1 Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
2 Axis Genetics, Babraham, Cambridge CB2 4AZ, UK
Correspondence
Nigel Dimmock
ndimmock{at}bio.warwick.ac.uk
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ABSTRACT |
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Present address: Department of Immunotherapeutics, GlaxoSmithKline Medicine and Research Centre, Stevenage SG1 2NY, UK.
Present address: Biovation Ltd, Babraham, UK.
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INTRODUCTION |
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There are several antigenic sites within gp120 and gp41 that correlate with virus neutralizing ability (reviewed by Burton, 1997). 669ELDKWA677 is an important cross-clade reactive epitope in the ectodomain of gp41 (
Muster et al., 1993; Sattentau et al., 1995
), while 746ERDRD750 is situated in a supposedly intravirion location within the 731PRGPDRPEGIEEEGGERDRDRS752 peptide (Gallagher et al., 1992
; Kennedy et al., 1986
; Modrow et al., 1987
), although its location inside the virion is controversial (Buratti et al., 1997
; Dalgleish et al., 1988
; Kennedy et al., 1986
; Niedrig et al., 1992
; Sattentau et al., 1995
). If it was accepted that the 731752 peptide contains a neutralizing epitope, this would almost certainly mean that the part of the C-terminal tail containing that epitope was looped back through the viral membrane to the exterior in free virions, or that it was normally hidden and was exposed intermittently. However, only weakly or non-neutralizing monoclonal antibodies (mAbs) have been raised (Dalgleish et al., 1988
; Niedrig et al., 1992
; Vella et al., 1993
), so the function of region 731752 as a neutralization site is regarded with some scepticism. Nonetheless, we show here that one of the ERDRD-specific mAbs neutralized virus in the presence of complement.
The antigenic properties of gp41 region 731752 were first described by Kennedy et al. (1986). The antibody response to this region in infected patients is generally poor (Davis et al., 1990
; Niedrig et al., 1992
; Vella et al., 1991
), although several groups have prepared 731752 peptide-specific neutralizing antisera using antigen-presenting systems (Evans et al., 1989
; McLain et al., 1995
, 1996a
, b
; Newton et al., 1995
) or synthetic peptides (Chanh et al., 1986
). However, this is not always the case (Kalyan et al., 1994
). Indeed, these and other immunological assays suggested that mAbs to the Kennedy peptide might react with HIV-1 particles, although interaction with broken virus or virus after it had attached to cell receptors could not be excluded (Niedrig et al., 1992
). Some of these apparently conflicting data were resolved with the realization that the Kennedy peptide contained, not one, but three epitopes: 734PDRPEG739 (Abacioglu et al., 1994
), 740IEEE743 and 746ERDRD750 (Vella et al., 1993
).
We have recently confirmed the presence of the IEEE and ERDRD epitopes in gp41 using epitope-purified polyclonal antibodies, but only antibody to ERDRD, a conformationally constrained epitope, was neutralizing. IEEE is a linear epitope and its cognate antibodies are non-neutralizing (Buratti et al., 1998). mAb C8 usually recognizes a denatured form of the PDRPEG epitope (Abacioglu et al., 1994
), but also reacts with virions that have escaped neutralization with ERDRD-specific antibody (McLain et al., 2001
). It is non-neutralizing. The IEEE sequence is both immunogenically and antigenically dominant over the ERDRD epitope, and this has provided an explanation for some of the confusion surrounding the immunology of the Kennedy peptide (Cleveland et al., 2000a
).
In this report we present data from a variety of approaches suggesting that a region of gp41, C-terminal to the main transmembrane region, is exposed on the exterior of infectious virions, possibly as a loop supported by two additional transmembrane regions. We therefore suggest that the structure of the HIV-1 gp41 protein should be revisited. Implicit in our findings are previously unsuspected HIV-1 functions mediated through the gp41 tail loop, and a new target for immunological and chemotherapeutic antiviral measures.
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METHODS |
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Antibodies.
Neutralizing antiserum was prepared by immunizing 68-week-old C3H/He-mg mice (H-2k; bred in-house) with CPMV-HIV/1 expressing the HIV-1 gp41 peptide 731PRGPDRPEGIEEEGGERDRDRS752 (McLain et al., 1995). Mice were injected subcutaneously on days 0 and 28 with 10 µg CPMV-HIV/1 or CPMV in aluminium hydroxide (Imject Alum: Pierce & Warriner), and bled on day 42. Other gp41 antibodies used were: mouse mAb 1575 to 740IEEE743, mAbs 1577 and 1583 to 746ERDRD750 (Vella et al., 1993
), mAb C8 to 734PDRPEG739 (Abacioglu et al., 1994
), and the human mAb 2F5 to amino acids 669ELDKWA674 (Muster et al., 1993
). Gp120 antibodies were the human CD4-binding site mAb b12 (Burton et al., 1994
), and the V3 rat mAb ICR41.1i (McKeating et al., 1992
).
