An antibody specific for the C-terminal tail of the gp41 transmembrane protein of human immunodeficiency virus type 1 mediates post-attachment neutralization, probably through inhibition of virus–cell fusion

Caroline J. Heap, Steven A. Reading and Nigel J. Dimmock

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK

Correspondence
Nigel J. Dimmock
ndimmock{at}bio.warwick.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Evidence has been presented which shows that part of the C-terminal tail of the gp41 transmembrane protein of human immunodeficiency virus type 1 (HIV-1) contains a neutralization epitope and is thus exposed on the external surface of the virion. Here, SAR1, a monoclonal antibody, which was stimulated by immunization with a plant virus expressing 60 copies of the GERDRDR sequence from the exposed gp41 tail, and has an unusual pattern of neutralization activity, giving little or no neutralization of free virions, but effecting modest post-attachment neutralization (PAN) of virus bound to target cells was investigated. Here, the properties of PAN were investigated. It was found that PAN could be mediated at 4 or 20 °C, but that at 20 °C maximum PAN required virus–cell complexes to be incubated for 3 h before addition of antibody. Further PAN appeared stable at 20 °C and could be mediated for at least 5 h at this temperature. In contrast, when virus–cell complexes formed at 20 °C but then shifted to 37 °C for various times before addition of SAR1, PAN was maximal after just 10 min, and was lost after 30 min incubation. Thus, PAN at 37 °C is transient and temperature-dependent. Since this scenario recalled the temperature requirements of virus–cell fusion, fusion of HIV-1-infected and non-infected cells was investigated, and it was found that SAR1 inhibited this process by up to 75 %, in a dose-dependent manner. However, antibodies to adjacent epitopes did not inhibit fusion. These data confirm the external location of the SAR1 epitope, implicate the gp41 C-terminal tail in the HIV-1 fusion process for the first time, and suggest that SAR1 mediates PAN by inhibiting virus-mediated fusion.


   INTRODUCTION
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INTRODUCTION
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The mature envelope protein of the virion of human immunodeficiency virus type 1 (HIV-1) is a trimer of heterodimers formed of the gp120 outer subunit and the gp41 transmembrane (tm) subunit. The gp120 serves to attach virus to the CD4 primary receptor and to the CXCR4 or CCR5 co-receptors on the surface of target cells, while the gp41 enables the viral and cell lipid bilayers to fuse together, resulting in the viral genome and associated proteins entering the cytoplasm and infecting the cell. Both gp120 and gp41 carry antibody neutralization epitopes (Levy, 1998).

The gp41 subunit of HIV-1 and simian immunodeficiency viruses comprises the ectodomain, the tm domain, and a long C-terminal tail of approximately 150 aa residues (Gallaher et al., 1992). There is structural information on the ectodomain (Caffrey et al., 1998; Chan et al., 1997; Malashkevitch et al., 1998; Tan et al., 1997; Weissenhorn et al., 1997), but not on the C-terminal tail. The limits of the tm domain are not known exactly (West et al., 2001). Conventionally the tail is regarded as being located entirely inside the virion or the infected cell, but it has been proposed that about 40 residues of the tail region are looped out to the external surface of the virion (Cleveland et al., 2003; McLain et al., 2001). The main evidence for this relates to the neutralization of infectivity by certain tail epitope-specific antibodies (Buratti et al., 1998; Chanh et al., 1986; Cheung, 2002; Cheung et al., 2005; 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), and since particles of infectious virus are by definition intact, and IgG does not cross lipid bilayers, it follows that the epitope is expressed on the outside of the virion. The neutralization epitope (centred on the sequence ERDRD) is constitutively exposed on the surface of virions (Cleveland et al., 2003), and two other non-neutralizing epitopes are sited close by. Interaction with mAbs to all these epitopes is abrogated by digestion with trypsin or thermolysin, and is consistent with their external location (Cleveland et al., 2003). To explain the external location of the epitope-bearing loop, we have hypothesized that the conventional tm region has a {beta}-turn at its centre that takes the tail back across the viral membrane to the exterior surface of the virion (Cleveland et al., 2003). Furthermore, we suggest that the loop is completed by a third potential tm region then takes the tail back inside the virion. All three proposed tm regions comprise approximately 10 residues and are theoretically compatible with predictions for {beta}-sheet structures (M. J. Hollier & N. J. Dimmock, unpublished data). Although short in length, there is a precedent for such tm regions in bacterial porins (Schirmer, 1998; Schirmer & Cowan, 1993). The approximately 40 aa residue external loop is thus defined by tm2 and 3. The gp41 tail C-terminal to tm3 comprises approximately 100 residues, is inside the virion, and can interact with internal components of the virion (e.g. Freed, 1998).

