AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 12, 1992 Mary Ann Liebert, Inc., Publishers
Antibody Epitopes Sensitive to the State of Human Immunodeficiency Virus Type 1 gp41 Oligomerization Map to a Putative a-Helical Region PANTELIS
POUMBOURIOS,1
DALE A.
McPHEE,2 and BRUCE E. KEMP1
ABSTRACT Two
antibodies, affinity-purified from human immunodeficiency virus-positive human plasma with synthetic
in the region gp41(566-596), were found to recognize oligomeric gp41 more strongly than the monomeric form in an immunoblot assay. In contrast, a murine anti-gpl60 monoclonal antibody, which maps within this sequence to gp41(581-596), recognized only monomeric gp41 after disruption of the oligomer with sodium dodecyl sulfate. This monoclonal anti-gpl60 antibody did not recognize chemically crosslinked oligomeric gp41 that had been treated with similar conditions used to disrupt the gp41 oligomer. These results indicate that this epitope is inaccessible to binding by this antibody when gp41 is oligomeric. Cyanogen bromide cleavage of gp41 resulted in a 17-kD fragment Thr-541-Met-631. A significant proportion of this fragment was oligomeric when derived from chemically crosslinked gp41. The region Ala-566-Gln-596, within the cyanogen bromide fragment, contains the oligomerization-sensitive epitopes as well as two lysine residues available for crosslinkage. This region is relatively conserved and has the propensity to form an amphipathic a-helix.
peptides
INTRODUCTION type 1 (HIV-1) enveand gpl20 are derived from the gp 160 precursor and mediate,the process of viral attachment and '~7 entry into target cells bearing the CD4 receptor. Several studies have shown that the HIV-1 envelope proteins exist as oligomeric structures. Tetrameric and trimeric forms of viral gp41 have been reported8'9 and viral gpl20 exists as a tetramer or trimer.lu Tetrameric, trimeric, and dimeric forms of vaccinia virus-expressed recombinant gpl60""13 and gpl2013 have also been demonstrated. Moreover, the glycoprotein precursors and transmembrane proteins of HIV type 2 (HIV-2) and simian immunodeficiency (SIV) are also oligomeric.14-16 Oligomerization of vaccinia-virus expressed recombinant gpl60 occurs immediately after synthesis in the endoplasmic reticulum," and with other viruses, this assembly step has been demonstrated to be essential for correct intracellular trans-
THElope glycoproteins gp41
human immunodeficiency virus
port.17~20
The ectodomain of gp41 appears to play a critical role in oligomerization in that deletion of the cytoplasmic and transmembrane domains from recombinant gpl60 does not effect dimerization. '2 Furthermore, co-infection of cells with vaccinia viruses expressing HIV-1 gpl60 and the envelope protein precursors of HIV-2 or SIV resulted in the formation of heterodimers, indicating that a functionally conserved region is involved in oligomerization for all three retro viruses.21 It has been suggested that this region may map to the relatively conserved ectodomains of HIV-1 gp41 and the transmembrane proteins of HIV-2 and SIV.21 Further evidence that gp41 plays an essential role in gpl60 oligomerization has come from a recent electron microscopic study which indicates that dimeric gpl60 is stabilized through interactions in the gp41 domain.22 Based on secondary structure predictions, Gallaher et al.23 reported that the external domain of gp41 contains a 33-amino acid sequence (residues 560-592) with the propensity to form an extended amphipathic a-helix. This may be analogous to the influenza virus hemagglutinin glycoprotein that contains a 54-
'St. Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, VIC 3065, Australia. 2A1DS Cellular Biology Laboratory, Macfarlane Burnet Centre for Medical Research, Fairfield Hospital, Fairfield, VIC 3078, Australia. 2055
2056
POUMBOURIOS ET AL.
amino acid amphipathic a-helix; hydrophobic amino acids in this structure mediate the packing of monomers in threefold symmetry to yield a trimeric molecule.24 In this study, we report that the state of gp41 oligomerization affects antibody epitopes that map to the gp41 putative helical region and that a 17-kD cyanogen bromide (CNBr) fragment containing this region can be crosslinked in gp41 oligomers.
MATERIALS AND METHODS Virus
Gradient-purified preparations of the HIV-1BRU isolate lysed (Commonwealth Serum Laboratories, Parkville, Victoria, Australia) were a kind gift of the National in 1% Triton X-100
Reference
Laboratory, Virology Department,
Fairfield
Hospi-
tal, Victoria, Australia.
Synthetic peptides Synthetic peptides gp41(566-586), gp41(577-596), gp41
(579-596), gp41(581-596), and gp41(583-596) corresponding to portions of the putative a-helical region —LLRAIEAQQH-
LLQLTVWGIKQLQARILAVERYL^2-,23
gp41(584-606)
peptides
and gp41(598-609), corresponding to the imregion of gp41, 25ÍRILAVERYLKDQQLLGIWGCSGKLICS0^,25'26 and a hybrid peptide comprising gp41(598-609). Mellitin(8-26), gryU^LGIWGCSGKLIC6^9-mellitin8VLTTGLPALISWIKRKRQQ26-NH2 were prepared by solid-phase synthesis using an Applied Biosystems Model 430 synthesizer as described elsewhere.27 The residue numbers encompassing the peptide sequences are shown in parentheses for each synthetic peptide. The amino acid sequences were derived from the HIV-1BRU isolate and numbering is in accordance with Myers et al.28 The synthetic sperm whale myoglobin (SWM) peptide, —EFIFEAIIHVLHSRHPC—, was used as a negative control peptide. In one experiment, peptide gp41 (577596), 1 mg/ml, 0.2 M borate buffer, pH 8, was N-acetylated with acetic anhydride (10 min at room temperature) and then purified by reversed phase chromatography on Sep-Pak C18 cartridges (Millipore Corp., Bedford, MA). munodominant
Antibodies
Monospecific anti-gp41 antibodies were purified from pooled HIV-1-positive human plasma using affinity chromatography. Synthetic peptides gp41(566-586), gp41(584-606) and gp41 (598-609), or gp41 (598-609)-mellitin(8-26) were coupled to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) as recommended by the manufacturer. Heat-inactivated pooled plasma was diluted 10-fold in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST) and agitated with immunosorbents for 1 h at room temperature .After exhaustive washing with PBST and PBS, antibodies were eluted sequentially with 0.1 M sodium acetate buffers, pH 4 and pH 3, respectively. Human a-(598-609) antibody was purified using
peptide gp41(598-609)-Sepharose or gp41 (598-609)-mellitin(8-26)-Sepharose while human a-(566-586) antibody was purified using gp41(566-586)-sepharose. The human a-(577596) antibody was affinity-purified by absorbing the pooled
HIV-1-positive human plasma to enzyme-linked immunosorbent assay (ELISA) plates (Nunc-Immunoplate Maxisorb, Nunc, Copenhagen, Denmark) coated with peptide gp41(577596) [10 |xg/ml, 200 u.1] followed by elution with 0.1 M acetate, pH 4 buffer. Purified human a-(566-586) and human a-(577596) antibodies were absorbed with peptide gp41(584-606)Sepharose immunoabsorbent to remove cross-reactive antibodies. Monoclonal antibody (mAb) PC5009, raised against recombinant gpl60, was obtained from Epitope Inc., Beaverton, OR. Monoclonal antibody 2A2/26, raised against viral gp41, was obtained from Agen Biomedicals Pty. Ltd., Brisbane, Australia (number H69-67-2A2/26). ELISA The epitope specificities of antibodies were determined from their binding to synthetic gp41 peptides in the ELISA as described elsewhere.29 Essentially"the same method was used in the competition ELISA except that a constant amount of antibody, giving 50% of maximal binding, was treated with serial dilutions of synthetic peptides for 18 h at room temperature before being added to synthetic peptide-coated ELISA plates.