Epitope-purified ERDRD-specific (EPES) IgG was prepared by adsorption to and elution from FHV-L1-B, a flock house virus fusion protein that expresses the gp41 sequence GERDRDR in its neutralizing conformation (Buratti et al., 1998). Wild-type FHV protein did not adsorb neutralizing antibody. Purified FHV-L1-B was produced in Escherichia coli as previously described, and immobilized on nitrocellulose (Buratti et al., 1996
). ERDRD-specific IgG was adsorbed and then eluted with glycine buffer solution, pH 2·5. This was predominantly IgG1, IgG2a and IgG2b (Stratagene IsoDetect). IgG was quantified using a solid-phase goat anti-mouse IgG ELISA or by determining the OD280.
Assay of HIV-1 neutralizing antibody.
Neutralization of all HIV-1 strains was measured by inhibition of infectious progeny production, p24 antigen production or syncytium formation using C8166 cells, as indicated in the text (Buratti et al., 1998; McLain & Dimmock, 1994
). Antibody was incubated with 2000 syncytium-forming units (s.f.u.) ml-1 HIV-1 for 1 h at 37 °C, and for complement-mediated neutralization was further incubated with an optimized dilution of guinea pig complement (Gibco BRL) for 1 h at 37 °C. C8166 cells (2x105) were infected for 1 h at 37 °C, washed and incubated for 3 days at 37 °C. Production of infectious progeny at 3 days after infection was determined by infectivity titration. P24 was captured from Empigen (Calbiochem)-treated TCF with solid-phase sheep anti-p24 antiserum (Aalto Bioreagents), and detected with a biotinylated mouse anti-p24 mAb (AIDS Reagent Project) by standard methodology. Syncytium production was determined by counting 50100 syncytia. Neutralization was calculated as the percentage reduction of infectivity due to antibody compared to that of non-neutralized virus.
Western blot of HIV-1 HXBH10 and HIV-1 CT-infected MT4 cells.
Virus or infected cells were boiled in reducing buffer [10 mM Tris pH 7·4, 5 % (w/v) dithiothreitol, 0·2 % (w/v) SDS, 7·5 % (w/v) glycerol and 0·004 % (w/v) bromophenol blue] for 2 min, and electrophoresed on a 1030 % SDS-PAGE gel. Polypeptides were transblotted to nitrocellulose, blocked and incubated with antibody in 10 % (w/v) defatted dried milk in TBS (20 mM Tris, 140 mM NaCl, pH 7·6). Bound antibody was detected with anti-species IgGhorseradish peroxidase (HRP; Bio-Rad) and 3,3'-diaminobenzidine (Sigma).
Binding of ERDRD-specific IgG to virus in solution.
Purified HIV-1 (5x106 s.f.u.) was incubated with 2 µg EPES IgG for 1 h at 37 °C. The virusantibody mix was then layered onto 4·5 ml of 20 % sucrose in TBS, and centrifuged at 155 000 g for 1 h to separate virus or putative virusantibody complexes from free antibody, which remains at the top of the tube (Jackson et al., 1999). The supernatant was carefully removed from the top, avoiding contamination of the virus pellet with free antibody. The pellet was resuspended and assayed for infectivity or the presence of bound antibody. For the latter, virus was heat inactivated in 1 % Empigen at 56 °C for 1 h, and incubated with solid-phase anti-species IgG in TBS containing 0·5 % BSA. Bound primary antibody was detected with biotinylated anti-species IgG, streptavidin-conjugated alkaline phosphatase, and a p-nitrophenyl phosphate substrate. There was no significant cross-reaction between human and mouse IgGs.
Binding of antibodies to paraformaldehyde-fixed virus.
Purified HIV-1 was fixed with 2 % paraformaldehyde and 3x104 s.f.u. captured using solid phase-bound human mAb b12 (0·1 µg per well). After washing, virus was incubated with a non-human gp120- or gp41-specific antibody, using normal IgG as a control. Binding of IgG was detected using species-specific anti-IgG as described above.
Removal of virus surface proteins with proteases.
Purified virus was incubated with 20 mg trypsin (Sigma) ml-1 for 30 min at 37 °C. The reaction was stopped by the addition of 20 % (v/v) foetal calf serum. Alternatively, virus was incubated with 10 µg thermolysin (Calbiochem) ml-1 for 1 h on ice, before terminating the reaction with 50 mM EDTA. Digested virus particles and free protein were separated by centrifugation. P24 antigen assay showed there was no loss due to protease digestion. Virus for electron microscopy was stained with 4 % sodium silicotungstate, pH 6·9.
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RESULTS |
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In the experiment shown in Fig. 3, purified virus alone, or virus mixed with various amounts of EPES IgG, was incubated for 1 h at 37 °C. Putative virusantibody complexes and virus were then separated from free antibody by layering over sucrose and centrifuging. The virus pellet was then resuspended and the extent of neutralization determined. Any unattached virus/virusantibody complexes or free antibody were removed by washing cells after a 1 h adsorption period at 37 °C. We found that all antibody concentrations tested were neutralizing, and that the extent of neutralization was only slightly less than virus neutralized by the standard procedure (virus+antibody with no centrifugation) (Fig. 3
). For example, at the highest antibody concentration there was 82 % neutralization of centrifuged virusantibody complexes and 88 % neutralization by the standard procedure. This demonstrated that the ERDRD epitope was exposed on the outer surface of free virus particles and was capable of binding its cognate neutralizing antibody.