The gp41 external tail loop includes the 22-residue Kennedy sequence, 731PRGPDRPEGIEEEGGERDRDRS752 (Chanh et al., 1986; numbering system of Ratner et al., 1985). This is a hydrophilic, antigenically complex region with no apparent structural organization. Confusingly both neutralizing and non-neutralizing antibodies recognize the Kennedy sequence, and 746ERDRD750 that forms the minimum epitope for a number of antibodies, appears to adopt a number of different conformations. These are recognized by a neutralizing epitope-purified, ERDRD-specific (EPES) polyclonal antibody (Buratti et al., 1998; Cleveland et al., 2000a, b, 2003; McLain et al., 2001), by mAbs 1577 and 1583, which neutralize only in the presence of complement (Cleveland et al., 2003; Vella et al., 1993), and by mAb SAR1, which neutralizes free virions poorly or not at all, but is active in post-attachment neutralization (PAN) (Reading et al., 2003). mAb C8 recognizes 734PDRPEG739 on the surface of infected cells (Abacioglu et al., 1994), but not virions and is thus non-neutralizing. Lastly the non-neutralizing mAb 1575 recognizes residues 740IEEE743. This is an immunodominant, non-conformational epitope that is present on virions and the surface of infected cells (Cleveland et al., 2000b; McLain et al., 2001; Vella et al., 1993).

mAb SAR1 is a novel IgG that was raised recently by immunizing mice with a plant virus chimera expressing the gp41 tail sequence 745GERDRDR751 (Reading et al., 2003). SAR1 recognizes gp41 expressed on the surface of infected cells, and binds to some, but not all, preparations of purified virions, suggesting that it may recognize non-infectious virions or degraded/immature forms of the envelope protein. In general, three types of neutralization activity can be distinguished: (i) standard neutralization (STAN), which takes place when virus and antibody are incubated together before they are added to target cells; (ii) PAN, which occurs when mAb is added to virions after they have attached to cells – this can be measured by inhibition of syncytium formation or p24 production; and (iii) neutralization of infectious progeny (NIP), which is the reduction in the amount of infectivity produced by the infected culture. SAR1 gives poor or no STAN, significant PAN and good NIP (Reading et al., 2003). Location of the SAR1 epitope was confirmed by failure of antibody to neutralize a mutant virus lacking the 144 C-terminal gp41 residues, and by competition with a mAb that recognizes an adjacent epitope (740IEEE743) (Reading et al., 2003). Here, we have further investigated the unusual pattern of SAR1 neutralization. We found that temperature requirements suggest that SAR1 effects PAN by inhibition of virion–cell fusion, and SAR1 was shown to be directly inhibitory in a virus-mediated cell–cell fusion assay. This is the first time that a region of the gp41 C-terminal tail has been implicated in fusion. Together these data support the view that part of the gp41 C-terminal tail lies outside the virion and the infected cell.


   METHODS
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METHODS
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Viruses and cells.
HIV-1 IIIB and the human T-cell lines, H9 and C8166, were obtained from the AIDS Reagent Project (NIBSC). Cells were grown in RPMI 1640 medium (Gibco-BRL), without antibiotics, but with 2 mM L-glutamine and 10 % (v/v) heat-inactivated fetal calf serum (LabTech International). Virus was produced by co-cultivation of persistently infected and non-infected H9 cells for 3 days. Medium was replaced 48 h before harvesting to minimize the influence of thermal decay on virions, and clarified culture fluid was stored in liquid nitrogen. Infectivity was assayed in C8166 cells (see below).

Antibodies.
mAb SAR1, a murine IgG2a, {kappa}, was produced by immunization with the plant cowpea mosaic virus chimera, CPMV-HIV/29 that expresses 745GERDRDR751 on its surface from the C-terminal tail of gp41 (Cleveland et al., 2000a; Reading et al., 2003). We also used the gp41 C-terminal tail mAbs: C8 to 734PDRPEG739 (Abacioglu et al., 1994), 1575 to 740IEEE743, 1577 and 1583 to 746ERDRD750 (Vella et al., 1993), and gp120 mAbs: ICR41.1i to a conformational epitope in the V3 loop (Cordell et al., 1991) and mAb b12 to the gp120 CD4-binding site (Barbas et al., 1992; Bender et al., 1993). IgG concentrations were determined by ELISA after capture of serial dilutions of purified IgG with immobilized goat anti-species IgG. IgG concentration was interpolated from a standard curve of IgG.