Polyaery lamide gel electrophoresis
and
immunoblot assays
Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) was carried out as described.30 The method was modified in experiments where different oligomeric forms of gp41 were produced by boiling 3.5 p»g of virus in the presence of varying SDS concentrations, 1% dithiothreitol, 10% glycerol, 0.01% bromophenol blue in 62.5 mM Tris, pH 6.8. These samples were electrophoresed on 8.5% polyacrylamide gels containing no SDS. In these experiments, the concentration of SDS in the electrode buffer was decreased to 0.025%. After separation, proteins were transferred to nitrocellulose and immunoblotted as described.31 A Bio-Rad MiniProtean II and Mini-Transblot system was used in all experiments. Coomassie Blue prestained high and low range molecular weight markers (Bio-Rad Laboratories, Richmond, CA) were used to estimate molecular weights.
Chemical
crosslinking
HIV-1BRU virus was treated with the lysine-specific, homobifunctional chemical crosslinker Bis (sulphosuccinimidyl) suberate (BS3, Pierce Chemicals). Optimal crosslinking was achieved at a ratio of 5 |xg of virus per 20 p.1 of 1 mM BS3 in PBS, pH 7.8, for 2 h at room temperature. Reactions were quenched with 100 mM glycine, pH 7.8, buffer for 45 min at room
temperature.
Staphylococcus aureus V8 protease and CNBr cleavage of crosslinked oligomeric gp41 BS3-crosslinked or untreated HIV-1BRU virus (5 u.g) was boiled in the presence of 0.2% SDS and 1% dithiothreitol for 3 min prior to cleavage with 5 u-g oí Staphylococcus aureus strain V8 protease (type XVII, Sigma) for 1 h at room temperature. Samples were supplemented with SDS to a final concentration of 1%, boiled for 3 min, subjected to SDS-PAGE on 8% to 20%
HIV
gp41 OLIGOMERIZATION
2057
gradient gels, transferred to nitrocellulose, and immunoblotted with anti-gp41 antibodies. Crosslinked or untreated HIV-1BRU virus (20 p,g) was cleaved with a 500-fold excess (wt/wt) of CNBr (25 mg/ml) in 70% (vol/vol) formic acid for 20 h at room temperature under nitrogen. Samples were dried by centrifuging under vacuum, dissolved in water, and then subjected to the same CNBr cleavage procedure. The resultant lyophilized samples were subjected to SDS-PAGE under reducing conditions on 7.5% to 15% gradient gels, transferred to nitrocellulose, and immunoblotted with anti-gp41 antibodies.
human
150 100
50
a-(598-609)
b 1
nd
2A2/Í
20000
10000 i
^
i
t| 0.014
.36
NI.
tl
N.I.
N.I
N.I.
nd
^^K-1-1-1—
N.I.I PC5009
1000
111
RESULTS
Previously, Pinter et al. (8) demonstrated that after treatment of HIV-1 virus with low SDS concentrations, SDS-PAGE, and transfer to nitrocellulose, the oligomeric forms of gp41 were preserved. We used this approach to monitor the effect of gp41 oligomerization on antibody recognition of epitopes in the immunodominant region and adjacent putative a-helical sequence. Two antibodies recognized the gp41 immunodominant region—the affinity-purified human a-(598-609) antibody recognized peptides gp41(584-606) and gp41 (598-609) (Fig. 1) while mAb 2A2/26 recognized only the extended immunodominant peptide gp41(584-606). In immunoblot assays, both antibodies detected tetrameric, trimeric, and monomeric forms of gp41 resulting from treatment of virus with 0.05% SDS (Fig. 2). Treatment of the virus with higher SDS concentrations (0.125% to 1%), caused tetrameric and trimeric gp41 to dissociate into monomers. At the higher SDS concentrations, a minor 85-kD band corresponding to gp41 dimers was also present. When unfractionated HIV-positive human plasma was used to immunoblot, multiple bands were observed at both 0.05% and 1% SDS (Fig. 2, HIV + ). Two human antibodies specific for an adjacent region, human a-(577-596) and human a-(566-586), were purified from HIV1-positive plasma using ELISA plates coated with the peptide
gp41(577-596) or a peptide gp41(566-586)-sepharose column, respectively. In the immunoblot assay, these human antibodies recognized predominantly tetrameric and trimeric gp41 and reacted only weakly with monomeric gp41 (Fig. 2). Human a-(577-596) recognized the peptide gp41(577-596) in the ELISA but its binding to the truncated peptide gp41(579-596) was diminished and further truncation of peptides at the amino terminus resulted in loss of binding (Fig. 1), suggesting that residues Gly-577-Lys-579 influence recognition. The binding of the human a-(577-596) antibody to plate-bound peptide was weakly inhibited by peptide gp41(577-596) in solution (100 u-g/ml peptide effecting 30% inhibition). Furthermore, aminoterminally truncated peptides had no inhibitory activity and chemical modification of lysine residues by N-acetylation of the peptide gp41(577-596) abrogated antibody binding. Human a-(566-586) recognized only the peptide sequence gp41(566586) in an ELISA. In contrast, mAb PC5009, a murine monoclonal antibody raised against recombinant gpl60, recognized only monomeric gp41 when virus was treated with either low (0.05%) or high (0.125% to 1%) SDS concentrations (Fig. 2), indicating that its epitope is unrecognizable in tetrameric or trimeric gp41 but is
100
I.
