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Originally published In Press as doi:10.1074/jbc.M000601200 on March 15, 2000

J. Biol. Chem., Vol. 275, Issue 21, 15809-15819, May 26, 2000
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Multimerization Potential of the Cytoplasmic Domain of the Human Immunodeficiency Virus Type 1 Transmembrane Glycoprotein gp41*

Sheau-Fen LeeDagger , Chin-Tien Wang§, Judy Y.-P. LiangDagger , Shi-Lan HongDagger , Chin-Cheng HuangDagger , and Steve S.-L. ChenDagger

From the Dagger  Division of Infectious Diseases, Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, Republic of China and § Institute of Clinical Medicine, National Yang-Ming University, School of Medicine, and Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei 11217, Taiwan, Republic of China

Received for publication, January 25, 2000, and in revised form, March 15, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We previously demonstrated that an envelope mutant of human immunodeficiency virus type 1 lacking the entire cytoplasmic domain interferes in trans with the production of infectious virus by inclusion of the mutant envelope into the wild-type envelope complex. We also showed that the envelope incorporation into virions is not affected when the wild-type envelope is coexpressed with the mutant envelope. These results suggest that an oligomeric structure of the cytoplasmic domain is functionally required for viral infectivity. To understand whether the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 has the potential to self-assemble as an oligomer, in the present study we fused the coding sequence of the entire cytoplasmic domain at 3' to the Escherichia coli malE gene, which encodes a monomeric maltose-binding protein. The expressed fusion protein was examined by chemical cross-linking, sucrose gradient centrifugation, and gel filtration. The results showed that the cytoplasmic domain of gp41 assembles into a high-ordered structural complex. The intersubunit interaction of the cytoplasmic domain was also confirmed by a mammalian two-hybrid system that detects protein-protein interactions in eucaryotic cells. A cytoplasmic domain fragment expressed in eucaryotic cells was pulled down by glutathione-Sepharose 4B beads via its association with another cytoplasmic domain fragment fused to the C terminus of the glutathione S-transferase moiety. We also found that sequences encompassing the lentiviral lytic peptide-1 and lentiviral lytic peptide-2, which are located within residues 828-856 and 770-795, respectively, play a critical role in cytoplasmic domain self-assembly. Taken together, the results from the present study indicate that the cytoplasmic domain of gp41 by itself is sufficient to assemble into a multimeric structure. This finding supports the hypothesis that a multimeric form of the gp41 cytoplasmic domain plays a crucial role in virus infectivity.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The envelope (Env)1 transmembrane (TM) glycoprotein gp41 of human immunodeficiency virus type 1 (HIV-1) is capable of directing oligomerization on its own. Scanning transmission electron microscopy showed that gp160 is a dimer stabilized by gp41 homo-oligomeric interaction (1). The Env protein of HIV-1 forms a hetero-oligomer with the Env proteins of HIV-2 and simian immunodeficiency virus (SIV) (2), suggesting that the Env proteins of primate immunodeficiency viruses share a conserved, high-ordered structural and functional assembly domain. Truncations in the gp41 cytoplasmic domain do not affect Env assembly as an oligomer (3), suggesting that the cytoplasmic tail may not have a role in Env oligomerization. Further truncation and mutagenesis studies showed that the N-terminal 68-129 residues of the gp41 ectodomain confer oligomer stability (3, 4). This region overlaps a highly conserved leucine zipper-like heptad repeat sequence adjacent to the N-terminal fusion peptide sequence. The leucine zipper-like motif plays a crucial role in viral infectivity and membrane fusion (5-8). The six-stranded coiled-coil structure, formed by the leucine zipper-like motif and the C-terminal alpha -helix located in the ectodomain of gp41 and proximal to the TM domain, represents a fusion active conformation of the TM core (9-11).

A striking feature of primate immunodeficiency virus Env proteins is their unusually long (about 150 amino acids) cytoplasmic domains. The cytoplasmic domain of HIV-1 gp41, spanning residues 706-856, plays various roles in virus replication, infectivity, transmissibility, and cytopathogenicity (5, 12-17). The C terminus of the cytoplasmic tail may play a role in viral uncoating or penetration of the viral core into host cells (18). The large helical hydrophobic moments of the two segments spanning residues 828-856 and 770-795 (19, 20), termed lentiviral lytic peptide (LLP)-1 and LLP-2, respectively, may have membrane-related functions (19). Structural modeling of these two regions reveals a high potential to form amphipathic alpha -helices (19-21). These putative alpha -helices are structurally similar to those formed by natural cytolytic peptides such as magainins, cecropins, melittin, etc., produced by certain amphibians and insects. The interactions of these cytolytic peptides with membranes are well studied (21, 22). Synthetic peptides representing LLP-1 and LLP-2 motifs lyse human erythrocytes and are toxic to procaryotic and eucaryotic cells when added exogenously to cultures (22-25). Expression of the C-terminal portion of the cytoplasmic domain where LLP-1 and LLP-2 are localized perturbs membrane permeability in Escherichia coli cells (26). Upon binding to the lipid bilayers, the LLP-1 peptide undergoes a transition from a random coil to an ordered lipid-associated helical conformation (23, 25). The membrane and pH-induced increase in the helical content may increase the likelihood of self-association and formation of a multimeric structure upon binding to the membrane. Nevertheless, the structural requirements for cytoplasmic domain-mediated viral entry and for permeable pathways and cytopathogenesis mediated by the LLPs of gp41 remain obscure.

We previously reported that an Env mutant, which lacks the whole cytoplasmic domain and the last two amino acids in the TM domain, encoded by an HIV-1 mutant provirus dominantly interferes with wild-type (wt) virus infectivity (27). Also, Env incorporation into virions is not affected when a wt provirus is coexpressed with this mutant provirus (27). The dominant interference phenotype conferred by this truncated Env is due to the formation of a dysfunctional hetero-oligomeric complex of the mutant with the wt Env (27). In a later study, we observed that this truncated Env mutant interferes with HIV-1 transmission when the env gene of the truncated mutant is targeted to HeLa-CD4 clones (28). Although previous mutagenesis studies did not reveal a plausible role for the HIV-1 cytoplasmic domain in Env assembly (3, 4), the dominant interference by this cytoplasmic domain truncated mutant (27, 28) suggests that the cytoplasmic domain, folding as an oligomeric structure, is functionally required for gp41-mediated viral entry.

