Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Correspondence
Nigel J. Dimmock
ndimmock{at}bio.warwick.ac.uk
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ABSTRACT |
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Present address: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA.
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INTRODUCTION |
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Conventionally, virion and cellular gp41 of HIV-1 and the related simian immunodeficiency virus (SIV) are viewed as having three domains: an ectodomain that contains the N-terminal fusion sequence and whose structure has been partially solved (Caffrey et al., 1998; Chan et al., 1997
; Malashkevitch et al., 1998
; Tan et al., 1997
; Weissenhorn et al., 1997
), a transmembrane domain of 22 aa and a long C-terminal tail of approximately 144 aa (Gallaher et al., 1989
). However, our group has argued that the structure of the C-terminal tail of the virion is more complex than generally appreciated and that the part of the tail including the Kennedy sequence, 731PRGPDRPEGIEEEGGERDRDRS752 (Chanh et al., 1986
; Kennedy et al., 1986
), is exposed on its outer surface. Part of the evidence for this is adduced from the neutralization of virus infectivity by antibodies to the Kennedy sequence (Buratti et al., 1998
; Chanh et al., 1986
; Cheung, 2002
; Cleveland et al., 2000a
, b
, 2003
; Dalgleish et al., 1988
; Durrani et al., 1998
; Evans et al., 1989
; Ho et al., 1987
; Kennedy et al., 1986
; McLain et al., 1995
, 1996a
, b
, 2001
; Newton et al., 1995
). Since particles of infectious virus are by definition intact and IgG does not cross lipid bilayers, it follows that the neutralizing epitope is expressed on the outside of the virion. More specifically, we have shown that antibody directed to a specific conformation of the tail sequence, 746ERDRD750, binds to virions and neutralizes infectivity (Cheung, 2002
; Cleveland et al., 2000b
, 2003
; McLain et al., 2001
). The ERDRD epitope is exposed constitutively and does not require contact with cell receptors or an elevated temperature (Cleveland et al., 2003
). Antibody-binding studies have revealed two other gp41 tail epitopes on the surface of the virion. One, formed by the sequence 740IEEE743, is recognized by mAb 1575 (Vella et al., 1993
). The other, formed by 734PDRPEG739, is recognized in Western blots by mAb C8 (Abacioglu et al., 1994
). However, mAb C8 also reacts with virions of a neutralization escape mutant selected by antibody to an adjacent epitope (McLain et al., 2001
).
Using the above data and standard protein structure prediction programs, we have proposed a model for the gp41 C-terminal tail of the HIV-1 virion (Cleveland et al., 2003). In brief, this suggests that the conventional transmembrane region has a central
turn that permits the current transmembrane region to form two short transmembrane sequences, the second of which takes the tail outside the virion. This external tail region is a predicted hydrophilic loop of approximately 40 residues that carries the three epitopes described above. A predicted third transmembrane region takes the most C-terminal part of the tail (approx. 100 residues) back inside the virion.
To date, work on the disposition of the C-terminal tail of gp41 in the plasma membrane of infected cells has concentrated on the interaction of gp41 with the matrix MA protein and the role of various sequences involved in trafficking of the envelope glycoprotein (see Discussion), and the gp41 tail has generally been assumed to be entirely cytoplasmic. However, using a panel of antibodies, we found here that the same region of the C-terminal tail that is exposed on the surface of virions is also exposed on the surface of HIV-1-infected T cells and HeLa cells infected with a gp41-expressing vaccinia virus recombinant, although the exposed part of the tail on the infected-cell surface was antigenically different from that of the virion. Furthermore, we have shown here that a virus-neutralizing, gp41 tail loop-specific antibody inhibited HIV-1-mediated cellcell fusion, confirming with this independent assay that part of the gp41 tail is exposed on the cell surface and demonstrating for the first time that the tail loop is involved in the fusion process itself or is closely apposed to the regions of the main gp41 ectodomain that are involved in the fusion process. These new data can be reconciled with the other reported activities and properties of the gp41 tail. The finding of part of the C-terminal tail on the surface of HIV-1-infected cells in a form that is both immunologically active and associated with cell fusion suggest that it merits investigation as a possible antiviral target.