1 CO
human
ct-(577-596)
_i_i_i_
human
a-(566-586)
J_I_L Oí
Oí
3
03
SYNTHETIC PEPTIDE
B 560
NH,-
577
566
584
596 598
609
LLRAIEÀQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLIC PUTATIVE ALPHA-HELICAL REGION
IMMUNODOMINANT REGION
FIG. 1. (A) Binding of antibodies to synthetic peptides in ELISA. Antibody titers against the various gp41 peptides were calculated from ELISA titration curves as the dilution of antibody required to give 50% of maximal binding. The titers have been multiplied by the dilution factor of the original plasma that occurred during purification. The binding of mAb PC5009 and human a-(577-596) to N-acetylated peptide gp41(577-596) is also shown (stippled bars), nd denotes not determined. tThe results of competition experiments with synthetic peptides are shown as insert panels for mAb PC5009 and human a-(577596). The results are given as u-g/ml of solution-phase synthetic peptides required to inhibit by 50% or 30%, respectively, the binding of mAb PC5009 or human a-(577-596) to plate-bound peptide gp41(577-596). N.I. denotes no inhibition at a starting inhibitor concentration of 100 Ltg/ml. (B) Sequence from which the synthetic peptides were derived.
recognized in monomeric gp41. In the ELISA, mAb PC5009, recognized peptide gp41(577-596), indicating that its epitope overlapped with that of the human a-(577-596) antibody. However, the fine specificity of mAb PC5009 and human a-(577-596) antibodies differed in that: (a) amino-terminal truncation of
peptides
did not affect mAb
PC5009-binding
except with peptide gp41(583-596) (Fig. 1), indicating that
Leu-58 l-Ala-583 is important for epitope-recognition; (b) solution phase peptide gp41(577-596) at 0.014 u.g/ml potently inhibited mAb PC5009-binding to the plate-bound homologous peptide. Amino-terminal truncation of the inhibitor peptide markedly diminished or abrogated inhibitory activity indicating that recognition of the shorter peptides was dependent on interaction with the plate surface; (c) N-acetylation of lysine
POUMBOURIOS ET AL.
2058
human
a-(598-609)
kD 205_
005
84—
41 gp414>. gp413». gp4l2*
47—
gp4>
"7-
-125
0-5
mAb 2A2/26 10
005
10
.»PC«» 0-05
-125
0-5
.."---„ .."--,,
1-0
0-05
1-0
0-05
10
H». 005
V0
%SDS
*#«gp4i
33—
P33
24—
p24
FIG. 2.
Reactivity of anti-gp41 antibodies with oligomeric forms of gp41 in immunoblot assay. SolubilizedHlV-lBR, virus was boiled for 2 min in the presence of the various concentrations of SDS indicated, electrophoresed on 8.5% polyacrylamide gels containing no SDS, transferred to nitrocellulose, and immunoblotted with the monoclonal and peptide-specific anti-gp41 antibodies or HIV-positive human plasma (HIV + ).
peptide gp41(577-596) had no effect on mAb PC5009-binding. To further investigate the role that gp41 oligomer dissociation and/or denaturation plays on epitope recognition by mAb PC5009, the gp41 oligomer was chemically crosslinked with BS3: This treatment resulted in two higher molecular weight species (—170 and 91 kD) which were detected by the human a-(598-609) antibody and correspond to the expected molecular weights of tetrameric and dimeric gp41, respectively (Fig. 3A, lane A). Polyclonal rabbit anti-gpl20 serum did not recognize either of these species (Fig. 4). Monoclonal antibody PC5009 did not recognize crosslinked gp41 (Fig. 3A, lane A). This result indicates that the epitope recognized by mAb PC5009 in gp41 is masked by crosslinking. Since N-acetylation of lysine residues does not alter the binding of this mAb (Fig. 1 ), modification of these residues during the crosslinking reaction alone would be unlikely to alter recognition by the antibody. Crosslinking of oligomeric gp41 may protect the epitope of mAb PC5009 from denaturation resulting in lack of recognition if a linear epitope is required for binding by this mAb. Alternatively this epitope may be inaccessible to binding-within oligomeric gp41. Proteolysis of uncrosslinked gp41 with Staphylococcus aureus strain V8 protease, specific for Glu and Asp residues, resulted in a fragment of gp41 recognized by human a-(598-609) and mAb PC5009 antibodies with an Mr of -25 kD (Fig. 3A, lane D). Proteolysis of crosslinked gp41 resulted in two discrete species with molecular weight of —95 kD and a less intense band at 65 kD (Fig. 3A, lane C) which were detected by the human a-(598-609) antibody. The 95-kD species is consistent with the tetrameric form of the 25-kD monomeric proteolyzed species. The identity of the 65-kD species is unknown but may result from incomplete proteolysis of dimeric gp41. Monoclonal antibody PC5009 did not recognize crosslinked gp41 nor the oligomeric, proteolytic fragments (Fig. 3A, lanes A and C). The precise cleavage sites for generating the 25-kD V8 fragment are residues of
not known. There is a cluster of acidic residues amino-terminal to the transmembrane domain (positions 652, 653, 659, 662,
664, 667, and 669). In contrast, only three acidic residues (Glu-565, Glu-589, and Asp-594) are present on the amino-
terminal side of the immunodominant region with Glu-565 and Glu-589 being located within the putative a-helical domain. The 25-kD proteolytic fragment corresponds in molecular weight to that expected for the glycosylated ectodomain of gp41, extending from the amino terminus at Ala-517 to the acidic region 652-669. Oligomeric gp41 was fragmented with cyanogen bromide to determine which regions were in close proximity in the oligomer and may therefore be involved in inter-gp41 contacts. CNBr treatment yielded a 17-kD band recognizable by the human a-(598-609) antibody in immunoblots (Fig. 3B, lane D). This corresponds to the M predicted for the 91-residue glycosylated fragment, Thr-541-Met-631, encompassing the putative a-helical region and adjacent immunodominant domain. A minor band was also present at a position corresponding to an Mr —23 kD and represents partial cleavage. This region contains four lysine residues at positions 579, 593, 606, 622 that are potentially available for crosslinking in the gp41 oligomer. Of these, Lys-579 and Lys-593 are located in the putative alpha-helical region. Cyanogen bromide cleavage of BS3-treated virus resulted in a discrete bands with A/r's of 17 and 36 kD, consistent with the monomer and dimer of the 17-kD fragment, respectively (Fig. 3B, lane C). In addition, a smear of minor bands with M/s ranging from —43 to 95 kD were also produced (Fig. 3B, lane C) and are likely to represent partial digestion products or higher-order forms of oligomeric 17-kD, 23-kD fragments, and mixed oligomers of the two. These results indicate that the sequence Thr-541-Met-631 containing the putative a-helical region can be crosslinked and thus contains residues that are in close proximity in the oligomeric structure. The CNBr digest of crosslinked gp41 contains a preponderance of monomeric 17-kD fragments (—60%) over the dimeric fragment indicating that lysine residues within the 17-kD fragment do not represent all of the crosslinking sites of gp41. On the other hand, in the V8 protease digests of crosslinked gp41, a 95-kD band was apparent and there was no 25-kD fragment remaining. These results are consistent with lysine residues outside the CNBr fragment being responsible for the additional crosslinking of gp41. —
