains mostly overlap with several conserved residues in p6. In this study, we investigated the importance of the highly conserved serine residue at position 40, which until now has not been assigned to any known function of p6.
Consistently with previous data, we found that mutation of Ser-40 has no effect on ALIX mediated rescue of HIV-1 L-domain mutants. However, the only feasible S40F mutation that preserves the overlapping pol open reading frame (ORF) reduces virus replication in T-cell lines and in human lymphocyte tissue cultivated ex vivo. Most intriguingly, L-domain mediated virus release is not dependent on the integrity of Ser-40. However, the S40F mutation significantly reduces the specific infectivity of released virions. Further, it was observed that mutation of Ser-40 selectively interferes with the cleavage between capsid (CA) and the spacer peptide SP1 in Gag, without affecting cleavage of other Gag products. This deficiency in processing of CA, in consequence, led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Moreover, the defects induced by the S40F mutation in p6 can be rescued by the A1V mutation in SP1 that generally enhances processing of the CA-SP1 cleavage site.
Overall, these data support a so far unrecognized function of p6 mediated by Ser-40 that occurs independently of the L-domain function, but selectively affects CA maturation and virus core formation, and consequently the infectivity of released virions.
KeywordsEquine Infectious Anemia Virus Virus Core Virus Release S40F Mutation Single Round Infection
The Gag polyprotein Pr55 of HIV-1 comprises the main structural components that are essential and sufficient for the formation of virus like particles (VLPs). Following synthesis in the cytoplasm, the Gag polyproteins are targeted to the plasma membrane where they assemble into immature budding particles. Concurrent with assembly and release of nascent virions, the Pr55 Gag precursor is cleaved by the autocatalytically activated viral protease (PR), generating the matrix (MA, p17), capsid (CA, p24), nucleocapsid (NC, p7), and the p6 protein. This processing ultimately leads to structural rearrangement of Gag molecules within the virion and the formation of the typical cone shaped core structure, characteristic for a mature infectious particle . MA mediates the plasma membrane targeting of Gag polyproteins and, after cleavage, lines the inner shell of the mature virion. CA forms the conical core shell encasing NC, which regulates packaging and condensation of the viral genome [2, 3, 4, 5, 6]. The C-terminal p6 domain of Pr55, the smallest known lentiviral protein, containing 52 amino acids,, comprises a quite complex structural and functional organization and contains two distinct late assembly (L-) domains that regulate efficient separation of assembled virions from the cell surface. L-domains function as docking sites for components of ESCRT (endosomal sorting complex required for transport), cellular multi-protein complexes that are normally involved in the endocytotic recycling of cell surface receptors and in cytokinesis [4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17].
The L-domain activity of p6 is mainly driven by the 7PTAP10 motif that is responsible for the recruitment of the primary budding factor Tsg101 (tumor susceptibility gene 101) to the virus assembly site [15, 18, 19, 20]. Another region of p6 involves the residues 36YPLASL41, comprising a cryptic YPXnL-type L-domain, which forms a degenerated version of the YPDL L-domain motif (YPXnL, n = 3) found in equine infectious anemia virus (EIAV). This secondary L-domain in p6 represents a binding site for another cellular budding factor, AIP1/ALIX (ALG-2 interacting protein 1/X, ALIX is used hereafter), a multifunctional ESCRT-associated regulator of intracellular protein transport [13, 21]. In addition to the interaction with cellular ESCRT components, p6 mediates the incorporation of the HIV-1 accessory protein Vpr into virus particles. This incorporation was shown to be dependent on three motifs in p6, the 15FRFG18 motif, the 34ELY36 motif, and the 41LXXLF45 motif [22, 23, 24].
Besides these well characterized interactions, p6 contains several highly conserved amino acids, some of which were shown or proposed to undergo posttranslational modification. In this study, we show that mutation of the highly conserved Ser residue in position 40 (Ser-40) leads to an irregular core assembly of the released virions, a reduced infectivity, and, thus, a disturbed virus replication capacity. The results support a novel function of p6 in virus maturation that occurs independently of L-domain function.