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The gp41 ERDRD epitope is exposed on virions at 4, 21 and 37 °C
The previous experiment was repeated using an ELISA format to determine if the exposure of the ERDRD epitope was temperature-dependent. Virus and EPES IgG were incubated together, and putative virusantibody complexes separated from free antibody by centrifugation through a sucrose spacer as described above. Similar amounts of ERDRD antibody were bound at 4 °C (120 %), 21 °C (133 %) and 37 °C (100 %). Thus the ERDRD region is exposed over a wide temperature range.
The gp41 ERDRD epitope on virus particles is sensitive to protease digestion
If the ERDRD epitope is exposed on the surface of virus as the data above suggest, it should be sensitive to digestion by proteases. Virtually complete removal of surface proteins from purified virus, as judged by PAGE analysis, was achieved by incubation with trypsin or thermolysin. Electron microscopy (Fig. 4a) showed that trypsin-digested particles were smooth and disaggregated whereas control particles had visible spikes/protrusions, and also tended to clump together. Virus digested in this way was then re-banded, and amounts of protease-treated virus and non-protease-treated control virus standardized by ELISA according to their internal virion p24 antigen content. These were then incubated with EPES IgG and virus/virusantibody complexes separated from free antibody by centrifugation through a sucrose spacer. Antibody bound to virions was detected by ELISA using solid-phase anti-mouse IgG. MAb 2F5, which recognizes an epitope in the ectodomain of gp41, was used in parallel. Digestion of HIV-1 with trypsin reduced the binding of EPES IgG to virus by 93·5 % and of mAb 2F5 by a similar amount (81 %) (Fig. 4b
). In an experiment with another protease, digestion with thermolysin destroyed all reactivity of mAb C8 to 734PDRPEG739, an upstream epitope, in Western blots (Fig. 4c
). The internal virion p24 protein was unaffected by protease. Data are consistent with the exposure of the PDRPEG and ERDRD epitopes on the surface of intact virions.
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Binding of monoclonal and polyclonal ERDRD-specific antibodies to infectious virus.
Antibody was reacted with purified virus, and putative virusantibody complexes separated from free antibody by centrifugation through sucrose. Virus was then disrupted with detergent and bound IgG captured and assayed. Fig. 5(a) shows that virions bound 7-fold more mAb 1583 (ERDRD-specific) than normal mouse IgG, and 12-fold more EPES IgG. Binding of mAb 2F5 to the ELDKWA epitope in the main ectodomain of gp41 (Muster et al., 1993
) is also shown. The integrity of virions was evidenced by their failure to bind p17-specific and p24-specific antibodies (data not shown).
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DISCUSSION |
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Immunological implications
One of the stumbling blocks to the appreciation that the Kennedy sequence of gp41 is part of an external loop structure was conflicting evidence about its ability to stimulate neutralizing antibodies. While we have consistently found that the gp41 731752 peptide, expressed on the surface of chimeric CPMV-HIV/1 particle elicited neutralizing antibodies in mice (Durrani et al., 1998; McInerney et al., 1999
; McLain et al., 1995
, 1996a
, b
), other formats have been less successful (Chanh et al., 1986
; Evans et al., 1989
), and only weakly or non-neutralizing mAbs have been raised (Dalgleish et al., 1988
; Evans et al., 1989
; Niedrig et al., 1992
; Pincus et al., 1993
; Vella et al., 1993
). Others have been unable to raise neutralizing polyclonal antibodies (Newton et al., 1995
; Pincus et al., 1993
), possibly because the ERDRD epitope was presented in a non-neutralizing conformation. However, the situation is more complex. In the first place, the Kennedy sequence in wild-type virions contains two functional epitopes, the linear IEEE epitope that elicits non-neutralizing antibodies, and the conformational ERDRD epitope that elicits neutralizing epitopes (Buratti et al., 1998
; Vella et al., 1993
). A third epitope, PDRPEG, is normally only detected by mAb C8 in Western blots (Abacioglu et al., 1994
), although it reacts with neutralization escape mutants selected with ERDRD-specific antibody (McLain et al., 2001
). Secondly, the IEEE epitope and upstream sequence expressed in the CPMV-HIV/1 chimera suppresses the production of neutralizing ERDRD-specific IgG (Cleveland et al., 2000a
). In rabbits, the immunogenic dominance of the IEEE epitope was so strong that no neutralizing or ERDRD-specific antibodies were detected at any time in animals immunized repeatedly with adjuvanted CPMV-HIV/1. However, rabbits immunized with another chimera, CPMV-HIV/29, that expresses a truncated form of the 731752 peptide, GERDRDR, readily made ERDRD-specific neutralizing IgG. The situation was similar, but less extreme, in mice. Thirdly, IEEE-specific antibodies were antigenically dominant over ERDRD-specific antibodies, preventing them binding to their epitope, and blocking neutralization of HIV-1 (Cleveland et al., 2000a
). Thus much of the conflicting data in the literature can be reconciled by our recent findings.