HIV
gp41 OLIGOMERIZATION
2059
DISCUSSION The results of the crosslinking studies reported here indicate that lysine residues within the 91-residue CNBr peptide Thr541-Met-631 are in close proximity in the gp41 oligomers. Antibodies present in HIV-positive sera recognize epitopes within this region. Those specific for the immunodominant epitope, gp41(598-609), recognize both monomeric and oligomeric gp41. In contrast, antibodies recognizing the nearby region gp41(566-596) show a strong preference for the oligomeric form and bind monomeric gp41 poorly. On the other hand, mAb PC5009 which also maps to this region, recognizes exclusively the monomeric form. Thus the state of gp41 oligomerization determines the binding of the human a-(566-586) and human a-(577-596) antibodies as well as mAb PC5009; this
human
mAb PC5009
(598-609) A B C a
kD
A
B
205— 117— 84—
C D
J
47—
B A
kD
D
B
205-
gp414i 117-
8447-
gp4i2>i*MPi gp41
3324-
16-
^F*^W|*
binding may be related to the presence or absence, respectively, of secondary structure in this region. Alternatively, mAb PC5009 binds to an epitope which is accessible on monomeric gp41 but is inaccessible when gp41 is oligomeric or crosslinked. The finding that synthetic peptides could be used to purify antibodies with preference for oligomeric forms of gp41 was unexpected. However, a number of studies indicate that interaction between an antibody or protein and a peptide can induce secondary structure in the peptide allowing a better "fit" of peptide into the antibody paratope or binding region of the protein. For example, the 19-residue homologue of the C-helix of myohemerythrin, which has no secondary structure in solution, was found to form a type II ß-turn when bound to antibody.32 Another example is the S-peptide of ribonuclease A which has no a-helicity at 25°C but regains full helical character upon binding to the S-protein.33 Analogous events may occur when antibodies present in human plasma bind to peptides allowing their affinity purification. However, preferential binding of human antibodies to oligomeric gp41 may be due to higher affinity binding to a more native epitope in oligomeric gp41 compared with lower affinity binding to denatured, monomeric gp41. Indeed, this region has the propensity to form an a-helix and this structure may be present in the oligomer but not the SDS-denatured monomer. These results are also consistent with these antibodies being elicited by a native oligomeric structure during infection. Our fragmentation experiments show that a significant proportion of a 17-kD CNBr fragment of gp41 is oligomeric when derived from crosslinked gp41 indicating that crosslinkage occurs between residues 541 and 631. This result suggests that lysines within this sequence of one gp41 monomer are in close proximity with corresponding lysines of an adjacent monomer and may constitute an oligomerization domain. While the majority of gp41 was crosslinked by BS3, a large proportion of the 17-kD fragment derived from crosslinked gp41 remained FIG. 3. (A) Immunoblot assays of Staphylococcus aureus strain V8 protease-digested crosslinked, oligomeric gp41. BS3 crosslinked or untreated HIV-1BRU virus (5 |xg) was boiled in the presence of 0.2% SDS and 1% dithiothreitol for 3 min and then digested with V8 protease (5 p,g, type XVII, Sigma Chemical) for 1 h at room temperature. Samples were supplemented with SDS to a final concentration of 1%, boiled for 3 min, and subjected to SDS-PAGE on 8% to 20% gradient gels. The separated proteins were transferred to nitrocellulose and immunoblotted with human a-(598-609) and PC5009 antibodies. Lanes A, undigested, BS3-crosslinked HIV-1BRU virus; lanes B, undigested, HIV-1BRU virus; lanes C, V8 proteasedigested BS3-crosslinked HIV-1BRU virus; lanes D, V8 protease-digested HIV-1BRU virus. (B) Immunoblot assay of CNBr-cleaved crosslinked, oligomeric gp41. Solubilized HIV1BRU virus was treated with the lysine-specific chemical crosslinker BS3. Crosslinked or untreated samples were then subjected to CNBr cleavage as described in Materials and Methods. The resultant lyophilized samples were subjected to SDS-PAGE under reducing conditions on 7.5% to 15% gradient gels, transferred to nitrocellulose, and immunoblotted with human a-(598-609) antibody. Lane A, BS3-crosslinked HIV1BRU virus; lane B, untreated HIV-1BRLJ virus; lane C, CNBrcleaved BS3-crosslinked HIV-lBRlJ virus; lane D, CNBrcleaved HIV-1BRU virus.
2060
POUMBOURIOS ET AL.
monomeric suggesting that additional sites in gp41 are also involved in crosslinkage. These additional sites appear to be included in the 25 kDa V8 proteolytic fragment since proteolysis of crosslinked gp41 gave quantitative recovery of higher molecular weight species and no remaining 25-kD fragment. A recent study of Neurath et al.34 indicated that antisera raised against a synthetic peptide corresponding to gp41 residues 637-666, which also has a-helical propensity,23 recognized exclusively monomeric gp41. The authors concluded that the epitope of this antibody may be obscured in the gp41 oligomer implicating involvement of this region in gp41 oligomerization. Two lysine residues (Lys-655 and Lys 665) present in the HIV-1BRU sequence raise the possibility that this region may also be involved in crosslinkage of the gp41 oligomer. Gallaher et al.23 proposed that gp41 and the transmembrane proteins of other retroviruses contain extended amphipathic a-helices analogous to the extended helix in the influenza virus
SUPPLEMENTARY DATA
rabbit
human
a-(598-609) M
a-rgp120 A
B
M
B
kD
-205
Antibody Epitopes Sensitive to the State of Human Immunodeficiency Virus Type 1 gp41 Oligomerization Map to a Putative a-Helical Region PANTELIS
POUMBOURIOS,1
DALE A.