Mutation of Ser-40 has no effect on folding of p6
While the PTAP L-domain is highly conserved, the conservation within the ALIX binding site varies to some extent (Figure 1). The ALIX-binding motif has been defined as (L)[FY]PX1-3LXX[IL] [21, 26, 27] and corresponds in p6 derived from HIV-1NL4-3  to 35LYPLASLRSL44, in which essential residues are in bold. Interestingly, the three amino acid motif 35LYP37 at the start of the binding region is only poorly conserved (Figure 1) while both, Leu-41 and Leu-44, are highly conserved. These two residues together, with the downstream Phe-45, comprise the LXXLF binding domain for the HIV-1 accessory protein Vpr. From previous structural and mutational analysis, it can be concluded that Thr-39 and Ser-40 do not directly participate in the binding of p6 to ALIX [21, 27, 29]. Consistently, Thr-39 is not conserved, while in contrast, Ser-40 is highly conserved among HIV-1 group M isolates, indicating a function other than ALIX binding.
To investigate a potential function of Ser-40 in p6, the residue was mutated in the infectious molecular clone HIV-1NL4-3  and an otherwise isogenic R5-tropic derivative thereof carrying the 005pf135 V3 loop region in Env . In order to obtain replication competent viruses, the mutation was introduced in a way that does not affect the overlapping pol-ORF. In particular, Ser-40 of p6 overlaps in the pol-ORF with the cleavage site between the transframe p6* protein and PR. The only possibility to leave the pol-ORF unaffected was to exchange Ser-40 for Phe, creating the mutant S40F.
Mutation of Ser-40 in HIV-1 compromises virus replication
Mutation of Ser-40 in p6 does not affect virus release but disturbs CA maturation
S40F mutation reduces specific infectivity of the virions
Mutation of Ser-40 has no effect on ALIX mediated virus release
To further uncouple the phenotype induced by S40F mutation from the underlying L-domain function of the ALIX binding site, we investigated whether ALIX can rescue the infectivity of the S40F mutant in the context of a functional PTAP motif. To this end, 293T cells were co-tranfected with HIV-1 encoding either wt p6 or the S40F mutant and ALIX. Release of infectious virions was determined 24 hours post transfection by single round infection of TZM-bl cells. Consistent with previous results, the S40F mutation reduced the infectivity of released virions by ~5-fold (Figure 6B, 1 and 3). While overexpression of ALIX had no significant influence on the infectivity of wt HIV-1 (Figure 6B, 2), ALIX also could not restore the reduced infectivity of the S40F mutant (Figure 6B, 4).
The S40F mutation does neither affect cleavage of Gag products, other than CA, nor incorporation of Env
It was shown previously that mutations of p6 in this area, in particular, mutations of Tyr-36 and Leu-41 produce mutants that fail to package Env proteins into virus particles . Since Ser-40 is located directly adjacent to Leu-41, we wanted to exclude that the reduced replication capacity and infectivity of the S40F mutant is due to reduced Env incorporation. To this end, purified virions standardized for p24 content were analyzed by Western blotting using Env specific antibodies. As shown in Figure 8B, the S40F mutation had no influence on Env incorporation into virus particles.
Electron microscopy analysis of p6 S40F mutants
To evaluate quantitatively this morphological phenomenon, cores of 100 - 120 virons were counted and the percentages of irregular core structures relative to wt virions were calculated (Figure 9B). Obviously, mutation of Ser-40 significantly increases the amount of virions containing aberrant, irregularly shaped virus cores. Moreover, the CA5 mutant, in which CA processing is blocked completely, shows the same phenotype as that of the S40F mutant, further supporting the notion that Ser-40 governs the processing of CA by a yet unidentified mechanism.
Defect in CA maturation of the S40F mutant can be rescued by mutation in the CA-SP1 cleavage site
Consequently, we wanted to examine whether this enhanced CA processing affects virus core assembly. Therefore, virion structure of the A1V mutants was analyzed by thin-section electron microscopy. Cores of 100 - 120 virons were counted and the percentages of irregular core structures relative to wt virions were calculated (Figure 10B). The S40F mutation again substantially increases the amount of virions containing aberrant, irregularly shaped virus cores. The A1V mutation had only marginal effects on the core morphology of wt HIV-1. However, in the case of the S40F mutant, introducing the A1V mutation largely improves virus core assembly (Figure 10B).