Structural implications
Data in this report suggest that the currently held view that the C-terminal tail of gp41 is contained entirely within the HIV-1 virion (e.g. Levy, 1998) is mistaken, and that a loop of gp41, C-terminal to the current transmembrane region, is exposed on the outside of the virion. Alternatively, the tails of only some virion envelope proteins may be exposed, while others are entirely intravirion. To have an exposed C-terminal loop, gp41 must cross the virion lipid bilayer an even number of times before the start of the exposed section. In addition, data showing that the gp41 tail interacts with the p17 MA protein suggest that a portion of tail is inside the virion (Bukrinskaya & Sharova, 1990
; Cosson, 1996
; Dorfman et al., 1994
; Freed & Martin, 1995a
, b
, 1996
; Mammano et al., 1995
; Murakami & Freed, 2000a
; Wyma et al., 2000
). Supporting evidence comes from the failure of antibodies to regions 799817 and 844863 to react with virions, suggesting that these sequences are inside the virion (unpublished data). If this is the case, gp41 must cross the lipid bilayer of the virion at least once more, making a minimum of three transmembrane regions, as we suggested earlier (McLain et al., 2001
). This proposal is consistent with secondary structural predictions. Using standard algorithms and a moving window of 7 amino acid residues, we found, as others have done before us, that the current transmembrane region (691712) has a significant bimodal hydropathicity (Eisenberg et al., 1982
; Kyte & Doolittle, 1982
). Thus in region 691712 there could be not one, but two short transmembrane spanning domains. There is also a peak of hydropathicity at 755763 and another overlapping LLP-2 (779797), which is known to have amphipathic properties (Venable et al., 1989
). The first two transmembrane regions (tm 1 and tm 2) identified above have 10 residues and have potential
-helical structure. If so they could traverse the membrane if (a) residues were drawn up into the lipid bilayer, (b) the bilayer narrowed locally, (c) the proposed transmembrane regions took up a more extended structure or (d) the transmembrane regions had a
-strand structure, which is a possibility according to total
-strand and antiparallel
-strand potential predictions (Lifson & Sander, 1979
). These also divide the current transmembrane region of HIV-1 into two domains. Such transmembrane
-strand structure is precedented by bacterial porin proteins that require a minimum of seven residues to cross the hydrophobic core of the membrane (Schirmer, 1998
; Schirmer & Cowan, 1993
).
Fig. 6 summarizes data showing that epitopes 734PDRPEG739, 740IEEE743 and 746ERDRD750 are exposed on the outside of virions. Since these epitopes appear to be situated in an external hydrophilic loop structure, there must be three (Fig. 7
a), or possibly four (Fig. 7b
), transmembrane domains to take the gp41 C-terminal tail out of the virion and then back again. The exact size of the loop is not known, as it depends on the number of residues that are exposed between the proposed tm 2 region and the 734PDRPEG739 epitope, and the position of tm 3 on the C-terminal side of the loop. The transmembrane domains in Fig. 7(a)
could be either short
-helices or
-strands as discussed above. We have not specified the tm 3 sequence in Fig. 7(a)
as there are at least two candidates (see above). However it is possible, as shown in Fig. 7(b)
, that tm 3 and tm 4 are derived from LLP-2 and LLP-1 respectively, and are
-helices of about 20 residues. These have been suggested to interact in an anti-parallel charge-complementary fashion (Venable et al., 1989
). Tm 4 would take the extreme C-terminal region back into the membrane and form an intravirion, closed loop.
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What is the likely function of the gp41 minor ectodomain? One approach is to determine the specific virus life-cycle event that is inhibited when ERDRD-specific IgG neutralizes infectivity. So far we know that this antibody does not block attachment of virus to the target cell (Cleveland et al., 2000b), but does inhibit virus-mediated cellcell fusion (L. Cheung & N. J. Dimmock, unpublished data). This fusion-inhibition had a similar dose-response to neutralization, suggesting cause and effect. Inhibition of fusion implies that the gp41 tail has a role in this process, but what this is remains to be determined. Much more experimentation is needed to reveal the complete structure and function of the gp41 C-terminal tail.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Arroyo, J., Boceta, M., González, M. E., Michel, M. & Carrasco, L. (1995). Membrane permeabilization by different regions of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41. J Virol 69, 40954102.[Abstract]
Berlioz-Torrent, C., Shacklett, B. L., Erdtmann, L., Delamarre, L., Bouchaert, I., Sonigo, P., Dokhelar, M. C. & Benarous, R. (1999). Interactions of the cytoplasmic domains of human and simian retroviral transmembrane proteins with components of the clathrin adapter complexes modulate intracellular and cell surface expression of envelope glycoprotein. J Virol 73, 13501361.