To further assess the evidence that the cytoplasmic domain participates in gp41-mediated tertiary structure formation, in the present study we set out to determine whether the cytoplasmic domain physically associates into a multimeric structure. We expressed the cytoplasmic domain as a fusion partner of a monomeric E. coli maltose-binding protein (MBP) (29) and examined the multimeric structure of this fusion protein. We also performed a mammalian two-hybrid assay and a glutathione S-transferase (GST) pull-down assay to determine the specific interactions between cytoplasmic domain subunits. Our results show that the cytoplasmic domain by itself is sufficient to assemble into a high-ordered multimer. The results also reveal that the two LLP sequences located in the cytoplasmic domain play a crucial role in cytoplasmic domain self-assembly. These findings are consistent with the hypothesis that a multimeric structure of the gp41 cytoplasmic domain is critical for viral infectivity.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- pMal-c2 (New England BioLabs, Beverly, MA), is a Ptac promoter-driven bacterial expression plasmid for expression of target genes fused downstream of the E. coli malE gene (30), which encodes a monomeric E. coli MBP. pSVE7-puro encodes the HXB2 Env protein under the control of the HIV-1 long terminal repeat (28). pSVE7-puro mutant plasmids, each separately encoding an Env protein with a deletion after residue 844, 813, 795, 775, or 752 to the C terminus of the cytoplasmic domain, were previously described (28). pSRS/SIV is a pSRS vector that encodes an StuI fragment (nucleotides 6128-10045), which encompasses the env, tat, and rev genes, from the SIVmac239 genome (31). pM and pVP16 are mammalian two-hybrid vectors that encode the Gal4 DNA-binding domain and the herpes simplex virus transcriptional activator VP16 activation domain, respectively. pG5CAT encodes five consensus Gal4-binding sites (UASG) and an adenovirus E1B minimal promoter upstream of a chloramphenicol acetyltransferase (cat) gene. pEBG is a mammalian elongation factor 1alpha promoter-driven GST expression vector (32). pRK5F is a cytomegalovirus promoter-driven expression plasmid in which a FLAG tag-coding sequence is inserted in the polycloning site (33).

Construction of Plasmids-- For construction of pMal-c2 plasmids that express segments corresponding to different regions of the cytoplasmic domain of the HIV-1 Env (see Fig. 1), wt, or mutant pSVE7-puro plasmids were primed with paired forward and reverse oligonucleotides in PCR. All amplification reactions used oligonucleotide 856rSalI (5'-CCGTCGACTTATAGCAAAATCCTTTCCA-3'; 8794-8775) as a reverse primer. The nucleotides underlined indicate the recognition sequence of the restriction enzyme as indicated in the name of the oligonucleotide. The numbers shown after the sequence indicate the nucleotide positions of the env sequence of the primer in the HXB2 proviral genome. The reverse primer 856rSalI contains a stop codon following the codon for Leu at residue 856 of the HIV-1 Env. To generate the coding sequences of 706-856, 760-856, 789-856, 805-856, and 816-856, wt pSVE7-puro was used as the template and oligonucleotides 706fEcoRI (5'-CGGAATTCAATAGAGTTAGGCAGGGATAT-3'; 8339-8359), 760fEcoRI (5'-CCGAATTCCTGCGGAGCCTGTGCC-3'; 8501-8516), 789fEcoRI (5'-CCGAATTCGGGTGGGAAGCCCTCAA-3'; 8588-8604), 805fEcoRI (5'-CCGAATTCCAGGAACTAAAGAATAGTGC-3'; 8636-8655), and 816fEcoRI (5'-GCGAATTCAATGCCACAGCCATAGC-3'; 8669-8685), respectively, were used as the forward primers. To obtain the coding sequences of 706-844, 706-813, 706-795, 706-775, and 706-752, oligonucleotide 706fEcoRI was used as the forward primer, and TM844, TM813, TM795, TM775, and TM752, respectively, of pSVE7-puro were used as the templates. To construct the coding sequences for 760-795 and 760-775, oligonucleotide 760fEcoRI was used as the forward primer, and TM795 and TM775, respectively, were used as the templates. To construct the 816-844 coding sequence, 816fEcoRI and TM844 were used as the forward primer and template, respectively. The 252-273 coding sequence was generated by PCR using 252fEcoRI (5'-CCGAATTCAGGCCAGTAGTATCAACT-3'; 6977-6994) and 273rSalI (5'-CCGTCGACTTATCTAATTACTACCTCTTCTTCTG-3'; 7042-7020) as primers and wt pSVE7-puro as the template. The reverse primer 273rSalI contains a stop codon following the codon for Arg at 273 of the HIV-1 Env. To obtain the coding sequence of the entire cytoplasmic domain of SIV Env, pSRS/SIV was primed with SIVenv716fEcoRI (5'-CGGAATTCAAGTTAAGGCAGGGG-3'; 9005-9019 of the SIVmac239 genome) and SIVenv879rSalI (5'-CCGTCGACTCACAAGAGAGTGAGCTC-3'; 9499-9482 of the SIVmac239 provirus) in PCR. Reverse primer SIVenv879rSalI contains a stop codon following the codon for Leu at 879 of the SIV Env. The EcoRI- and SalI-digested DNA fragments were inserted in the corresponding sites of pMal-c2 to produce various pMal-c2 expression plasmids. For construction of mammalian two-hybrid plasmids that encode the cytoplasmic domain, the EcoRI-SalI fragment isolated from pMal/706-856 that encodes the entire cytoplasm domain-coding region was cloned into the corresponding sites in the polycloning sequence of pM to produce pM-Env and into pVP16 to produce pVP16-Env. To increase the sensitivity of detection in the mammalian two-hybrid system, the stretch of the DNA sequence containing the Gal4-responsive element and the adenovirus E1b minimal promoter in pG5CAT was cloned by PCR using UASGfSacI (5'-CCGAGCTCGAGGACAGTACTCCGCTCGG-3') and UASGrBglII (5'-CCAGATCTTTTAGCTTCCTTAGCTCCTGAAAA-3') as primers. The SacI-BglII DNA fragment was then inserted in the polycloning sites of the pGL3-basic vector (Promega, Madison, WI), which lacks any eucaryotic enhancer/promoter sequence upstream to a luciferase-coding sequence, to produce the pGL3(UASG) reporter vector. For construction of pEBG/706-856 that encodes a GST-cytoplasmic domain fusion protein, oligonucleotides 706fSpeI (5'-CGGACTAGTAATAGAGTTAGGCAGGGATAT-3'; 8339-8359) and 856rAsp718 (5'-CCCGGTACCTTATAGCAAAATCCTTTCCA-3'; 8794-8775) were used as primers in PCR. For construction of pRK5F/706-856, which encodes a cytoplasmic domain fragment N-terminal to a FLAG tag, a PCR reaction using oligonucleotides 706fEcoRI(Met) (5'-CGGAATTCGCCGCCATGAATAGAGTTAGGCAGGGATAT-3') and 856rSalI(no stop) (5'-CCGTCGACTAGCAAAATCCTTTCCA-3') as primers was performed. pRK5F/706-856 encodes a Kozak sequence 5' to the ATG initiation codon, which is followed by the cytoplasmic domain-coding sequence. The cytoplasmic domain coding sequence was fused in frame to the FLAG tag-coding sequence.