Since enhancing the CA processing rate rescues the defect of viral core assembly, we subsequently wanted to analyze whether this improved core formation also affects the infectivity of the virions. To measure the specific infectivity, HeLa TZM-bl cells were infected with individual virus stocks standardized for p24 content and infectivity was determined by β-galactosidase assay. The A1V mutation alone enhances the specific infectivity of the virons by 4-fold (Figure 10C). Introducing the A1V mutation enhances the specific infectivity of the virions of the otherwise attenuated S40F mutant to almost wt levels (Figure 10C), indicating that the deficiency in CA processing is the major determinant for the reduced infectivity of the S40F mutant.
In this study we demonstrate that mutation of the highly conserved Ser-40 interferes with Gag processing and virus core formation. Although Ser-40 in HIV-1 p6 is highly conserved among HIV-1 isolates, it is not involved in any of the functional motifs described so far, neither the ALIX nor the Vpr binding site. Therefore, it was legitimate to speculate that Ser-40 is involved in another, until now unrecognized function of p6. However, it should be noted that the position of Ser-40 in the nucleotide sequence of HIV-1 overlaps with the p6*/PR cleavage site in the overlapping pol-ORF, which limits the probability of mutations in this respective area and might contribute to the high conservation of this amino acid. Nevertheless, our findings of a compromised replication capacity and reduced infectivity of the Ser-40 mutant viruses support the assumption that Ser-40 has an important function directly associated with p6.
Notably, virus release kinetic was not reduced for the S40F mutant providing first evidence that the L-domain function of p6 is not affected by mutation of Ser-40. This was supported further by the observation that the ability of ALIX to rescue HIV-1ΔPTAP L-domain mutant viruses was not influenced by the S40F mutation. Although Ser-40 is located within the ALIX binding region in p6, previous structural investigations indicated that Ser-40 itself does not participate in the binding of p6 to ALIX [21, 27, 29]. The fact that the Ser-40 mutant was still fully active in terms of L-domain function further supports the notion, that the mutation introduced into p6 did not disturb the overall structure of the molecule in this respective region. In consistency, structural calculations indicated that the non-conservative exchange of Ser-40 to Phe does not change the ability of the molecule to adopt a helical structure. In fact, the C-terminal α-helix is conserved in the S40F mutant, as it was established by NMR studies of the C-terminal peptides sp623-52.
Yet, as shown previously, the phenotypes induced by mutations in the ALIX binding site are depending on the type of amino acid that is mutated . Mutation of the 35LYP37 sequences in the ALIX binding site reduces release and infectivity of HIV-1 virions, which otherwise exhibit normal Gag processing [27, 36]. In contrast, mutations of Leu-41 and Leu-44 have no impact on virus release but increase the ratio of p25 to mature p24, similar to the phenotype we observed for mutation of Ser-40 . Unlike the typical phenotype of L-domain mutants, these mutants interfere somehow specifically with the final step in maturation of CA. It is currently not clear, whether this phenotype is in some way associated with the ALIX-p6 interaction. While mutations of Leu-41 and Leu-45 disrupt binding to ALIX, mutation of Ser-40 apparently has no influence on this interaction. Thus, it might also be possible, that this area in p6 harbors another function that, independent of L-domain activity, requires a so far unrecognized cellular interaction partner.