McPHEE,2 and BRUCE E. KEMP1
ABSTRACT Two
antibodies, affinity-purified from human immunodeficiency virus-positive human plasma with synthetic
in the region gp41(566-596), were found to recognize oligomeric gp41 more strongly than the monomeric form in an immunoblot assay. In contrast, a murine anti-gpl60 monoclonal antibody, which maps within this sequence to gp41(581-596), recognized only monomeric gp41 after disruption of the oligomer with sodium dodecyl sulfate. This monoclonal anti-gpl60 antibody did not recognize chemically crosslinked oligomeric gp41 that had been treated with similar conditions used to disrupt the gp41 oligomer. These results indicate that this epitope is inaccessible to binding by this antibody when gp41 is oligomeric. Cyanogen bromide cleavage of gp41 resulted in a 17-kD fragment Thr-541-Met-631. A significant proportion of this fragment was oligomeric when derived from chemically crosslinked gp41. The region Ala-566-Gln-596, within the cyanogen bromide fragment, contains the oligomerization-sensitive epitopes as well as two lysine residues available for crosslinkage. This region is relatively conserved and has the propensity to form an amphipathic a-helix.
peptides
INTRODUCTION type 1 (HIV-1) enveand gpl20 are derived from the gp 160 precursor and mediate,the process of viral attachment and '~7 entry into target cells bearing the CD4 receptor. Several studies have shown that the HIV-1 envelope proteins exist as oligomeric structures. Tetrameric and trimeric forms of viral gp41 have been reported8'9 and viral gpl20 exists as a tetramer or trimer.lu Tetrameric, trimeric, and dimeric forms of vaccinia virus-expressed recombinant gpl60""13 and gpl2013 have also been demonstrated. Moreover, the glycoprotein precursors and transmembrane proteins of HIV type 2 (HIV-2) and simian immunodeficiency (SIV) are also oligomeric.14-16 Oligomerization of vaccinia-virus expressed recombinant gpl60 occurs immediately after synthesis in the endoplasmic reticulum," and with other viruses, this assembly step has been demonstrated to be essential for correct intracellular trans-
THElope glycoproteins gp41
human immunodeficiency virus
port.17~20
The ectodomain of gp41 appears to play a critical role in oligomerization in that deletion of the cytoplasmic and transmembrane domains from recombinant gpl60 does not effect dimerization. '2 Furthermore, co-infection of cells with vaccinia viruses expressing HIV-1 gpl60 and the envelope protein precursors of HIV-2 or SIV resulted in the formation of heterodimers, indicating that a functionally conserved region is involved in oligomerization for all three retro viruses.21 It has been suggested that this region may map to the relatively conserved ectodomains of HIV-1 gp41 and the transmembrane proteins of HIV-2 and SIV.21 Further evidence that gp41 plays an essential role in gpl60 oligomerization has come from a recent electron microscopic study which indicates that dimeric gpl60 is stabilized through interactions in the gp41 domain.22 Based on secondary structure predictions, Gallaher et al.23 reported that the external domain of gp41 contains a 33-amino acid sequence (residues 560-592) with the propensity to form an extended amphipathic a-helix. This may be analogous to the influenza virus hemagglutinin glycoprotein that contains a 54-
'St. Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, VIC 3065, Australia. 2A1DS Cellular Biology Laboratory, Macfarlane Burnet Centre for Medical Research, Fairfield Hospital, Fairfield, VIC 3078, Australia. 2055
2056
POUMBOURIOS ET AL.
amino acid amphipathic a-helix; hydrophobic amino acids in this structure mediate the packing of monomers in threefold symmetry to yield a trimeric molecule.24 In this study, we report that the state of gp41 oligomerization affects antibody epitopes that map to the gp41 putative helical region and that a 17-kD cyanogen bromide (CNBr) fragment containing this region can be crosslinked in gp41 oligomers.
MATERIALS AND METHODS Virus
Gradient-purified preparations of the HIV-1BRU isolate lysed (Commonwealth Serum Laboratories, Parkville, Victoria, Australia) were a kind gift of the National in 1% Triton X-100
Reference
Laboratory, Virology Department,
Fairfield
Hospi-
tal, Victoria, Australia.
Synthetic peptides Synthetic peptides gp41(566-586), gp41(577-596), gp41
(579-596), gp41(581-596), and gp41(583-596) corresponding to portions of the putative a-helical region —LLRAIEAQQH-
LLQLTVWGIKQLQARILAVERYL^2-,23
gp41(584-606)
peptides
and gp41(598-609), corresponding to the imregion of gp41, 25ÍRILAVERYLKDQQLLGIWGCSGKLICS0^,25'26 and a hybrid peptide comprising gp41(598-609). Mellitin(8-26), gryU^LGIWGCSGKLIC6^9-mellitin8VLTTGLPALISWIKRKRQQ26-NH2 were prepared by solid-phase synthesis using an Applied Biosystems Model 430 synthesizer as described elsewhere.27 The residue numbers encompassing the peptide sequences are shown in parentheses for each synthetic peptide. The amino acid sequences were derived from the HIV-1BRU isolate and numbering is in accordance with Myers et al.28 The synthetic sperm whale myoglobin (SWM) peptide, —EFIFEAIIHVLHSRHPC—, was used as a negative control peptide. In one experiment, peptide gp41 (577596), 1 mg/ml, 0.2 M borate buffer, pH 8, was N-acetylated with acetic anhydride (10 min at room temperature) and then purified by reversed phase chromatography on Sep-Pak C18 cartridges (Millipore Corp., Bedford, MA). munodominant
Antibodies
Monospecific anti-gp41 antibodies were purified from pooled HIV-1-positive human plasma using affinity chromatography. Synthetic peptides gp41(566-586), gp41(584-606) and gp41 (598-609), or gp41 (598-609)-mellitin(8-26) were coupled to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) as recommended by the manufacturer. Heat-inactivated pooled plasma was diluted 10-fold in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST) and agitated with immunosorbents for 1 h at room temperature .After exhaustive washing with PBST and PBS, antibodies were eluted sequentially with 0.1 M sodium acetate buffers, pH 4 and pH 3, respectively. Human a-(598-609) antibody was purified using
peptide gp41(598-609)-Sepharose or gp41 (598-609)-mellitin(8-26)-Sepharose while human a-(566-586) antibody was purified using gp41(566-586)-sepharose. The human a-(577596) antibody was affinity-purified by absorbing the pooled
HIV-1-positive human plasma to enzyme-linked immunosorbent assay (ELISA) plates (Nunc-Immunoplate Maxisorb, Nunc, Copenhagen, Denmark) coated with peptide gp41(577596) [10 |xg/ml, 200 u.1] followed by elution with 0.1 M acetate, pH 4 buffer. Purified human a-(566-586) and human a-(577596) antibodies were absorbed with peptide gp41(584-606)Sepharose immunoabsorbent to remove cross-reactive antibodies. Monoclonal antibody (mAb) PC5009, raised against recombinant gpl60, was obtained from Epitope Inc., Beaverton, OR. Monoclonal antibody 2A2/26, raised against viral gp41, was obtained from Agen Biomedicals Pty. Ltd., Brisbane, Australia (number H69-67-2A2/26). ELISA The epitope specificities of antibodies were determined from their binding to synthetic gp41 peptides in the ELISA as described elsewhere.29 Essentially"the same method was used in the competition ELISA except that a constant amount of antibody, giving 50% of maximal binding, was treated with serial dilutions of synthetic peptides for 18 h at room temperature before being added to synthetic peptide-coated ELISA plates.