PCR Amplification-- PCR was performed in 100 µl of buffer containing 50 ng of templates, 0.2 mM of each dNTPs, 2 µM each of the forward and reverse primers, and 5 units of Pfu DNA polymerase (Stratagene, La Jolla, CA). In addition to an initial denaturation at 94 °C for 4 min, the samples were subjected to the following amplification programs: (a) five cycles of 94 °C for 30 s, 45 °C for 30 s, and 72 °C for 60 s; (b) five cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 60 s; (c) five cycles of 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 60 s; and (d) fifteen cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 60 s using a GeneAmp PCR System 2400 (Perkin-Elmer, Norwalk, CT). A final extension step was allowed to proceed at 72 °C for 4 min before cooling down to room temperature.

DNA Sequencing-- All pMal-c2 expression plasmids were autosequenced using the malE primer 5'-GGTCGTCAGACTGTCGATGAAGCC-3' (New England BioLabs) to confirm the cytoplasmic domain and 252-273 coding sequences. The env gene in pEBG/706-856 and pRK5F/706-856 were sequenced by oligonucleotides 5'-CAGCAAGTATATAGCATGGCC-3' and Sp6 primer 5'-GATTTAGGTGACACTATAG-3', respectively.

Expression and Purification of Recombinant Fusion Proteins-- The pMal constructs were used to transform competent NM522 or AD494 cells (Novagen, Inc., Madison, WI). MBP fusion proteins were isopropylthiogalactopyranoside-induced and expressed as described previously (34). The bacterial pellets were sonicated in 12 mM phosphate buffer, pH 7.4, containing 3.2 mM KCl and 137 mM NaCl (referred to as PBS hereafter) supplemented with 1 mM phenylmethylsulfonyl fluoride in a 1:10 ratio of the volume of the sonication buffer to that of the initial culture. A final concentration of 0.2% glucose was added to the initial cultures prior to isopropylthiogalactopyranoside induction when purification of MBP fusion proteins was desired. Fusion proteins were purified on an amylose resin affinity column (New England BioLabs) in PBS according to the procedure recommended by the manufacturer.

Sucrose Gradient Centrifugation-- Fusion proteins MBP/706-856 and MBP/252-273 and standard molecular mass markers (obtained from Amersham Pharmacia Biotech and Sigma) in a total volume of 0.5 ml were sedimented in parallel by linear 10-45% sucrose gradient centrifugation. The gradients prepared in PBS were centrifuged at 40,000 rpm in a Beckman SW41 rotor at 4 °C for 20 h. After centrifugation, proteins were collected from the bottom of the gradients. Gradient performance was checked by reading the refraction index of sucrose using a Fisher refractometer. Aliquots of fractionated samples were separated by SDS-7.5% polyacrylamide gel electrophoresis (PAGE) followed by Western blotting analysis.

Chemical Cross-linking-- Purified MBP/706-856 was incubated with different concentrations of a cross-linker glutaraldehyde in PBS with a total volume of 40 µl on ice for 30 min and then quenched with the addition of a final concentration of 100 mM glycine, pH 8.0. The mixture was kept on ice for 30 min prior to SDS-7.5% PAGE followed by Western blotting.

Size Exclusion Chromatography-- Aliquots (0.2 ml) containing 25-100 µg of purified fusion proteins or molecular mass markers were fractionated by gel filtration through a Superose 6 HR column (10 × 300 mm, Amersham Pharmacia Biotech) and eluted with PBS. The column was run with an Amersham Pharmacia Biotech fast protein liquid chromatography system equipped with a UV monitor at a flow rate of 0.5 ml/min at room temperature, and samples were collected at 1-min intervals. Aliquots of fractionated fusion proteins were analyzed by Western blotting using various antibodies to detect fusion proteins. Aliquots of fractionated molecular markers were also measured for protein concentrations using the Bio-Rad protein assay kit. Alternatively, purified MBP fusion proteins and molecular markers were passed over the Superose 6 column and eluted with PBS containing 0.2% Triton X-100. Because Triton X-100 interferes with UV absorption at 280 nm, aliquots of fractionated markers were assessed for protein concentrations using the Bio-Rad protein assay kit.

A Mammalian Two-hybrid Assay-- 293 cells were cotransfected with the two-hybrid paired plasmids pM-Env and pVP16-Env along with pG5CAT or pGL3(UASG) by the calcium phosphate coprecipitation method as described previously (27, 35). Plasmids of pM and pVP16 were added into transfections in which pM-Env or pVP16-Env was not included to keep the total amounts of DNA in all transfections constant. When HeLa cells were examined, LipofectAMINE (Life Technologies, Inc.) was used in transfection according to the procedure recommended by the manufacturer. For pG5CAT transfection, cell lysates were assayed for CAT activity as described previously (27). For pGL3(UASG) transfection, cell lysates were measured for luciferase activity using a luciferase assay reagent (Promega). The luminescent light produced was quantitated on a Lumac Biocounter M2500 (Landgraaf, the Netherlands).