Maturation of the Gag processing intermediate p25 to mature CA p24, e. g. the cleavage of the CA-SP1 junction by the PR, appears to be one of the last steps of Gag processing [41, 42]. It was previously demonstrated that mutating the junction between CA and SP1, in order to block cleavage of p25, leads to the production of noninfectious viral particles with aberrant core morphology . In addition, treatment of virus producing cells with 3-O-(3'-3'dimethylsuccinyl) betulinic acid (BVM, Bevirimat, also known as PA-457 or DSB), a specific inhibitor that blocks PR-mediated cleavage between CA and SP1, disturbed viral core formation . Intriguingly, mutation of Ser-40 leads to an almost identical aberrant virus core morphology as shown previously for CA5 mutants. Both are characterized by misshapen core structures and the formation of an electron dense lateral body near the viral membrane. Previous studies already indicated that maturation of HIV-1 virions, leading to the typical cone shaped cores, is regulated by the sequential, and highly ordered proteolytic cleavage of Gag . Apparently, the last step of Gag processing - the cleavage of the CA-SP1 junction - is required for capsid condensation. However, pulse chase data indicate that the kinetic of cleavage of the CA-SP1 junction is delayed, but not completely blocked inasmuch as the mature CA accumulates over time. This indicates a rather dynamic process in which the mutation of Ser-40 somehow delays the kinetic of CA maturation. This phenomenon correlates with deficiencies in CA processing, infectivity, and core morphology.
Recently published results from Müller et al. demonstrate that even low amounts of Gag processing intermediates interfere with HIV particle maturation in a trans-dominant manner, with the final cleavage between p24 and SP1 being of particular importance . This explains why the rather subtle effect on CA maturation detected for Ser-40 mutants by Western blotting and pulse chase analysis results in a substantial reduction of virus core formation.
Interestingly, the effect of the S40F mutation appears to be specific for the CA-SP1 cleavage inasmuch as i) no other Gag processing deficiency could be detected (Figure 6) and ii) enhancing CA processing by introducing the A1V mutation could restore the deficient core formation, and, consequently, enhance infectivity. Thus, it can be concluded that Ser-40 somehow regulates the cleavage of the CA-SP1 junction and the subsequent capsid condensation.
The molecular mechanism behind how Ser-40 regulates the processing of Gag, in particular the cleavage of the CA-SP1 junction, is still elusive so far. The previously described defects in Gag processing commonly observed for L-domain mutants are believed to be linked to the overall process of virus budding inasmuch as PR activation and subsequent Gag processing occur concomitantly with and shortly after release of virus particles [45, 46]. In the case of Ser-40, this can be excluded, as the mutant S40F exhibits wt budding. However, Ser-40 in p6 and the CA-SP1 junction are separated by 123 amino acids in Pr55 and it remains elusive so far, how both proteins can affect each other, either in the context of Pr55 or after Gag processing. Our NMR experiments demonstrated that the C-terminal structure of p6 is not influenced by the S40F mutation. Therefore, one possibility of the effect observed would be that the mutation affects the Gag structure prior to initiation of Gag processing, thereby reducing the cleavage efficiency of the weakest cleavage site in Pr55. A prerequisite for this scenario would be that p6 represents a structured Gag domain and thus influence the folding the Pr55 polyprotein. Even though the 283 residue N-terminal part of HIV-1 Gag including MA and CA has been solved by NMR , the structure of the complete Pr55 has not been determined hitherto. Although S40F does not appear to affect the folding of the mature p6 protein, we can not exclude that this mutation indeed affect the overall structure of the PR55 polyprotein, which in turn would reduce the processing efficiency, a phenotype we clearly observed for the S40F mutant as a novel function of p6.
Currently, there is no evidence of an intra-molecular interaction between these domains in the Pr55 polyprotein. The p6 domain of the Pr55 represents a docking site of several cellular and viral factors. Thus, since an intramolecular interaction between CA and p6 appears to be unlikely, it is conceivable to hypothesize that p6 harbors another interaction domain of a yet unknown factor that, independently of the L-domains, regulates processing of CA.
Overall, these data support a so far unrecognized function of p6 that occurs independently of the L-domain function, does not affect virus release, but selectively affects CA maturation, virus core formation, and thus, infectivity.
Peptide synthesis and purification
The synthesis, purification and molecular characterization of p6 and the related fragments derived from HIV-1NL4-3 have been described in detail previously .