Polyaery lamide gel electrophoresis
and
immunoblot assays
Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) was carried out as described.30 The method was modified in experiments where different oligomeric forms of gp41 were produced by boiling 3.5 p»g of virus in the presence of varying SDS concentrations, 1% dithiothreitol, 10% glycerol, 0.01% bromophenol blue in 62.5 mM Tris, pH 6.8. These samples were electrophoresed on 8.5% polyacrylamide gels containing no SDS. In these experiments, the concentration of SDS in the electrode buffer was decreased to 0.025%. After separation, proteins were transferred to nitrocellulose and immunoblotted as described.31 A Bio-Rad MiniProtean II and Mini-Transblot system was used in all experiments. Coomassie Blue prestained high and low range molecular weight markers (Bio-Rad Laboratories, Richmond, CA) were used to estimate molecular weights.
Chemical
crosslinking
HIV-1BRU virus was treated with the lysine-specific, homobifunctional chemical crosslinker Bis (sulphosuccinimidyl) suberate (BS3, Pierce Chemicals). Optimal crosslinking was achieved at a ratio of 5 |xg of virus per 20 p.1 of 1 mM BS3 in PBS, pH 7.8, for 2 h at room temperature. Reactions were quenched with 100 mM glycine, pH 7.8, buffer for 45 min at room
temperature.
Staphylococcus aureus V8 protease and CNBr cleavage of crosslinked oligomeric gp41 BS3-crosslinked or untreated HIV-1BRU virus (5 u.g) was boiled in the presence of 0.2% SDS and 1% dithiothreitol for 3 min prior to cleavage with 5 u-g oí Staphylococcus aureus strain V8 protease (type XVII, Sigma) for 1 h at room temperature. Samples were supplemented with SDS to a final concentration of 1%, boiled for 3 min, subjected to SDS-PAGE on 8% to 20%
HIV
gp41 OLIGOMERIZATION
2057
gradient gels, transferred to nitrocellulose, and immunoblotted with anti-gp41 antibodies. Crosslinked or untreated HIV-1BRU virus (20 p,g) was cleaved with a 500-fold excess (wt/wt) of CNBr (25 mg/ml) in 70% (vol/vol) formic acid for 20 h at room temperature under nitrogen. Samples were dried by centrifuging under vacuum, dissolved in water, and then subjected to the same CNBr cleavage procedure. The resultant lyophilized samples were subjected to SDS-PAGE under reducing conditions on 7.5% to 15% gradient gels, transferred to nitrocellulose, and immunoblotted with anti-gp41 antibodies.
human
150 100
50
a-(598-609)
b 1
nd
2A2/Í
20000
10000 i
^
i
t| 0.014
.36
NI.
tl
N.I.
N.I
N.I.
nd
^^K-1-1-1—
N.I.I PC5009
1000
111
RESULTS
Previously, Pinter et al. (8) demonstrated that after treatment of HIV-1 virus with low SDS concentrations, SDS-PAGE, and transfer to nitrocellulose, the oligomeric forms of gp41 were preserved. We used this approach to monitor the effect of gp41 oligomerization on antibody recognition of epitopes in the immunodominant region and adjacent putative a-helical sequence. Two antibodies recognized the gp41 immunodominant region—the affinity-purified human a-(598-609) antibody recognized peptides gp41(584-606) and gp41 (598-609) (Fig. 1) while mAb 2A2/26 recognized only the extended immunodominant peptide gp41(584-606). In immunoblot assays, both antibodies detected tetrameric, trimeric, and monomeric forms of gp41 resulting from treatment of virus with 0.05% SDS (Fig. 2). Treatment of the virus with higher SDS concentrations (0.125% to 1%), caused tetrameric and trimeric gp41 to dissociate into monomers. At the higher SDS concentrations, a minor 85-kD band corresponding to gp41 dimers was also present. When unfractionated HIV-positive human plasma was used to immunoblot, multiple bands were observed at both 0.05% and 1% SDS (Fig. 2, HIV + ). Two human antibodies specific for an adjacent region, human a-(577-596) and human a-(566-586), were purified from HIV1-positive plasma using ELISA plates coated with the peptide
gp41(577-596) or a peptide gp41(566-586)-sepharose column, respectively. In the immunoblot assay, these human antibodies recognized predominantly tetrameric and trimeric gp41 and reacted only weakly with monomeric gp41 (Fig. 2). Human a-(577-596) recognized the peptide gp41(577-596) in the ELISA but its binding to the truncated peptide gp41(579-596) was diminished and further truncation of peptides at the amino terminus resulted in loss of binding (Fig. 1), suggesting that residues Gly-577-Lys-579 influence recognition. The binding of the human a-(577-596) antibody to plate-bound peptide was weakly inhibited by peptide gp41(577-596) in solution (100 u-g/ml peptide effecting 30% inhibition). Furthermore, aminoterminally truncated peptides had no inhibitory activity and chemical modification of lysine residues by N-acetylation of the peptide gp41(577-596) abrogated antibody binding. Human a-(566-586) recognized only the peptide sequence gp41(566586) in an ELISA. In contrast, mAb PC5009, a murine monoclonal antibody raised against recombinant gpl60, recognized only monomeric gp41 when virus was treated with either low (0.05%) or high (0.125% to 1%) SDS concentrations (Fig. 2), indicating that its epitope is unrecognizable in tetrameric or trimeric gp41 but is
100
I.