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METHODS |
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Antibodies.
The following antibodies specific for the gp41 C-terminal tail were used: mAb C8 to 734PDRPEG739 (Abacioglu et al., 1994; numbering system of Ratner et al., 1985
), mAb 1575 to 740IEEE743 and mAb 1577 and mAb 1583 to 746ERDRD750 (Vella et al., 1993
); for the gp41 ectodomain: mAb 2F5 (Muster et al., 1993
); for the gp120 CD4-binding site: mAb b12 (Burton et al., 1994
); and for p17: mAb 4C9 (Ferns et al., 1987
) and mAb MH-1 (J. Cottingham, unpublished data). Epitope-purified ERDRD-specific (EPES) IgG to the gp41 C-terminal tail was prepared as previously described using a cowpea mosaic virus (CPMV)HIV chimera (Cleveland et al., 2003
), except that mice were immunized with the chimera (CPMVHIV/29) expressing the HIV-1 gp41 tail peptide 745GERDRDR751. Serum antibody was purified on a flock house virus (FHV) coat fusion protein (FHV-L2-A) expressing the gp41 tail sequence 740IEEEGGERDRDR751 in its neutralizing conformation (Buratti et al., 1998
). EPES antibody consisted predominantly of IgG1, IgG2a and IgG2b. Antibodies were quantified by solid-phase ELISA or by measurement of A280.
ELISA for gp41 expressed on infected cells.
C8166 cells were infected with HIV-1 IIIB [0·005 syncytium-forming units (s.f.u.) per cell] and incubated at 37 °C for 3 days. At this time, approximately 40 syncytia, with 14 % of cells (but not syncytia) stained with trypan blue. Of the mock-infected cells, 3 % stained with trypan blue. HeLa cells were infected with recombinant or wt vaccinia viruses at an m.o.i. of 10 for 18 h at 37 °C. There was no visible cytopathology at this time. Antibodies were reacted with cells prior to fixation or after fixation with 2 % paraformaldehyde (BDH) at 4 °C. Cells were permeabilized with 0·2 % saponin (CalBiochem) for 5 min at 4 °C as required. U-bottomed microtitre plates (Greiner Labortechnik Ltd) were blocked with 3 % Marvel skimmed milk (Premier Brands Ltd) in PBS containing 1 % Tween 20. Cells were aliquotted at 5x104 cells per well, pelletted and antibody added for 1 h at room temperature. After washing, cell-bound IgG was detected with HRP-conjugated anti-mouse IgG and o-phenylenediamine according to the manufacturer's instructions (Sigma).
Assay for the fusion of HIV-1-infected cells with non-infected cells.
This was based on an earlier method (Armstrong et al., 1996). Briefly, C8166 cells were infected with HIV-1 IIIB (0·005 s.f.u. per cell) and incubated at 37 °C for 3 days. Cells (6x104 in 50 µl) were then incubated with antibody in medium or with medium alone (50 µl) for 1 h at 37 °C. Non-infected cells (6x105 cells in 50 µl) were added and incubation continued for 3·5 h to allow fusion to take place. A replicate culture was kept at 4 °C as a negative control. To help visualize syncytia and nuclei, cells were incubated with 0·2 % Wright's stain in methanol, rehydrated and then stained with 0·05 % aqueous Giemsa. At least 1000 cells were counted per sample using low-power microscopy. A syncytium was defined as a cell containing three or more nuclei. Syncytia contained 38 nuclei, with a mean of 4±1 nuclei per syncytium.
Neutralization of HIV-1 infectivity.