Assessment of Cytoplasmic Domain Subunit Association by a GST Pull-down Assay-- COS-1 cells were transfected with either pEBG/706-856 or pRK5F/706-856 or cotransfected with pEBG/706-856 and pRK5F/706-856 by the DEAE-dextran method as described previously (6). Plasmids of pEBG and pRK5F were added into transfections in which pEBG/706-856 or pRK5F/706-856 was not included to keep the total amount of DNA in all transfections constant. Two days after transfection, cells were washed once with 10 mM Tris-HCl, pH 7.5, containing 1 mM EDTA (TE buffer) and scraped off from the plates. The washed cells were then resuspended in 1.0 ml of TE butter containing 10% (w/v) sucrose, and complete protease inhibitor mixture (Roche Molecular Biochemicals). The resuspended cells were sonicated on ice twice, each time for 15 s, and then centrifuged at 2,700 rpm in a Sorvall RMC 14 centrifuge (DuPont) at 4 °C for 10 min. Aliquots of postnuclear supernatants were incubated with 50 µl of washed glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) at 4 °C for 2 h. The beads were washed with PBS and then boiled at 95 °C for 3 min. The proteins released from beads were resolved by SDS-15% PAGE followed by Western blotting using mouse monoclonal antibody (mAb) Chessie 8, which recognizes residues 727-732 of the HIV-1 Env protein.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction, Expression, and Sucrose Gradient Centrifugation of MBP Fusion Proteins-- To obtain a clearer picture of the role of the gp41 cytoplasmic domain in the viral life cycle, the autonomous structural and oligomerization domains in this region were explored. The entire cytoplasmic domain-coding sequence from residues 706-856 of the Env of the HXB2 strain was cloned by PCR and fused downstream of the E. coli malE gene in pMal-c2 (Fig. 1). The fusion protein encoded by the malE gene contains 461 residues including MBP, lacZ-alpha , and the residues encoded by the sequence downstream to the lacZ-alpha gene but before the termination codon. As a control for oligomerization, pMal/252-273, which encodes residues 252-273, located in gp120, also known as the epitope of mAb Chessie 13, was also constructed by PCR. The MBP fusion proteins 706-856 and 252-273 contained a total of 542 and 413 residues, respectively. The pMal plasmids were used to transform competent NM522 cells, and the fusion proteins were expressed following isopropylthiogalactopyranoside induction. The apparent molecular masses of MBP, MBP/706-856, and MBP/252-273 were 45, 58, and 42 kDa, respectively, as determined by SDS-7.5% PAGE followed by Western blotting using mAb anti-MBP (36) (data not shown).


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  		Fig. 1.   Schematic representation of recombinant MBP fusion proteins encoding different regions of the HIV-1 gp41 cytoplasmic domain. DNA fragments encompassed by the EcoRI and SalI linkers corresponding to different regions of the gp41 cytoplasmic domain were generated by PCR and inserted into pMal-c2 using the EcoRI and SalI cloning sites in the vector.

To determine whether the cytoplasmic domain assembled as an oligomeric form, the expressed fusion protein MBP/706-856 and molecular mass markers were sedimented in parallel through linear 10-45% sucrose gradient centrifugation. After sedimentation, gradients were fractionated from the bottom, and aliquots of the fractions were resolved by SDS-7.5% PAGE followed by Western blotting using pooled anti-HIV antisera. MBP/706-856 sedimented to fractions 7-17 and with a peak approximately at fractions 11-13 (Fig. 2A, top panel). To assess the apparent molecular mass of MBP/706-856, a method previously described by Wang et al. (37) to determine the oligomeric state of p53 was employed. Fig. 2B is the plot of the apparent molecular mass of the predominant MBP/706-856 form as determined by the sedimentation of the markers. The vertical and horizontal lines relate the sedimentation of the predominant form of MBP/706-856 to its apparent molecular mass of 370 kDa, suggesting that MBP/706-856 assembles as a hexamer. To determine the sedimentation position of the monomeric form of the cytoplasmic domain fusion protein, the cell extract that contained MBP/706-856 was treated with 1% SDS, which is known to effectively break apart interactions of subunits in a protein complex, prior to sucrose gradient analysis. The SDS-dissociated MBP/706-856 predominantly sedimented to fractions 21-23, whereas albumin cosedimented (Fig. 2A, bottom panel). Thus, proteins in fractions 21-23 represented the monomeric form of MBP/706-856.


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  		Fig. 2.   Sucrose gradient centrifugation of MBP fusion proteins. A, density gradient analysis of MBP/706-856. Cell extracts that contained MBP/706-856, treated with (bottom panel) or without (top panel) a final concentration of 1% SDS at 4 °C for 30 min, were analyzed by linear 10-45% sucrose gradient centrifugation followed by Western blotting using anti-HIV. The following standard globular proteins with sedimentation coefficients and molecular masses shown in parentheses were sedimented in parallel: thyroglobulin (19.4 S; 670 kDa), ferritin (17.6 S; 440 kDa), catalase (11.1 S; 232 kDa), beta -amylase (8.9 S; 200 kDa), alcohol dehydrogenase (7.6 S; 150 kDa), albumin (4.5 S; 67 kDa), and carbonic anhydrase (3.2 S; 29 kDa). The peaks of sedimentation of molecular markers with their molecular masses, determined by the Bio-Rad protein assay kit from at least three separate runs, are marked at the top of the gel. B, molecular mass plot of standard markers. A plot of log molecular masses in kDa of molecular markers versus their sedimentation fraction numbers was established according to the method described by Wang et al. (37). The position of MBP/706-856 is related to its apparent molecular mass by horizontal and vertical lines. C, sucrose gradient analysis of MBP/252-273. MBP/252-273 treated with or without SDS was analyzed by sucrose gradient centrifugation followed by Western blotting using Chessie 13 mAb.

To determine whether multimerization of MBP/706-856 was specific to the cytoplasmic domain, the MBP/252-273 fusion protein expressed was subjected to sucrose gradient centrifugation followed by Western blotting using Chessie 13 mAb. MBP/252-273 predominantly sedimented to fractions 21-23 regardless of whether it was treated with (Fig. 2C, bottom panel) or without SDS (Fig. 2C, top panel) prior to sucrose gradient analysis. These observations indicate that multimerization of the cytoplasmic domain fusion protein (Fig. 2A) is specific for MBP fusion to the cytoplasmic domain.

Purification and Characterization of MBP Fusion Proteins-- To study the biochemical characteristics of MBP fusion proteins, the expressed MBP and MBP fusion proteins were purified using an amylose affinity column and then analyzed by SDS-PAGE followed by Coomassie Brillant Blue staining. MBP, MBP/706-856, and MBP/252-273 each predominantly migrated as a major band in gel electrophoresis (Fig. 3A). To rule out the possibility that the multimerization potential of the cytoplasmic domain observed in sucrose gradient analysis (Fig. 2A) was due to its cosedimentation with cellular proteins, the purified MBP/706-856 fusion protein was also analyzed by sucrose gradient centrifugation. Purified MBP/706-856 predominantly sedimented to fractions 11-13 (Fig. 3B), which was consistent with the sedimentation pattern of unpurified MBP/706-856 (Fig. 2A).