Blacklow, S. C., Lu, M. & Kim, P. S. (1995). A trimeric subdomain of the simian immunodeficiency virus envelope glycoprotein. Biochemistry 34, 1495514962.[Medline]
Bukrinskaya, A. G. & Sharova, N. K. (1990). Unusual features of protein interaction in human immunodeficiency virus (HIV) virions. Arch Virol 110, 287293.[Medline]
Buratti, E., Tisminetzky, S. G., Scodeller, E. S. & Baralle, F. E. (1996). Conformational display of two neutralizing epitopes of HIV-1 gp41 in the flock house virus capsid protein. J Immunol Methods 197, 718.[CrossRef][Medline]
Buratti, E., Tisminetzky, S. G., D'Agaro, P. & Baralle, F. E. (1997). A neutralizing monoclonal antibody previously mapped exclusively on human immunodeficiency virus type 1 gp41 recognizes an epitope in p17 sharing the core sequence IEEE. J Virol 71, 24572462.[Abstract]
Buratti, E., McLain, L., Tisminetzky, S. G., Cleveland, S. M., Dimmock, N. J. & Baralle, F. E. (1998). The neutralizing antibody response against a conserved region of HIV-1 gp41 (amino acid residues 731752) is uniquely directed against a conformational epitope. J Gen Virol 79, 27092716.[Abstract]
Burton, D. R. (1997). A vaccine for HIV type 1: the antibody perspective. Proc Natl Acad Sci U S A 94, 1001810023.
Burton, D. R., Pyati, J., Koduri, R. & 15 other authors (1994). Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science 266, 10241027.[Medline]
Celma, C. C. P., Manrique, J. M., Affranchino, J. L., Hunter, E. & González, S. A. (2001). Domains in the simian immunodeficiency virus gp41 cytoplasmic tail required for envelope incorporation into particles. Virology 283, 253261.[CrossRef][Medline]
Chan, D. C., Fass, D., Berger, J. & Kim, P. S. (1997). Core structure of gp41 from the HIV envelope glycoprotein. Cell 89, 263273.[Medline]
Chanh, T. C., Dreesman, G., Kanda, P., Linette, G. P., Sparrow, J. T., Ho, D. D. & Kennedy, R. C. (1986). Induction of anti-HIV neutralizing antibodies by synthetic peptides. EMBO J 5, 30653071.[Abstract]
Chernomordik, L., Chanturiya, A. N., Suss-Toby, E., Nora, E. & Zimmerberg, J. (1994). An amphipathic peptide from the C-terminal region of the human immunodeficiency virus envelope glycoprotein causes pore formation in membranes. J Virol 68, 71157123.[Abstract]
Cleveland, S. M., Buratti, E., Jones, T. D., North, P., Baralle, F. E., McLain, L., McInerney, T. L., Durrani, Z. & Dimmock, N. J. (2000a). Immunogenic and antigenic dominance of a non-neutralizing epitope over a highly conserved neutralizing epitope in the gp41 transmembrane envelope glycoprotein of HIV-1: its deletion leads to a strong neutralizing antibody response. Virology 266, 6678.[CrossRef][Medline]
Cleveland, S. M., Jones, T. D. & Dimmock, N. J. (2000b). Properties of a neutralizing antibody that recognizes a conformational form of epitope ERDRD in the C-terminal tail of human immunodeficiency virus type 1. J Gen Virol 81, 12511260.
Comardelle, A. M., Norris, C. H., Plymale, D. R. & 8 other authors (1997). A synthetic peptide corresponding to the carboxyterminus of human immunodeficiency virus type 1 transmembrane protein induces alterations in the ionic permeability of Xenopus laevis oocytes. AIDS Res Hum Retrovir 13, 15251532.[Medline]
Cosson, P. (1996). Direct interaction between the envelope and matrix proteins of HIV-1. EMBO J 15, 57835788.[Abstract]
Dalgleish, A. G., Chanh, T. C., Kennedy, R. C., Kanda, P., Clapham, P. R. & Weiss, R. A. (1988). Neutralization of diverse strains of HIV-1 by monoclonal antibodies raised against a gp41 synthetic peptide. Virology 165, 209215.[CrossRef][Medline]
Davis, D., Chaudri, B., Stephens, D. M., Carne, C. A., Willers, C. & Lachmann, P. J. (1990). The immunodominance of epitopes within the transmembrane protein (gp41) of human immunodeficiency virus type 1 may be related by the host's previous exposure to similar epitopes on unrelated antigens. J Gen Virol 71, 19751983.[Abstract]
Di Fiore, P. P. & Gill, G. N. (1999). Endocytosis and mitogenic signalling. Curr Opin Cell Biol 11, 483488.[CrossRef][Medline]
Dorfman, T., Mammano, F., Haseltine, W. A. & Göttlinger, H. G. (1994). Role of the matrix protein in the virion association of the human immunodeficiency virus type 1 envelope protein. J Virol 68, 16891696.[Abstract]
Durrani, Z., McInerney, T. L., McLain, L., Jones, T., Bellaby, T., Brennan, F. R. & Dimmock, N. J. (1998). Intranasal immunization with a plant virus expressing a peptide from HIV-1 gp41 stimulates better mucosal and systemic HIV-1-specific IgA and IgG than oral immunization. J Immunol Methods 220, 93103.[CrossRef][Medline]
Edwards, T. G., Hoffman, T. L., Baribaud, F. & 7 other authors (2001). Relationships between CD4 independence, neutralization sensitivity, and exposure of a CD4-induced epitope in a human immunodeficiency virus type 1 envelope protein. J Virol 75, 52305239.