2D1H Total Correlation Spectroscopy (TOCSY), Correlation Spectroscopy (COSY) and Nuclear Overhauser enhancement spectroscopy (NOESY) NMR experiments were performed at 600.13 MHz on a Bruker Avance 600 MHz instrument equipped with an UltraShield Plus magnet and a triple resonance cryoprobe with gradient unit. Individual samples were dissolved in 600 μl 50% aqueous TFE-d2 at concentrations between 1-2 mM. The 2D NMR experiments were performed at 300 K without spinning with mixing times of 110 ms for the TOCSY experiments and 250 ms for the NOESY experiments, respectively. Efficient suppression of the water signal was achieved with application of excitation sculpting in the 1D 1H and the 2D 1H TOCSY and NOESY NMR experiments. 1H signal assignments of the NMR spectra were achieved by identification of the individual spin systems in the 2D 1H TOCSY spectra, combined with observations of sequence-specific short-distance crosspeaks (Hα-HN i, i+1) in the 2D 1H-1H NOESY spectra [48, 49]. Readily recognizable spin systems were used as starting points for correlation of the individual spin systems observed in the TOCSY and NOESY spectra with individual residues in the peptide sequences. Acquisition of data, processing and spectral analysis were performed with Bruker Topspin 1.3 software.
Antibody specific for FLAG was obtained from Sigma, the ribosomal P antigen specific antiserum from Immunovision Inc., the CA specific antiserum from Seramun. The p6 specific antibody was described earlier . The anti-mouse, anti-rabbit, and anti-human IgG antibodies coupled to horseradish peroxidase (HRP) were obtained from Amersham.
Amino acid exchanges at Ser-40 in p6 were introduced by site-directed mutagenesis using oligonucleotides containing the indicated changes (S40F, ΔYP, and ΔPTAP) and the Quick Change® site directed mutagenesis kit (Stratagene). The mutations were introduced in the X4-tropic HIV-1NL4-3 infectious molecular clone  and isogenic R5-tropic derivative thereof . In order to avoid taking biosafety measures, the mutations were also introduced in two HIV-1NL4-3 based subgenomic expression vectors giving rise to noninfectious VLPs: the pNLenv, in which env was deleted , and a an HIV-1 expression construct that carries a primer binding site deletion, as well as two point mutations in the active site of the RT coding region (pΔR ). All introduced mutations did not lead to mutations in the overlapping pol-ORF.
HeLa SS6, HeLa TZM-bl and 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. CEM cells were maintained in RPMI 1640 supplemented with 10% (v/v) inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. All media and compounds were provided by Gibco.
Preparation and cultivation of primary cells
Human tonsils, removed during routine tonsillectomy, were received a few hours after excision from the Olgahospital, Stuttgart, Germany, prepared and infected as described earlier [32, 33]. After washing the tonsils, human lymphocyte aggregate cultures (HLAC) were prepared by dividing the tonsils into tissue blocks of 2-3 mm and grinding the tissue through the sieve of a cell strainer (70 μm, BD Falcon) with a syringe plunger. Cells were seeded in a 96 well plate at a concentration of 2 × 106 cells per well. HLACs were cultured in RPMI 1640 supplemented with 15% (v/v) inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, 2.5 μg/ml Fungizone, 1 mM sodium pyruvate, 1% (v/v) MEM non-essential amino acid solution and 50 μg/ml gentamicin.
Western blot for protein analysis
HeLa SS6 cells were transiently transfected with the appropriate DNA using Lipofectamine 2000™ (Invitrogen) according to the manufacturer's protocol. For ALIX cotransfection, 293T cells were transfected with equal amounts of both DNAs and cells were harvested 24 h post transfection. Cells were lysed in cold RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholat, 0.1% Na-SDS, 5 mM EDTA, DNase, 1 mM PMSF and complete protease inhibitor cocktail (Boehringer Mannheim)), and the lysates were cleared by centrifugation at 16000 × g and 4°C for 10 min. RIPA-soluble proteins and VLPs were separated in 10% SDS/PAA gels, according to Laemmli , transferred onto PVDF membranes (GE Healthcare) and probed with specific antibodies, followed by enhanced chemiluminescence detection. For internal controls, blots were stripped and re-incubated with the appropriate antibody.