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  		Fig. 3.   Characterization of MBP fusion proteins. A, purification of MBP and MBP fusion proteins. Isopropylthiogalactopyranoside-induced MBP and MBP fusion proteins were purified by an amylose affinity column, and the purified proteins (7.5 µg) were analyzed by SDS-PAGE followed by Coomassie Brillant Blue staining. The migration positions of molecular mass markers are also shown. B, sucrose gradient analysis of purified MBP/706-856. 50 µg of purified MBP/706-856 were analyzed by 10-45% sucrose gradient centrifugation. Aliquots of the indicated fractions were analyzed by Western blotting using anti-HIV. C, cross-linking of MBP/706-856. Purified MBP/706-856 (0.15 µg) was reacted with different concentrations of glutaraldehyde as indicated and then analyzed by Western blotting with anti-MBP. The arrowheads indicate the migration of different forms of MBP/706-856 in the gel.

Analysis of the Quaternary Structure of the Cytoplasmic Domain Fusion Protein-- To determine whether disulfide linkages were involved in multimerization of the cytoplasmic domain fusion protein, purified MBP/706-856 was analyzed by denaturing SDS-PAGE in the presence or absence of dithiothreitol followed by Western blotting using anti-MBP. MBP/706-856 migrated to the same position in SDS-PAGE in the presence or absence of dithiothreitol (data not shown), indicating that disulfide bond linkages are not involved in MBP/706-856 multimerization.

To confirm the multimeric structure of MBP/706-856, the purified fusion protein (0.15 µg) was cross-linked with different concentrations of glutaraldehyde and then analyzed by Western blotting using anti-MBP. MBP/706-856 was cross-linked to a dimer and a trimer in the presence of 0.1% of glutaraldehyde (Fig. 3C, lane 3). When the concentration of glutaraldehyde was increased to 0.5%, the amount of the trimeric form became more significant, and a tetrameric form became predominant (Fig. 3C, lane 4). When the concentration of glutaraldehyde was at or above 0.5%, high ordered multimers of MBP/706-856, which migrated near the top of the gel, were also evident.(Fig. 3C, lane 5). Purified MBP was not cross-linked by up to 1% glutaraldehyde (data not shown). These observations indicate the specificity of cross-linking of MBP/706-856 by glutaraldehyde.

Gel Filtration of MBP Fusion Proteins-- To further study the high ordered multimeric structure of MBP/706-856, the purified protein and molecular mass markers were analyzed by fast protein liquid chromatography using a Superose 6 HR column equilibrated with PBS. These molecular mass markers eluted in tight peaks, at times within 1% difference when reanalyzed under the same conditions (data not shown). MBP/706-856 eluted at 15.4 min, whereas blue dextran eluted at 16.4 min (Fig. 4A, top panel). In Western blotting analysis using anti-HIV, MBP/706-856 began to appear at fraction 17 and waned as time elapsed (Fig. 4A, bottom panel). Aliquots of fractionated MBP/706-856 and molecular markers were also assessed for protein concentrations using the Bio-Rad protein detection kit. The fractionation peak of MBP/706-856 was located to fraction 17. Also, the fractionation peak of blue dextran at fraction 18 corresponded to the elution peak of 15.4 min. The difference in UV monitoring and fractionation was due to the time lag between the UV recording and the actual sample collection. When MBP/252-273 was analyzed by gel filtration, the purified 252-273 fusion protein eluted at 33.5 min, which corresponded to the appearance of protein in fractions 35-37 as detected by Western blotting using mAb Chessie 13 (Fig. 4B).


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  		Fig. 4.   Fast protein liquid chromatography analysis of MBP fusion proteins. A, gel filtration chromatography of MBP/706-856. Purified MBP/706-856 was injected into a Superose 6 HR size exclusion column and eluted with PBS. The protein elution pattern as measured by UV absorption at 280 nm is shown (top panel). The elution peaks of the molecular mass standards with their molecular masses, determined from at least three separate runs, are marked at the top of the elution profile with arrows. These molecular markers are: blue dextran (2,000 kDa), thyroglobulin (670 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), albumin (67 kDa), and ovalbumin (43 kDa). Aliquots of fractionated MBP/706-856 were analyzed by Western blotting using anti-HIV (bottom panel). Also, aliquots of fractionated molecular markers were measured for protein concentrations, and the fractionation peaks of the markers with their molecular masses are marked at the top of the bottom panel. B, gel filtration of MBP/252-273. Purified MBP/252-273 was fractionated by a Superose 6 column and eluted with PBS (top panel). The result of Western blotting using Chessie 13 is also shown (bottom panel). C, gel filtration of MBP fusion proteins in the presence of Triton X-100. Purified MBP/706-856 and MBP/252-273 and molecular markers were passed over a Superose 6 column and eluted with PBS containing 0.2% Triton X-100. Aliquots of fractionated MBP/706-856 and MBP/252-273 were analyzed by Western blotting using mAb Chessie 8 and Chessie 13, respectively. Fractionated molecular markers were assessed for protein concentrations, and the fractionation peaks of the markers with their molecular masses are marked by arrowheads at the top of the gel. The molecular mass markers used here are the same as those described in A. D, molecular mass plot of standard markers. A plot of log values of the molecular masses of protein markers versus their elution fractions in the Superose 6 column is shown. The fraction number of the elution peak of MBP/706-856 is related to its apparent molecular mass by horizontal and vertical lines.

To minimize the chance that the 706-856 fusion protein, which is prone to self-assembly, might form a high-ordered structure during gel filtration, purified 706-856 and 252-273 fusion proteins and molecular markers were fractionated and eluted with PBS containing 0.2% Triton X-100. MBP/706-856 eluted to a peak at fraction 30, whereas MBP/252-273 eluted to a peak at fraction 36 (Fig. 4C). Aliquots of protein markers were also assessed for protein concentrations. The peaks of markers eluted with PBS containing Triton X-100 (Fig. 4C) matched the peak numbers eluted in the absence of Triton X-100 (Fig. 4A, bottom panel), indicating that Triton X-100 does not affect the elution of these molecular markers. A standard log plot of molecular mass for the column run under this condition was established from analysis of molecular standards. The vertical and horizontal lines relate the elution of MBP/706-856 to a molecular mass of approximately 340 kDa (Fig. 4D), indicating that the predominant form of the 706-856 fusion protein is a hexamer.