Edwards, T. G., Wyss, S., Reeves, J. D., Zolla-Pazner, S., Hoxie, J. A., Doms, R. W. & Baribaud, F. (2002). Truncation of the cytoplasmic domain induces exposure of conserved regions in the ectodomain of human immunodeficiency virus type 1 envelope protein. J Virol 76, 26832691.
Eisenberg, D. & Wesson, M. (1990). The most highly amphiphilic alpha-helices include two amino acid segments in human immunodeficiency virus glycoprotein 41. Biopolymers 29, 171177.[Medline]
Eisenberg, D., Weiss, R. M. & Terwilliger, T. C. (1982). The helical hydrophobic moment: a measure of the amphiphilicity of a helix. Nature 299, 371374.[Medline]
Evans, D. J., McKeating, J. A., Meredith, J. M., Burke, K. L., Katrak, K., Ferguson, M., Minor, P. D., Weiss, R. A. & Almond, J. W. (1989). An engineered poliovirus chimaera elicits broadly reactive HIV-1 neutralizing antibodies. Nature 339, 385388.[CrossRef][Medline]
Freed, E. O. & Martin, M. A. (1995a). The role of the human immunodeficiency virus type 1 envelope glycoproteins in virus infection. J Biol Chem 270, 2388323886.
Freed, E. O. & Martin, M. A. (1995b). Virion incorporation of envelope glycoproteins with long but not short cytoplasmic tails is blocked by specific single amino acid substitutions in the human immunodeficiency virus type 1 matrix. J Virol 69, 19841989.[Abstract]
Freed, E. O. & Martin, M. A. (1996). Domains on the human immunodeficiency virus type 1 matrix and gp41 cytoplasmic tail required for envelope incorporation into virions. J Virol 70, 341351.[Abstract]
Fultz, P. N., Vance, P. J., Endres, M. J. & 8 other authors (2001). In vivo attenuation of simian immunodeficiency virus by disruption of a tyrosine-dependent sorting signal in the envelope glycoprotein cytoplasmic tail. J Virol 75, 278291.
Gallagher, W. R., Henderson, L. A., Fermin, C. & 7 other authors (1992). Membrane interactions of human immunodeficiency virus: attachment, fusion, and cytopathology. Adv Membrane Fluidity 6, 113142.
Gawrisch, K., Han, K.-H., Yang, J., Bergelson, L. D. & Ferretti, J. A. (1993). Interaction of peptide fragment 828848 of the envelope glycoprotein of human immunodeficiency virus type 1 with lipid bilayers. Biochemistry 32, 31123118.[Medline]
Haffar, O. K., Dowbenko, D. & Berman, P. W. (1988). Topogenic analysis of the HIV-1 envelope glycoprotein, gp160, in microsomal membranes. J Cell Biol 107, 16771687.[Abstract]
Haffar, O. K., Dowbenko, D. & Berman, P. W. (1991). The cytoplasmic tail of HIV-1 gp160 contains regions that associate with cellular membranes. Virology 180, 439441.[Medline]
Heilker, R., Spiess, M. & Crottet, P. (1999). Recognition of sorting signals by clathrin adapters. Bioessays 7, 558567.[CrossRef]
Iwatani, Y., Ueno, T., Nishimura, A., Zhang, X., Hattori, T., Ishimoto, A., Ito, M. & Sakai, H. (2001). Modification of virus infectivity by cytoplasmic tail of HIV-1 TM protein. Virus Res 74, 7587.[CrossRef][Medline]
Jackson, N. A. C., Levi, M., Wahren, B. & Dimmock, N. J. (1999). Mechanism of action of a 17 amino acid microantibody specific for the V3 loop that neutralizes free HIV-1 virions. J Gen Virol 80, 225236.[Abstract]
Kalyan, N. K., Lee, S.-G., Wilhelm, J. & 7 other authors (1994). Immunogenicity of recombinant influenza virus haemagglutinin carrying peptides from the envelope protein of human immunodeficiency virus type 1. Vaccine 12, 753760.[CrossRef][Medline]
Kennedy, R. C., Henkel, R. D., Pauletti, D., Allan, J. S., Lee, T. H., Essex, M. & Dreesman, G. R. (1986). Antiserum to a synthetic peptide recognizes the HTLV-III envelope glycoprotein. Science 231, 15561559.[Medline]
Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157, 105132.[Medline]
Levy, J. A. (1998). HIV and the Pathogenesis of AIDS, 2nd edn. Herndon, VA: ASM Press.
Li, Q., Yafal, A. G., Lee, Y. M.-H., Hogle, J. & Chow, M. (1994). Poliovirus neutralization by antibodies to internal epitopes of VP4 and VP1 results from reversible exposure of these sequences at physiological temperature. J Virol 68, 39653970.[Abstract]
Lifson, S. & Sander, M. (1979). Antiparallel and parallel -strands differ in amino acid residue preferences. Nature 282, 109111.[Medline]
Lu, M., Blacklow, S. C. & Kim, P. S. (1995). A trimeric structural domain of the HIV-1 transmembrane glycoprotein. Nat Struct Biol 2, 10741082.