1 CO
human
ct-(577-596)
_i_i_i_
human
a-(566-586)
J_I_L Oí
Oí
3
03
SYNTHETIC PEPTIDE
B 560
NH,-
577
566
584
596 598
609
LLRAIEÀQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLIC PUTATIVE ALPHA-HELICAL REGION
IMMUNODOMINANT REGION
FIG. 1. (A) Binding of antibodies to synthetic peptides in ELISA. Antibody titers against the various gp41 peptides were calculated from ELISA titration curves as the dilution of antibody required to give 50% of maximal binding. The titers have been multiplied by the dilution factor of the original plasma that occurred during purification. The binding of mAb PC5009 and human a-(577-596) to N-acetylated peptide gp41(577-596) is also shown (stippled bars), nd denotes not determined. tThe results of competition experiments with synthetic peptides are shown as insert panels for mAb PC5009 and human a-(577596). The results are given as u-g/ml of solution-phase synthetic peptides required to inhibit by 50% or 30%, respectively, the binding of mAb PC5009 or human a-(577-596) to plate-bound peptide gp41(577-596). N.I. denotes no inhibition at a starting inhibitor concentration of 100 Ltg/ml. (B) Sequence from which the synthetic peptides were derived.
recognized in monomeric gp41. In the ELISA, mAb PC5009, recognized peptide gp41(577-596), indicating that its epitope overlapped with that of the human a-(577-596) antibody. However, the fine specificity of mAb PC5009 and human a-(577-596) antibodies differed in that: (a) amino-terminal truncation of
peptides
did not affect mAb
PC5009-binding
except with peptide gp41(583-596) (Fig. 1), indicating that
Leu-58 l-Ala-583 is important for epitope-recognition; (b) solution phase peptide gp41(577-596) at 0.014 u.g/ml potently inhibited mAb PC5009-binding to the plate-bound homologous peptide. Amino-terminal truncation of the inhibitor peptide markedly diminished or abrogated inhibitory activity indicating that recognition of the shorter peptides was dependent on interaction with the plate surface; (c) N-acetylation of lysine
POUMBOURIOS ET AL.
2058
human
a-(598-609)
kD 205_
005
84—
41 gp414>. gp413». gp4l2*
47—
gp4>
"7-
-125
0-5
mAb 2A2/26 10
005
10
.»PC«» 0-05
-125
0-5
.."---„ .."--,,
1-0
0-05
1-0
0-05
10
H». 005
V0
%SDS
*#«gp4i
33—
P33
24—
p24
FIG. 2.
Reactivity of anti-gp41 antibodies with oligomeric forms of gp41 in immunoblot assay. SolubilizedHlV-lBR, virus was boiled for 2 min in the presence of the various concentrations of SDS indicated, electrophoresed on 8.5% polyacrylamide gels containing no SDS, transferred to nitrocellulose, and immunoblotted with the monoclonal and peptide-specific anti-gp41 antibodies or HIV-positive human plasma (HIV + ).
peptide gp41(577-596) had no effect on mAb PC5009-binding. To further investigate the role that gp41 oligomer dissociation and/or denaturation plays on epitope recognition by mAb PC5009, the gp41 oligomer was chemically crosslinked with BS3: This treatment resulted in two higher molecular weight species (—170 and 91 kD) which were detected by the human a-(598-609) antibody and correspond to the expected molecular weights of tetrameric and dimeric gp41, respectively (Fig. 3A, lane A). Polyclonal rabbit anti-gpl20 serum did not recognize either of these species (Fig. 4). Monoclonal antibody PC5009 did not recognize crosslinked gp41 (Fig. 3A, lane A). This result indicates that the epitope recognized by mAb PC5009 in gp41 is masked by crosslinking. Since N-acetylation of lysine residues does not alter the binding of this mAb (Fig. 1 ), modification of these residues during the crosslinking reaction alone would be unlikely to alter recognition by the antibody. Crosslinking of oligomeric gp41 may protect the epitope of mAb PC5009 from denaturation resulting in lack of recognition if a linear epitope is required for binding by this mAb. Alternatively this epitope may be inaccessible to binding-within oligomeric gp41. Proteolysis of uncrosslinked gp41 with Staphylococcus aureus strain V8 protease, specific for Glu and Asp residues, resulted in a fragment of gp41 recognized by human a-(598-609) and mAb PC5009 antibodies with an Mr of -25 kD (Fig. 3A, lane D). Proteolysis of crosslinked gp41 resulted in two discrete species with molecular weight of —95 kD and a less intense band at 65 kD (Fig. 3A, lane C) which were detected by the human a-(598-609) antibody. The 95-kD species is consistent with the tetrameric form of the 25-kD monomeric proteolyzed species. The identity of the 65-kD species is unknown but may result from incomplete proteolysis of dimeric gp41. Monoclonal antibody PC5009 did not recognize crosslinked gp41 nor the oligomeric, proteolytic fragments (Fig. 3A, lanes A and C). The precise cleavage sites for generating the 25-kD V8 fragment are residues of
not known. There is a cluster of acidic residues amino-terminal to the transmembrane domain (positions 652, 653, 659, 662,
664, 667, and 669). In contrast, only three acidic residues (Glu-565, Glu-589, and Asp-594) are present on the amino-
terminal side of the immunodominant region with Glu-565 and Glu-589 being located within the putative a-helical domain. The 25-kD proteolytic fragment corresponds in molecular weight to that expected for the glycosylated ectodomain of gp41, extending from the amino terminus at Ala-517 to the acidic region 652-669. Oligomeric gp41 was fragmented with cyanogen bromide to determine which regions were in close proximity in the oligomer and may therefore be involved in inter-gp41 contacts. CNBr treatment yielded a 17-kD band recognizable by the human a-(598-609) antibody in immunoblots (Fig. 3B, lane D). This corresponds to the M predicted for the 91-residue glycosylated fragment, Thr-541-Met-631, encompassing the putative a-helical region and adjacent immunodominant domain. A minor band was also present at a position corresponding to an Mr —23 kD and represents partial cleavage. This region contains four lysine residues at positions 579, 593, 606, 622 that are potentially available for crosslinking in the gp41 oligomer. Of these, Lys-579 and Lys-593 are located in the putative alpha-helical region. Cyanogen bromide cleavage of BS3-treated virus resulted in a discrete bands with A/r's of 17 and 36 kD, consistent with the monomer and dimer of the 17-kD fragment, respectively (Fig. 3B, lane C). In addition, a smear of minor bands with M/s ranging from —43 to 95 kD were also produced (Fig. 3B, lane C) and are likely to represent partial digestion products or higher-order forms of oligomeric 17-kD, 23-kD fragments, and mixed oligomers of the two. These results indicate that the sequence Thr-541-Met-631 containing the putative a-helical region can be crosslinked and thus contains residues that are in close proximity in the oligomeric structure. The CNBr digest of crosslinked gp41 contains a preponderance of monomeric 17-kD fragments (—60%) over the dimeric fragment indicating that lysine residues within the 17-kD fragment do not represent all of the crosslinking sites of gp41. On the other hand, in the V8 protease digests of crosslinked gp41, a 95-kD band was apparent and there was no 25-kD fragment remaining. These results are consistent with lysine residues outside the CNBr fragment being responsible for the additional crosslinking of gp41. —