Assay for PAN.
Virus 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. Plates (96-well, Gibco-BRL) were treated with 50 µg poly-L-lysine (Sigma) ml–1 to anchor cells, and seeded with 5x104 C8166 cells per well (McLain & Dimmock, 1994). Cells were inoculated with approximately 50 syncytium forming units (s.f.u.) per well usually for 1 h at 20 °C, but this was varied according to protocols described later. After removing virus and rinsing cells, medium, or medium containing mAb SAR1 (200 µl per well in five replicates) was added. Antibody was incubated with infected cells according to protocols presented later. When required, antibody was removed and replaced with fresh medium. It takes 3 days at 37 °C for primary syncytia to develop fully; secondary syncytia do not appear until after this time. Syncytia contain three or more nuclei, and their identity has been confirmed by cytostaining. However, they are readily recognized under low-power microscopy. The same virus titre is obtained when culture fluid p24 is assayed by ELISA (data not shown). PAN was calculated as the percentage reduction in syncytium formation in cultures treated with antibody compared to syncytium formation in the virus control without antibody (approx. 50 s.f.u. per well counted). Values were corrected for the very few syncytia (mean <1 per well) that occurred in cell controls.

Fusion of HIV-1-infected cells with non-infected cells.
This method was essentially as previously described (Armstrong et al., 1996). C8166 cells (1x107 cells in 10 ml) were infected with HIV-1 (5x104 s.f.u.) for 2 days at 37 °C. The medium was changed and cells incubated for a further day. Washed cells (6x104 in 100 µl) were mixed with an equal volume of medium or medium containing antibody for 1 h at 37 °C. A 10-fold excess of non-infected C8166 cells (6x105 in 50 µl) was then added and fusion allowed to proceed for 3·5 h at 37 °C. Infected and non-infected cells without mAb, were incubated in parallel at 4 °C to control the level of spontaneous cell–cell fusion. All cells were then washed with PBS and stained with chilled 0·2 % Wright's stain in methanol. These were rehydrated, pelleted and stained further with 0·05 % Giemsa in distilled water. After washing, at least 1000 cells from each sample were counted by low-power microscopy. Syncytia are defined as cells containing three or more nuclei. The baseline number of syncytia was subtracted from all samples and the percentage inhibition of cell–cell fusion calculated.

Binding of soluble recombinant (sr) CD4 to HIV-1.
SrCD4 expressed by CHO cells, was generously provided by W. Meier (Biogen Inc, Cambridge, MA). Purified virus (5x105 s.f.u.) was incubated with 0·5 µg srCD4 in 1 ml for 1 h at 20 °C in 100 mM sodium bicarbonate, pH 8·5. The mix (100 µl per well) was then transferred to a 96-well plate (Immulon 2; Dylan). Other wells contained virus alone, srCD4 alone or buffer. The plate was incubated overnight at 20 °C. Plates were washed with Tris-buffered saline (TBS; 50 mM Tris, 140 mM NaCl, pH 7·6), blocked with 3 % BSA (Sigma) in TBS, and incubated overnight at 20 °C with envelope-specific IgGs in TBS containing 0·05 % Tween 20 (TBS-T) and 1 % BSA. Virus was also incubated with an irrelevant IgG. After washing, the plate was immersed overnight at 4 °C in 3 % paraformaldehyde. Bound IgG was assayed with biotinylated anti-species IgG, and colour developed with streptavidin-linked alkaline phosphatase (Amersham Life Science) and p-nitrophenyl phosphate in diethanolamine buffer (Pierce & Warriner) with MgCl2, pH 9·8. Controls gave a reading of <0·2 OD405 units, when the virus with SAR1 gave approximately 1·0 OD405 units.