McInerney, T. L., Brennan, F. R., Jones, T. D. & Dimmock, N. J. (1999). Analysis of the ability of five adjuvants to enhance immune responses to a chimeric plant virus displaying a HIV-1 peptide. Vaccine 17, 13591368.[CrossRef][Medline]
McKeating, J. A., Cordell, J. A., Dean, C. J. & Balfe, P. (1992). Synergistic interaction between ligands binding to the CD4 binding site and V3 domain of human immunodeficiency virus type 1 gp120. Virology 191, 732742.[Medline]
McLain, L. & Dimmock, N. J. (1994). Single- and multi-hit kinetics of immunoglobulin G neutralization of human immunodeficiency virus type 1 by monoclonal antibodies. J Gen Virol 75, 14571460.[Abstract]
McLain, L., Porta, C., Lomonossoff, G. P., Durrani, Z. & Dimmock, N. J. (1995). Human immunodeficiency virus type 1 neutralizing antibodies raised to a gp41 peptide expressed on the surface of a plant virus. AIDS Res Hum Retrovir 11, 327334.[Medline]
McLain, L., Durrani, Z., Wisniewski, L. A., Porta, C., Lomonossoff, G. P. & Dimmock, N. J. (1996a). A plant virusHIV-1 chimera stimulates antibody that neutralizes HIV-1. In Vaccine 96, pp. 311316. Edited by F. Brown, D. R. Burton, J. Collier, J. Mekalonos & E. Norrby. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
McLain, L., Durrani, Z., Wisniewski, L. A., Porta, C., Lomonossoff, G. P. & Dimmock, N. J. (1996b). Stimulation of neutralizing antibodies to human immunodeficiency virus type 1 in three strains of mice immunized with a 22-mer amino acid peptide expressed on the surface of a plant virus. Vaccine 14, 799810.[CrossRef][Medline]
McLain, L., Brown, J. L., Cheung, L., Reading, S. A., Parry, C., Jones, T. D., Cleveland, S. M. & Dimmock, N. J. (2001). Different effects of a single amino acid substitution on three epitopes in the gp41 C-terminal loop of a neutralizing antibody escape mutant of human immunodeficiency virus type 1. Arch Virol 146, 157166.[CrossRef][Medline]
Mammano, F., Kondo, E., Sodroski, J., Bukovsky, A. & Göttlinger, H. G. (1995). Rescue of human immunodeficiency virus type 1 matrix protein mutants by envelope glycoproteins with short cytoplasmic tails. J Virol 69, 38243830.[Abstract]
Manrique, J. M., Celma, C. C. P., Affranchino, J. L., Hunter, E. & González, S. A. (2001). Small variations in the length of the cytoplasmic domain of the simian immunodeficiency virus transmembrane protein drastically affect envelope incorporation and virus entry. AIDS Res Hum Retrovir 17, 16151624.[CrossRef][Medline]
Miller, M. A., Garry, R. F., Jaynes, G. J. & Montelaro, R. C. (1991). A structural correlation between lentivirus transmembrane proteins and natural cytolytic peptides. AIDS Res Hum Retrovir 7, 511519.[Medline]
Miller, M. A., Cloyd, M. W., Liebmann, J., Rinaldo, C. R., Islam, K. R., Wang, S. Z. S., Mietzner, T. A. & Montelaro, R. C. (1993). Alterations in cell membrane permeability by the lentiviral peptide (LLP-1) of HIV-1 transmembrane protein. Virology 196, 89100.[CrossRef][Medline]
Modrow, S., Hahn, B. H., Shaw, G. M., Gallo, R. C., Wong-Staal, F. & Wolf, H. (1987). Computer assisted analysis of envelope protein sequences of seven human immunodeficiency virus isolates: prediction of antigenic epitopes in conserved and variable regions. J Virol 61, 570578.[Medline]
Mulligan, M. J., Yamshchikov, G. V., Ritter, G. D., Gao, F., Jin, M. J., Nail, C. D., Spies, C. P., Hahn, B. H. & Compans, R. W. (1992). Cytoplasmic domain truncation enhances fusion activity by the exterior glycoprotein complex of human immunodeficiency virus type 2 in certain cell types. J Virol 66, 39713975.[Abstract]
Murakami, T. & Freed, E. O. (2000a). Genetic evidence for an interaction between human immunodeficiency virus type 1 matrix protein and alpha-helix 2 of the gp41 cytoplasmic tail. J Virol 74, 35483554.
Murakami, T. & Freed, E. O. (2000b). The long cytoplasmic tail of gp41 is required in a cell type-dependent manner for HIV-1 envelope glycoprotein incorporation into virions. Proc Natl Acad Sci U S A 97, 343348.