HIV
gp41 OLIGOMERIZATION
2059
DISCUSSION The results of the crosslinking studies reported here indicate that lysine residues within the 91-residue CNBr peptide Thr541-Met-631 are in close proximity in the gp41 oligomers. Antibodies present in HIV-positive sera recognize epitopes within this region. Those specific for the immunodominant epitope, gp41(598-609), recognize both monomeric and oligomeric gp41. In contrast, antibodies recognizing the nearby region gp41(566-596) show a strong preference for the oligomeric form and bind monomeric gp41 poorly. On the other hand, mAb PC5009 which also maps to this region, recognizes exclusively the monomeric form. Thus the state of gp41 oligomerization determines the binding of the human a-(566-586) and human a-(577-596) antibodies as well as mAb PC5009; this
human
mAb PC5009
(598-609) A B C a
kD
A
B
205— 117— 84—
C D
J
47—
B A
kD
D
B
205-
gp414i 117-
8447-
gp4i2>i*MPi gp41
3324-
16-
^F*^W|*
binding may be related to the presence or absence, respectively, of secondary structure in this region. Alternatively, mAb PC5009 binds to an epitope which is accessible on monomeric gp41 but is inaccessible when gp41 is oligomeric or crosslinked. The finding that synthetic peptides could be used to purify antibodies with preference for oligomeric forms of gp41 was unexpected. However, a number of studies indicate that interaction between an antibody or protein and a peptide can induce secondary structure in the peptide allowing a better "fit" of peptide into the antibody paratope or binding region of the protein. For example, the 19-residue homologue of the C-helix of myohemerythrin, which has no secondary structure in solution, was found to form a type II ß-turn when bound to antibody.32 Another example is the S-peptide of ribonuclease A which has no a-helicity at 25°C but regains full helical character upon binding to the S-protein.33 Analogous events may occur when antibodies present in human plasma bind to peptides allowing their affinity purification. However, preferential binding of human antibodies to oligomeric gp41 may be due to higher affinity binding to a more native epitope in oligomeric gp41 compared with lower affinity binding to denatured, monomeric gp41. Indeed, this region has the propensity to form an a-helix and this structure may be present in the oligomer but not the SDS-denatured monomer. These results are also consistent with these antibodies being elicited by a native oligomeric structure during infection. Our fragmentation experiments show that a significant proportion of a 17-kD CNBr fragment of gp41 is oligomeric when derived from crosslinked gp41 indicating that crosslinkage occurs between residues 541 and 631. This result suggests that lysines within this sequence of one gp41 monomer are in close proximity with corresponding lysines of an adjacent monomer and may constitute an oligomerization domain. While the majority of gp41 was crosslinked by BS3, a large proportion of the 17-kD fragment derived from crosslinked gp41 remained FIG. 3. (A) Immunoblot assays of Staphylococcus aureus strain V8 protease-digested crosslinked, oligomeric gp41. BS3 crosslinked or untreated HIV-1BRU virus (5 |xg) was boiled in the presence of 0.2% SDS and 1% dithiothreitol for 3 min and then digested with V8 protease (5 p,g, type XVII, Sigma Chemical) for 1 h at room temperature. Samples were supplemented with SDS to a final concentration of 1%, boiled for 3 min, and subjected to SDS-PAGE on 8% to 20% gradient gels. The separated proteins were transferred to nitrocellulose and immunoblotted with human a-(598-609) and PC5009 antibodies. Lanes A, undigested, BS3-crosslinked HIV-1BRU virus; lanes B, undigested, HIV-1BRU virus; lanes C, V8 proteasedigested BS3-crosslinked HIV-1BRU virus; lanes D, V8 protease-digested HIV-1BRU virus. (B) Immunoblot assay of CNBr-cleaved crosslinked, oligomeric gp41. Solubilized HIV1BRU virus was treated with the lysine-specific chemical crosslinker BS3. Crosslinked or untreated samples were then subjected to CNBr cleavage as described in Materials and Methods. The resultant lyophilized samples were subjected to SDS-PAGE under reducing conditions on 7.5% to 15% gradient gels, transferred to nitrocellulose, and immunoblotted with human a-(598-609) antibody. Lane A, BS3-crosslinked HIV1BRU virus; lane B, untreated HIV-1BRLJ virus; lane C, CNBrcleaved BS3-crosslinked HIV-lBRlJ virus; lane D, CNBrcleaved HIV-1BRU virus.
2060
POUMBOURIOS ET AL.
monomeric suggesting that additional sites in gp41 are also involved in crosslinkage. These additional sites appear to be included in the 25 kDa V8 proteolytic fragment since proteolysis of crosslinked gp41 gave quantitative recovery of higher molecular weight species and no remaining 25-kD fragment. A recent study of Neurath et al.34 indicated that antisera raised against a synthetic peptide corresponding to gp41 residues 637-666, which also has a-helical propensity,23 recognized exclusively monomeric gp41. The authors concluded that the epitope of this antibody may be obscured in the gp41 oligomer implicating involvement of this region in gp41 oligomerization. Two lysine residues (Lys-655 and Lys 665) present in the HIV-1BRU sequence raise the possibility that this region may also be involved in crosslinkage of the gp41 oligomer. Gallaher et al.23 proposed that gp41 and the transmembrane proteins of other retroviruses contain extended amphipathic a-helices analogous to the extended helix in the influenza virus
SUPPLEMENTARY DATA
rabbit
human
a-(598-609) M
a-rgp120 A
B
M
B
kD
-205