Multimerization Potential of the SIV Cytoplasmic Domain-- To assess whether the potential to assemble into a high-ordered structure is unique to the HIV-1 Env cytoplasmic domain, the multimerization ability of the 164 residues of the SIV cytoplasmic domain fused to the C terminus of MBP was determined by a Superose 6 column and eluted with PBS. The SIV cytoplasmic domain fusion protein eluted at 15.5 min (Fig. 5A, top panel). The apparent molecular mass 59 kDa of the anti-MBP-reactive species observed (Fig. 5A, bottom panel) represented the authentic MBP/SIV cytoplasmic domain fusion protein, as judged by comparing the predicted molecular mass of the cytoplasmic domain of the SIV TM protein and the apparent molecular mass of the species observed. The SIV cytoplasmic domain fusion protein predominantly eluted to fractions 30 and 31 in the presence of 0.2% Triton X-100 (Fig. 5B), corresponding to an apparent molecular mass of 310-340 kDa (calculated from Fig. 4D). This observation indicates that the MBP/SIV cytoplasmic domain fusion protein also assembles as a hexamer.


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  		Fig. 5.   Gel filtration of MBP/SIV cytoplasmic domain fusion protein. A, analysis in the absence of detergent. The entire cytoplasmic domain of SIVmac239 Env from residues 716 to 879 was expressed as a MBP fusion protein, and the amylose affinity-purified protein was applied to a Superose 6 column equilibrated with PBS (top panel). Aliquots of the indicated fractions were resolved by SDS-PAGE followed by Western blotting using anti-MBP (bottom panel). The molecular mass markers used here are the same as those described in Fig. 4A. B, gel filtration in the presence of Triton X-100. The purified protein was injected into the column and eluted with PBS containing 0.2% Triton X-100. Aliquots of fractions were analyzed by Western blotting using anti-MBP. The fractionation peaks of molecular markers are also marked at the top of the gel.

Assessment of in Vivo Cytoplasmic Domain Intersubunit Interactions-- To show the in vivo intermolecular interaction of the cytoplasmic domain, a two-hybrid system that detects protein-protein interactions in mammalian cells was employed. The entire cytoplasmic domain-coding sequence was fused in frame to the C terminus of the Gal4 DNA-binding domain in pM and of the VP16 activation domain in pVP16 to yield pM-Env and pVP16-Env, respectively. 293 cells were cotransfected with these two plasmids and pG5CAT. CAT activity was increased 9.4-fold in cells cotransfected with both pM-Env and pVP16-Env compared with cells cotransfected with the control plasmids pM and pVP16 (Fig. 6A, compare lanes 4 and 1). CAT activity was not significantly increased when pM or pVP16 encoding the entire 1-132 residues of the matrix protein was cotransfected with its partner that encoded the cytoplasmic domain (Fig. 6A, lanes 2 and 3). To examine whether activation of CAT activity by cytoplasmic domain intermolecular interaction was cell type-specific, pM-Env and pVP16-Env were cotransfected with pG5CAT into HeLa cells. CAT activity in cells transfected with pM-Env and pVP16-Env was increased 6.1-fold compared with cells cotransfected with the negative control pairs pM-53 and pVP16-CP (Fig. 6B, compare lanes 4 and 1). To quantitate the effect of the intermolecular interaction mediated by the cytoplasmic domain, pGL3(UASG), which encodes the Gal4-responsive element upstream of a luciferase gene, was cotransfected with pM-Env and pVP16-Env. A 10-fold increase in luciferase activity was observed in cells transfected with pM-Env and pVP16-Env compared with cells transfected with the two-hybrid plasmids in which the cytoplasmic domain was fused to only one of the pM or pVP16 vectors (Fig. 6C).


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  		Fig. 6.   In vivo intermolecular interaction of the cytoplasmic domain. A, detection of cytoplasmic domain subunit-subunit interaction in 293 cells using a CAT reporter vector. 293 cells were cotransfected with 10 µg each of pG5CAT and the two-hybrid plasmids as indicated. Cell lysates were prepared 2 days after transfection and assayed for CAT activity. B, assessment of intermolecular interaction of the cytoplasmic domain in HeLa cells. HeLa cells were cotransfected with pG5CAT and the two-hybrid plasmids as indicated, and CAT activity was assayed. C, quantitation of the cytoplasmic domain intersubunit interaction in 293 cells using a luciferase reporter gene. 293 cells were cotransfected with 10 µg each of pGL3(UASG) along with the hybrid plasmids as indicated, and luciferase activity was assayed. Results from at least three individual experiments were averaged, and standard deviations were calculated. The transfections marked as "control" used the two-hybrid pair in which the cytoplasmic domain-coding sequence is fused to either the pM or pVP16 plasmid.

To assess whether the cytoplasmic domain expressed in vivo could physically associate into a complex, COS-1 cells were transfected with pEBG/706-856 or pRK5F/706-856 or cotransfected with both pEBG/706-856 and pRK5F/706-856. pEBG/706-856 and pRK5F/706-856 encode the entire cytoplasmic domain fused to the C and N termini of the GST and the FLAG tag, respectively. PBS-washed transfected cells were sonicated in TE hypotonic buffer containing 10% sucrose. Aliquots of postnuclear supernatant from each transfection were analyzed by Western blotting using Chessie 8. The apparent molecular masses encoded by pEBG/706-856 and pRK5F/706-856 were 41 and 16 kDa, respectively (Fig. 7, lanes 2 and 3, respectively). The 706-856/FLAG tag fusion protein expressed alone did not bind to glutathione-Sepharose beads (Fig. 7, lane 7) but did bind to the beads when coexpressed with GST/706-856 (Fig. 7, lane 8).


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  		Fig. 7.   Assessment of cytoplasmic domain intersubunit association by a GST pull-down assay. COS-1 cells were transfected with pEBG/706-856 or pRK5F/706-856 or cotransfected with both pEBG/706-856 and pRK5F/706-856. After transfection, aliquots of postnuclear supernatants (200 µl) were concentrated by addition of five volumes of cold acetone and then analyzed by Western blotting using Chessie 8 (lanes 1-4). Additional aliquots (400 µl) of postnuclear supernatants were incubated with glutathione-Sepharose 4B beads, and the proteins bound to the beads were analyzed by SDS-15% PAGE followed by Western blotting using Chessie 8 (lanes 5-8).

Mapping of Regions That Mediate Cytoplasmic Domain Multimerization-- To determine regions in the cytoplasmic domain crucial for multimerization, the self-assembly potential of a series of pMal mutants encoding deletions from either the N or C terminus of the cytoplasmic domain was examined by gel filtration using the PBS elution system. The N-terminal truncation fusion proteins including 760-856, 789-856, and 805-856 all eluted in the void volume (Fig. 8), indicating that these fusion proteins assemble as multimers. When the C-terminal truncation fusion proteins including 706-844 and 706-813 were analyzed, they all eluted in the void volume (Fig. 8). A fraction of the 706-795 fusion protein eluted into the void volume and another fraction eluted with a peak at fraction 23 (Fig. 8). These observations indicate that these C-terminal deletion fusion proteins assemble as multimers. The 706-775 fusion protein eluted with a peak at fraction 23 (Fig. 8), indicating that this fusion protein assembles as a low-ordered oligomer. In contrast, the 706-752 fusion protein predominantly eluted to fractions 35 to 37 (Fig. 8). These observations indicate that MBP/706-752 forms a monomer and also suggest that the region 753-775 may contain an oligomerization signal.