Metabolic labeling and immunoprecipitation
For pulse chase experiments, adherent cultures of transfected HeLa SS6 cells were washed once with PBS and starved for 30 min in methionine-free, serum-free RPMI 1640. Cells were pulse-labeled for 15 min with [35S]-methionine (3 mCi/ml; Amersham Life Sciences) and chased for up to 4 h while shaking at 37°C in D-MEM, supplemented with 10% FCS and 10 mM methionine. At the indicated time points, cells and supernatants were collected by centrifugation for 1 min at 16000 × g. Virions were pelleted through a 20% (w/v) sucrose cushion and lysed in Triton wash buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 0.1% Triton X-100, 1 mM PMSF). Cells were lysed in RIPA buffer as described above, containing additionally 5 mM N-ethylmaleimide and 20 μM carbobenzoxyl-Leu-Leu-leucinal (zLLL; Sigma). Gag proteins from precleared cell lysates and lysed VLPs were recovered by immunoprecipitation using a mixture of polyclonal rabbit anti-p6 and anti-p24 antibodies prebound to protein G-Sepharose (GE Healthcare). Samples were separated by SDS-PAGE on a 10% (w/v) acryl amide ProSieve gel (Cambrex Bioscience), backed with Gel Bond film (FMC Bioproducts). Following fixation for 1 h in 50% methanol and 10% acetic acid, gels were rinsed with water, soaked in 1 M sodium salicylic acid solution with 10% glycerol for 5 h and dried. Radioactivity in dried gels was quantified using AIDA imaging software (Raytest).
Virus containing cell culture supernatant was harvested after 48 h and, after removal of residual cells by centrifugation, passed through a 0.45 μm pore-size filter. Virus was pelleted through 20% (w/v) sucrose (16000 × g, 4°C, 90 min). Virus stocks were normalized for p24 content as quantified by a enzyme-linked immunosorbent assay (ELISA, Aalto, Dublin, Ireland) and aliquots were stored at -80°C.
Infection of cells
For infection of T cell cultures, 1 × 107 cells were incubated with 20 or 50 ng of p24, respectively, and supernatant was collected every second day post infection. Virus replication was assessed by quantification of the virus-associated RT activity by [32P]-TTP incorporation using an oligo(dT)-poly(A) template as described . For testing each virus in the HLAC from one donor, 1 ng of p24 was applied to 2 × 106 cells in 96 well format, and virus replication was assessed, as described for T cell cultures, every third day post infection.
Viral infectivity assay
HeLa TZM-bl cells were seeded in 96 well format (4000 cells per well) and infected with standardized amount of p24. The next day, fresh medium with 100 μg/ml dextran sulphate was added to prevent further spread of virus infection, and cells were incubated for further two days. Infection was detected using a galactosidase screen kit from Tropix as recommended by the manufacturer. β-Galactosidase activity was quantified as relative light units per second using an Orion Microplate Luminometer (Berthold).
Transmission electron microscopy (TEM)
Transfected HeLa SS6 cells were processed for transmission electron microscopy in the following way: 24 h post transfection, cells were placed in cellulose capillary tubes , cultivated for one more day, then fixed in 2.5% glutaraldehyde for 1 h at 37°C and stored for further preparation at 4°C. Tubes were collected by centrifugation and sealed by immersion in low-melting-point agarose. The samples were post fixed with OsO4 (1% in distilled water, 1 h), tannic acid (0.1% in Hepes 0,05 M, 30 min) and uranyl acetate (1% in distilled water, 2 h) followed by stepwise dehydration in a graded ethanol series and embedding in epon resin, which was subsequently polymerized. Thin sections were prepared with an ultramicrotome (Ultracut S; Leica, Wetzlar, Germany) and counterstained with uranyl acetate and lead citrate. The sections were examined using a TEM 902 (Carl Zeiss SMT AG) at 80 kV, and the images were digitized using a slow-scan charge-coupled-device camera (Pro Scan; Scheuring, Germany). The evaluation of the capsid morphology was performed by using these images or directly on the screen.
We thank Dr. Henning Heumann and the surgical staff of the Olgahospital, Stuttgart, for generous assistance in obtaining post-tonsillectomy samples, Raymond Sowder for HPLC purification, and Victor Wray for critical reading of the manuscript. This work was supported by a grant IE-S08T06 from the German Human Genome Research Project, by grants SFB 643-A1, SCHU1125/3, and SCHU 1125/5, from the German Research Council to US.
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