Muster, T., Steindl, F., Purtscher, M., Trkola, A., Klima, A., Himmler, G., Rüker, F. & Katinger, H. (1993). A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J Virol 67, 66426647.[Abstract]
Newton, S. M. C., Joys, T. M., Anderson, S. A., Kennedy, R. C., Hovi, M. E. & Stocker, B. A. D. (1995). Expression and immunogenicity of an 18-residue epitope of HIV-1 gp41 inserted in the flagellar protein of a Salmonella live vaccine. Res Microbiol 146, 203216.[CrossRef][Medline]
Niedrig, M., Bröker, M., Walter, G., Stüber, W., Harthus, H.-P., Mehdi, S., Gelderblom, H. R. & Pauli, G. (1992). Murine monoclonal antibodies directed against the transmembrane protein gp41 of human immunodeficiency virus type 1 enhance its infectivity. J Gen Virol 73, 951954.[Abstract]
Piller, S. C., Dubay, J. W., Derdeyn, C. A. & Hunter, E. (2000). Mutational analysis of conserved domains within the cytoplasmic tail of gp41 from human immunodeficiency virus type 1: effects on glycoprotein incorporation and infectivity. J Virol 74, 1171711723.
Pincus, S. H., Messer, K. G., Schwartz, D. H., Lewis, G. K., Graham, B. S., Blattner, W. A. & Fisher, G. (1993). Differences in the antibody response to human immunodeficiency virus type 1 envelope protein (gp160) in infected laboratory workers and vaccinees. J Clin Invest 91, 19871996.[Medline]
Ratner, L., Haseltine, W., Patarca, R. & 16 other authors (1985). Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313, 277284.[Medline]
Sattentau, Q. J., Zolla-Pazner, S. & Poignard, P. (1995). Epitopes exposed on functional, oligomeric gp41 molecules. Virology 206, 713717.[Medline]
Sauter, M. M., Pelchen-Matthews, A., Bron, R. & 8 other authors (1996). An internalization signal in the simian immunodeficiency virus transmembrane protein cytoplasmic domain modulates expression of envelope glycoproteins on the cell surface. J Cell Biol 132, 795811.[Abstract]
Schirmer, T. (1998). General and specific porins from bacterial outer membranes. J Struct Biol 121, 101109.[CrossRef][Medline]
Schirmer, T. & Cowan, S. W. (1993). Prediction of membrane-spanning -strands and its application to maltoporin. Protein Sci 2, 13611363.
Sodroski, J., Goh, W. C., Rosen, C., Campbell, K. & Haseltine, W. A. (1986). Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity. Nature 322, 470474.[Medline]
Spies, C. P., Ritter, G. D., Mulligan, M. J. & Compans, R. W. (1994). Truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein alters the conformation of the external domain. J Virol 68, 585591.[Abstract]
Srinivas, S. K., Srinivas, R. V., Anatharamaiah, G. M., Segrest, J. P. & Compans, R. W. (1992). Membrane interactions of synthetic peptides corresponding to amphipathic helical segments of the human immunodeficiency virus type 1 with lipid bilayers. J Biol Chem 267, 71217127.
Vella, C., Minor, P. D., Weller, I. V. D., Jenkins, O., Evans, D. & Almond, J. (1991). Recognition of poliovirus/HIV chimeras by antisera from individuals with HIV infection. AIDS 5, 425430.[Medline]
Vella, C., Ferguson, M., Dunn, G., Meloen, R., Langedijk, H., Evans, D. & Minor, P. D. (1993). Characterization and primary structure of a human immunodeficiency virus type 1 (HIV-1) neutralization domain as presented by a poliovirus type 1/HIV-1 chimera. J Gen Virol 74, 26032607.[Abstract]
Venable, R. M., Pastor, R. W., Brooks, B. R. & Carson, F. W. (1989). Theoretically determined three-dimensional structure for amphipathic segments of the HIV-1 gp41 envelope protein. AIDS Res Hum Retrovir 5, 722.[Medline]
Vzorov, A. N. & Compans, R. W. (2000). Effect of the cytoplasmic domain of the simian immunodeficiency virus envelope protein on incorporation of heterologous envelope proteins and sensitivity to neutralization. J Virol 74, 82198225.
Weissenhorn, W., Dessen, A., Harrison, S. C., Skehel, J. J. & Wiley, D. C. (1997). Atomic structure of the ectodomain from HIV-1 gp41. Nature 387, 426430.[CrossRef][Medline]
Wilk, T., Pfeiffer, T. & Bosch, V. (1992). Retained in vitro infectivity and cytopathogenicity of HIV-1 despite truncation of the C-terminal tail of the env gene product. Virology 189, 167177.[Medline]
Wyma, D. J., Kotov, A. & Aiken, C. (2000). Evidence for a stable interaction of gp41 with Pr55Gag in immature human immunodeficiency virus type 1 particles. J Virol 74, 93819387.
Yu, X., Yuan, X., McLane, M. F., Lee, T.-H. & Essex, M. (1993). Mutations in the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein impair the incorporation of Environ proteins into mature virions. J Virol 67, 213221.[Abstract]
Zhang, H., Dornadula, G., Alur, P., Laughlin, M. A. & Pomerantz, R. J. (1996). Amphipathic domains in the C terminus of the transmembrane protein (gp41) permeabilize HIV-1 virions: a molecular mechanism underlying natural endogenous reverse transcription. Proc Natl Acad Sci U S A 93, 1251912524.
Zingler, K. & Littman, D. R. (1993). Truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein increases Environ incorporation into particles and fusogenicity and infectivity. J Virol 67, 28242831.[Abstract]
Received 5 June 2002;
accepted 23 October 2002.