Infectivity was assayed by the production of syncytia in C8166 cell monolayers. There is a linear relationship between the number of syncytia and the amount of inoculum, meaning that each syncytium is the product of a single infectious unit (McLain & Dimmock, 1994). The assay thus reflects a single cycle of replication. Virus (300 s.f.u. in 50 µl) was incubated with an equal volume of antibody or medium for 1 h at 37 °C. This was then incubated with C8166 cells (6x105 in 50 µl) for 1 h at 37 °C. Cells were washed, resuspended in 1·2 ml medium, aliquotted at 200 µl per well into 96-well plates (Gibco-BRL) and incubated at 37 °C. It took 3 days for primary syncytia to develop fully; secondary syncytia did not appear until after this time. Virus controls had approximately 50 s.f.u. per well. Syncytia contained three or more nuclei and their identity was confirmed by cytostaining. However, they were readily recognized unstained under low-power microscopy. The same virus titre was obtained when culture fluid p24 was assayed by ELISA (data not shown). Neutralization was expressed as the percentage reduction in syncytium count (five replicates) in wells containing the virusantibody mix compared with that of the virus control without antibody. Values were corrected for the few syncytia (mean <1 per well) that occurred in cell controls.
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RESULTS |
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DISCUSSION |
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It is unlikely that the gp41 tail exposure observed above in HIV-1-infected C8166 cells was due to attached progeny virions, as mAb C8 reacted positively with these cells but does not react with virions (see below and McLain et al., 2001). Furthermore, HeLa cells infected with the vaccinia gp41 recombinant produced no virions, but gave the same reactivity with the panel of tail-specific antibodies as HIV-1-infected C8166 cells. gp41 is a transmembrane anchored glycoprotein and is not shed, and cells do not have receptors for gp41. It is also unlikely that cells had disrupted and were revealing cytoplasmic gp41, as neither vaccinia recombinant-infected HeLa cells nor HIV-1-infected C8166 cells reacted with p17-specific antibodies until after they had been permeabilized by saponin. Cytopathology was minimal in both cell systems.
There was a striking difference between cell-surface and virion gp41 tail loop antigenicity as shown by mAb C8, which recognized cell-expressed gp41 but not virion-associated gp41. However, C8 reacts with both cell and virion gp41 in Western blots (Abacioglu et al., 1994; McLain et al., 2001
), suggesting that the C8 epitope on the cell surface is non-conformational, while the virion epitope has a different conformation or is not available to antibody. It is not known when in the course of virion budding or maturation the change in the C8 epitope takes place. It appears that either the cell conformer of gp41 is incorporated into nascent virions and is later converted into the virion conformation or virions selectively incorporate a minority species of gp41 from the cell with a different antigenicity.
The other evidence that placed part of the gp41 tail on the outside of the cell was the dose-dependent inhibition of HIV-mediated cellcell fusion by the virion-neutralizing EPES IgG. This was important independent confirmation of the ELISA data above. Recently we found that another ERDRD-specific IgG, mAb SAR1, inhibits HIV-1-mediated cellcell fusion (C. H. Heap, S. A. Reading and N. J. Dimmock, unpublished data). mAb SAR1 sees a different epitope conformation of ERDRD from EPES IgG as it gives post-attachment neutralization, but, unlike EPES IgG, it gives little or no neutralization of free virions (Reading et al., 2003). Exactly how the external gp41 tail loop is involved in fusion and how its cognate antibody inhibits the fusion process is not clear. It appears that the tail loop either functions in the fusion process directly or is close enough to the fusogenic regions of the main gp41 ectodomain for bound antibody to interfere sterically with the fusion process. However, mAbs C8 and 1575, which bind to epitopes adjacent to ERDRD, do not inhibit fusion, although they might be expected to provide a similar steric barrier to ERDRD-specific IgG. This argues for specificity of the gp41 tail loop in the fusion process. Neither the possible direct involvement of the tail loop in the fusion process nor its proximity to the fusogenic regions of the gp41 ectodomain have been recognized before, although gp41 mutations are known to affect fusogenicity (Mulligan et al., 1992
; Sodroski et al., 1986
; Wilk et al., 1992
; Zingler & Littman, 1993
). It may also be relevant that alterations to the C-terminal tail of HIV-1 and SIV can affect the conformation of both the gp41 ectodomain and gp120 (Edwards et al., 2001
, 2002
; Spies et al., 1994
; Vzorov & Compans, 2000
).