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  		Fig. 8.   Gel filtration of MBP fusion proteins containing different regions of the cytoplasmic domain. Purified MBP fusion proteins as indicated were passed over a Superose 6 column and eluted with PBS. Aliquots of the fractions as indicted were resolved by SDS-PAGE followed by Western blotting analysis. MBP/760-856, 789-856, 805-856, and 706-813 were analyzed by anti-HIV, whereas MBP/706-844, 706-795, 706-775, and 706-752 were analyzed by Chessie 8.

The Two LLP alpha -Helices Mediate Cytoplasmic Domain Multimerization-- To assess whether the two alpha -helical LLP regions located in the gp41 cytoplasmic domain might mediate multimerization, the purified fusion proteins 816-856 and 760-795, which encompass the LLP-1 and LLP-2, respectively, were analyzed by gel filtration using the PBS elution system. MBP/816-856 eluted in the void volume (Fig. 9, first panel) and MBP/760-795 eluted into fractions 17-29 with two protein peaks: one in the void volume and another at fraction 25 (Fig. 9, third panel). These observations indicate that these two LLP sequences contain sufficient information to convert the monomeric MBP into a high-ordered multimeric complex. To further examine the role of LLP-1 and LLP-2 in multimerization, deletions were made in the C terminus of these two sequences. MBP/816-844 eluted into fractions 35-37 (Fig. 9, second panel). When MBP/760-775 was analyzed, a minor form eluted into fractions 29-31, which represented the low-ordered oligomeric form of the fusion protein (Fig. 9, fourth panel). Also, a major form eluted into fractions 35-37, which represented the monomeric structure of MBP/760-775 (Fig. 9, fourth panel). Collectively, these studies indicate that the two LLP sequences play a crucial role in self-assembly into a high-ordered structure.


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  		Fig. 9.   Gel filtration of MBP fusion proteins containing LLP sequences. Purified fusion proteins were passed through a Superose 6 column and eluted by PBS. Aliquots of samples in the fractions as indicated were analyzed by Western blotting using anti-MBP.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Extensive studies have been conducted to determine the oligomeric state of the HIV-1 Env, and reported results are conflicting. Evidence has been presented to suggest that the Env gp160 precursor forms a dimer shortly after its synthesis and during transport to the cell surface (1, 3, 38). However, studies involving chemical cross-linking of viral gp41 or Env proteins expressed in mammalian cells showed that the Env assembles as a tetramer (39-42). A trimeric form of HIV-1 Env has also been reported (43, 44). Circular dichroism and sedimentation analysis of a synthetic peptide corresponding to the leucine zipper-like motif show a tetrameric form (45). The leucine zipper-like motif also possesses the ability to convert monomeric MBP and protein A to become a tetramer or trimer when fused to these otherwise monomeric proteins (46, 47). Synthetic peptides or recombinant proteins representing the N-terminal alpha -helix forms a stable trimeric coiled-coil heterodimeric structure when they are complexed with the C-terminal alpha -helical peptide (9-11, 48, 49).

In the present study, we analyzed the multimerization potential of the HIV-1 gp41 cytoplasmic domain using bacterial and eucaryotic expression systems. The expressed MBP-cytoplasmic domain fusion protein assembled into a soluble high- ordered structure, as judged by sucrose gradient centrifugation, chemical cross-linking, and gel filtration (Figs. 2A, 3B, 3C, 4A, and 4C). The in vivo intermolecular interaction of the cytoplasmic domain was demonstrated by a mammalian two-hybrid assay (Fig. 6). Using a GST pull-down assay, we also found that the cytoplasmic domain assembles as a multimer in eucaryotic cells (Fig. 7).

MBP/706-856 is predominantly cross-linked at either a high protein concentration (1.3 µg; data not shown) or a low protein concentration (0.15 µg; Fig. 3C) to a tetrameric form and also structures higher ordered than the tetrameric form. The lower efficiency of recovery of multimers higher ordered than the tetramer could be due to the lower efficiency in protein transfer to nitrocellulose membranes and protein cross-linking of high molecular masses compared with low molecular masses. The predominant form of MBP/706-856 sediments as a hexamer in sucrose gradient centrifugation (Fig. 2, A and B). Because MBP assumes a spherical structure in solution (50), it would be expected that all cytoplasmic domain fusion proteins would also assume a spherical shape. As determined by gel filtration in the presence of 0.2% Triton X-100, MBP/706-856 assembles as a hexamer (Fig. 4, C and D). Consistent with this observation, the SIV Env cytoplasmic domain fusion protein also assembles into a hexamer in the presence of Triton X-100 (Fig. 5B). In previous determinations of the oligomeric structure of the HIV Env protein, up to 1% of Triton X-100 was used to lyse HIV-1 Env expressing cells and was included in buffer to prepare sucrose gradients (2, 4, 6). These studies indicate that Triton X-100 at the concentration used in our study (0.2%) would not affect the native oligomeric structures of MBP/706-856 and MBP/SIV716-879. Our preliminary results suggest that an HIV-1 Env cytoplasmic domain fragment expressed by a eucaryotic expression system tends to associate with cellular membranes.2 Thus, determination of MBP fusion proteins in the presence of Triton X-100 gives a reasonable estimate to the multimerization state of MBP/706-856 and MBP/SIV716-879, which may more accurately reflect the biological oligomeric state of the cytoplasmic domain in the membrane-associated HIV-1 Env protein than the estimate determined in aqueous solution.