The ERDRD sequence in the exposed gp41 tail loop appears to give rise to a number of different epitopes. One is recognized by the virion-neutralizing EPES IgG (Cheung, 2002; Cleveland et al., 2000b
, 2003
; McLain et al., 2001
), another by mAb SAR1 that gives little or no neutralization of free virions but gives post-attachment neutralization (Reading et al., 2003
), and a third by mAbs 1577 and 1583, which are non-neutralizing except in the presence of complement (Cleveland et al., 2003
). However, 1577 and 1583 may not see the same epitope (Vella et al., 1993
). Only the 1577 and 1583 epitopes were apparently destroyed by paraformaldehyde, as already mentioned. Others, using cell sorting, also found that 1583 did not react with fixed infected cells (Sattentau et al., 1995
). However, paraformaldehyde fixation does not destroy the 1583 epitope on virions (Cleveland, 1999
). The reason for the difference in stability of this gp41 epitope expressed on cells and virions is not clear. It is interesting that a short sequence like ERDRD can express such a range of different epitopes, but, apart from differences in conformation, it may be that ERDRD is only the core epitope and that other residues contribute to antibody reactivity.
The proposal that part of the gp41 tail is on the outer surface of the cell membrane is not inconsistent with the reported interactions of the gp41 tail with the p17 protein (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
), providing that these are mediated by the more C-terminal portion of the tail (approx. 100 residues) and that this is back inside the virion. The C-terminal domain of gp41 of HIV-1, HIV-2 and SIV has been implicated in other viral properties, including the incorporation of the envelope glycoprotein into virions (Celma et al., 2001
; Iwatani et al., 2001
; Manrique et al., 2001
; Murakami & Freed, 2000b
; Piller et al., 2000
; Yu et al., 1993
; Zingler & Littman, 1993
), fusogenicity (Mulligan et al., 1992
; Sodroski et al., 1986
; Wilk et al., 1992
; Zingler & Littman, 1993
) and infectivity (Celma et al., 2001
; Iwatani et al., 2001
; Piller et al., 2000
). Our proposal that part of the gp41 C-terminal tail is looped out also has implications for the trafficking of gp41, in particular for the internalization of gp41 after it has been inserted in the plasma membrane. This activity is directed by sequences such as Yxx
and LL (Di Fiore & Gill, 1999
; Fultz et al., 2001
; Heilker et al., 1999
; Sauter et al., 1996
; Berlioz-Torrent et al., 1999
). The proposed looping out of part of the gp41 tail places the most N-terminal region of the tyrosine-sorting signal (719Yxx
722) outside the membrane and hence renders it inoperative. There is, however, a second potential tyrosine sorting signal (775Yxx
778), which would be cytoplasmic if residues 753763 became the third transmembrane region and could function as an internalization signal as discussed previously (Cleveland et al., 2003
).
The data described here suggest that HIV-1-infected cells might be susceptible to antibodies that recognize the PRDPEG, IEEE or ERDRD epitopes exposed on the cell surface, either directly or through antibody-dependent cellular cytotoxicity and/or complement-mediated cellular cytotoxicity. ERDRD would be the epitope of choice as EPES IgG neutralizes 91 % of virus infectivity and inhibits nearly 90 % of cellcell fusion that leads to syncytium formation and cell death (Fig. 3). However, IEEE is highly immunogenic and is known to exist in only one conformation (Cleveland et al., 2000a
). Both EPES- and IEEE-specific antibodies are readily stimulated by plant virus chimeras expressing short gp41 sequences (Cheung, 2002
; Cleveland et al., 2000a
) and these chimeras can be cost-effectively produced (Porta & Lomonossoff, 1998
). However, it will be necessary to establish if the gp41 of primary virus strains is expressed in the same way as that of the T-cell-line-adapted virus studied here.
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ACKNOWLEDGEMENTS |
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Received 13 July 2004;
accepted 8 September 2004.