The oligomerization state of peptides capable of binding to membranes may be altered by changes in the environment. A 33-residue identical to the extended fusion domain of HIV-1 Env dissociates from a higher oligomer to a lower oligomer upon binding to membranes (51). A peptide corresponding to residues 789-815, a segment with a leucine zipper-like 4-3 heptad repeat sequence positioned between the two LLP motifs, penetrates deeply into the hydrophobic milieu of the phospholipid membranes (52). This peptide forms an oligomeric structure in solution but dissociates into a monomer upon binding to phospholipid membranes (52). Our results showed that in a membrane-mimetic environment the recombinant MBP/706-856 and MBP/SIV716-879 fusion proteins form a hexamer. A comparison of the elution profiles of molecular markers eluted in the presence and absence of Triton X-100 showed that Triton X-100 has no effect on the elution of these molecular standards, including dimeric thyroglobulin and tetrameric catalase (compare Fig. 4, A, bottom panel, and C). Thus, micelle formation in the presence of Triton X-100 does not seem to have an effect on the molecular shapes of these proteins. The elution of MBP/706-856 and MBP/SIV Env cytoplasmic domain fusion proteins into the void volume in the absence of the detergent during gel filtration (Figs. 4A and 5A) indicates that the cytoplasmic domains of HIV-1 and SIV have a strong tendency to self-assemble as a structure even higher ordered than a membrane-mimetic hexameric structure. The formation of a high-ordered multimer in aqueous solution is an intrinsic property of the cytoplasmic domain and is not due to high protein concentration-induced aggregation. This notion is supported by the observation that MBP/706-856 elutes into the void volume at both 0.5 mg/ml and 50 ng/ml in gel filtration (data not shown).

Because elution in the absence of Triton X-100 gave better separation of the multimeric MBP/706-856 form from the low range molecular markers than elution in the presence of the detergent (Fig. 4, compare A with C), elution in the absence of Triton X-100 was employed to determine the multimerization potential of the gp41 cytoplasmic tail subdomains. Except for MBP/706-752, which predominantly eluted to fractions 35-37, all of the N- and C-terminal deletion fusion proteins eluted in the void volume or to fractions corresponding to multimeric forms under PBS elution conditions (Fig. 8). In addition, deletions in the C terminus of LLP-1 and LLP-2 significantly affected the ability of LLP-1 and LLP-2 sequences to self-assemble into a multimer (Fig. 9). The differential elution patterns of fusion proteins in gel filtration thus reflect the differential multimerization abilities of these cytoplasmic domain sequences. Amphipathic alpha -helices have been characterized as structure motifs important for protein-protein interactions (53-56). However, not all of the amphipathic alpha -helices are able to self-assemble as multimers. Peptides representing the C-helix located in the ectodomain of gp41 do not form a coiled-coil structure in solution but form a hetero-oligomer with the N-peptides when the C- and N-peptides are mixed together (48, 57). Our findings thus reveal a unique feature of the two LLP motifs located in the HIV-1 gp41 cytoplasmic domain in mediating self-assembly.

The HIV-1 LLP-1 peptides bind strongly to planar membranes, resulting in an increase in bilayer conductance and a decrease in bilayer stability (58). The LLP-1 peptide can also alter the ionic permeability of Xenopus laevis oocytes (59). Thus, the toxic characteristic of LLPs has been proposed to result from the insertion of multimeric complexes of peptides into procaryotic or eucaryotic membranes, forming hydrophilic pores and leading to osmotic disintegrity (22, 60). The finding that the gp41 cytoplasmic domain possesses the potential to self-assemble into a multimer may provide better insight into the structural role of the cytoplasmic domain in gp41-induced cytopathicity.

Previously, we reported that a cytoplasmic domain truncated Env mutant is able to dominantly interfere with wt virus infectivity by inclusion of the mutant Env into the wt Env complex (27). A deletion of 12 amino acids from the C terminus of the gp41 cytoplasmic domain, mutant TM844, results in virus replication with a strikingly slower kinetic than that of the wt virus (16). However, the fusion ability of this mutant is not significantly affected compared with that of the wt Env (16). Also, assembly and release of virions and incorporation of TM844 mutant Env into mature virions are normal compared with these processes in the wt virus (16). Previously, we showed that this deletion Env mutant is able to trans-dominantly interfere with wt Env-mediated virus infectivity (28). In the present study, we found that the region between residues 816 and 844 is unable to self-associate (Fig. 8). These results point out the possibility that cytoplasmic domain multimerization may play a crucial role in a step post-gp41 ectodomain-mediated membrane fusion.

Previous HIV-1 Env truncation studies failed to denote the potential role of gp41 cytoplasmic domain in multimerization (3, 4). The reason for this failure is that in addition to the cytoplasmic domain, which possesses the multimerization potential, the gp41 ectodomain also contains N- and C-terminal alpha  helices which themselves are able to assemble into a six-stranded coiled-coil (9-11, 48, 49). Thus, deletions of the cytoplasmic domain do not affect multimerization of the C-terminal truncated Env proteins. To the best of our knowledge, the present study is the first to demonstrate that the cytoplasmic domain of viral Env glycoproteins is sufficient to mediate self-assembly into a high ordered multimeric structure. The results of our study may provide insight into the potential structural role of the HIV-1 gp41 cytoplasmic domain in postbinding, pore formation, and cytopathogenicity, and thus may help elucidate the process of Env-mediated virus-cell fusion.

    ACKNOWLEDGEMENTS

We are indebted to Hai-Yung Hsieh for technical help. We are also grateful to Wan-Jr Syu (National Yang-Ming University, Taipei, Taiwan, Republic of China) for providing a mouse mAb directed against the E. coli MBP, to Jiann-Shiun Lai (Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China) for providing pEBG, and to Eric Hunter (University of Alabama at Birmingham, AL) for providing pSRS/SIV. We also thank Douglas Platt for his careful reading and editing of the manuscript.

    FOOTNOTES

* This work was supported by Department of Health Grants DOH87-TD-1035 and DOH88-TD-1085 and the National Science Council Grant 89-2320-B-001-019) and by grants from the Institute of Biomedical Sciences at Academia Sinica (Taipei, Taiwan, Republic of China).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Div. of Infectious Diseases, Inst. of Biomedical Sciences, Academia Sinica, 128, Section 2, Yen-Chiu-Yuan Road, Taipei 11529, Taiwan, Republic of China. Tel.: 886-2-2652-3933; Fax: 886-2-2785-8847; E-mail: schen@ibms.sinica. edu.tw.

Published, JBC Papers in Press, March 15, 2000, DOI 10.1074/jbc.M000601200

2 S.F. Lee and S. S.-L. Chen, unpublished data.

    ABBREVIATIONS

The abbreviations used are: Env, envelope; TM, transmembrane; HIV-1, human immunodeficiency virus type 1; SIV, simian immunodeficiency virus; LLP, lentiviral lytic peptide; wt, wild-type; MBP, maltose-binding protein; GST, glutathione S-transferase; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; CAT, chloramphenicol acetyltransferase; mAb, monoclonal antibody.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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