   RESULTS
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Temperature-dependence of PAN
Fig. 1 is a schematic showing how the gp41 tail loop SAR1 epitope (GERDRDR) is thought to be exposed on the outside of the virion, with the epitope region occupying the more C-terminal part of the loop (Cleveland et al., 2003; M. J. Hollier & N. J. Dimmock, unpublished data). However, while SAR1 mediates PAN, it has little ability to mediate STAN (Reading et al., 2003). Thus, the SAR1 epitope may be occluded by the main gp41 ectodomain or gp120 outer domain of the envelope protein (not shown in Fig. 1), or may undergo conformational changes as a result of virus–receptor interaction. In an attempt to gain insight into these problems, we investigated the temperature-dependence of PAN. Accordingly cells were infected at 4, 20 or 37 °C for 2 h, and after removing unattached virus, SAR1 was added for a further 2 h, keeping the temperatures unchanged. Antibody was then removed, and cells incubated at 37 °C for 3 days. Fig. 2(a) shows that optimum PAN was obtained only when cells were infected at 4 or 20 °C, and that PAN was much reduced when cells were infected at 37 °C. These data suggested that after 2 h incubation at 37 °C, the SAR1 epitope was not available, had changed conformation, or was still present but had lost the ability to mediate neutralization.



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Fig. 1. A model of the gp41 C-terminal tail of an HIV-1 virion. The gp41 ectodomain and gp120 are not shown. The postulated external loop of approximately 40 residues and three transmembrane domains (tm1–3) are labelled. The Kennedy sequence 731–752 is shown, and the minimum antibody epitopes are in blue, red and green and underlined. The position of the LLP-1 peptide and its possible interaction with the membrane are indicated (modified after Cleveland et al., 2003).

 


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Fig. 2. Temperature requirements for PAN by SAR1. (a) Temperature optimum: C8166 cells were infected with HIV-1 NL-4.3 at 4 ({blacktriangleup}), 20 ({blacktriangledown}) or 37 °C ({blacksquare}) for 2 h. After washing, cell–virus complexes were incubated at the same temperatures in medium containing SAR1 for 2 h. (b) Optimization of time of incubation of virus–cell complexes at 20 °C before addition of antibody: virus was incubated with cells at 20 °C for 1·25–5 h, as shown on the right. Cultures were then washed, and incubated with SAR1 at 20 °C for 30 min. In both experiments antibody was then replaced with medium at 37 °C and incubation continued for 3 days. Syncytia were counted, and PAN calculated as the percentage inhibition relative to a control incubated in the absence of antibody. Typically there were 40–50 syncytia in each virus control well. Curves were derived using GraphPad Prism non-linear regression analysis fitted to a one site-binding hyperbola. The figures shown are representative of two replicate experiments. Error bars are the standard error of the mean.

 
PAN increases when virus–cell complexes are incubated for 3 h at 20 °C before the addition of SAR1
Although SAR1 binds to some virion preparations, it gives weak or no STAN (Reading et al., 2003). One interpretation is that SAR1 is binding only to non-infectious virions or to virus with degraded or immature forms of the envelope protein, and that the SAR1 epitopes on free infectious virus particles are hidden or not in the appropriate conformation. To investigate the latter, we incubated virus with cells at 20 °C at intervals for up to 5 h before pulsing with SAR1 at the same temperature for 30 min. Fig. 2(b) shows that PAN became maximal after 3 h incubation before the addition of antibody. Thus, it takes 3 h at 20 °C for the SAR1 epitope to become maximally exposed or to attain its optimum conformation. There was no loss of PAN when incubation was continued at 20 °C for up to 5 h showing that the epitope was stably exposed. The presence of cells is essential, as incubating virus and SAR1 without cells for up to 20 h did not increase neutralization (Reading et al., 2003). The time of incubation at 20 °C of virus–cell complexes with SAR1 required to give maximum PAN was then determined. mAb was added after virus and cells had been incubated at 20 °C for 3 h. Fig. 3 shows that 10 min incubation at 20 °C with SAR1 gave no PAN, while a 30 min pulse, or a 2 h pulse (data not shown) gave maximum PAN. Thus at 20 °C, SAR1 PAN requires significant time for incubation of virus and cells (3 h), and of cell–virus complexes with antibody (30 min).



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Fig. 3. Time required for virus–cell complexes to be incubated with SAR1 for maximum PAN. Cells and virus were incubated together for 3 h at 20 °C as in Fig. 2, and then washed and pulsed with mAb for 10, 20 and 30 min at 20 °C (as shown on the right). Antibody was then replaced with medium and incubation continued for 3 days. Other information is in the legend to Fig. 2. The figure shown is representative of two replicate experiments.

 
Temperature shift to define rate of loss of PAN at 37 °C
The time taken for virus to become refractory to PAN by SAR1 at 37 °C was determined by infecting cells for 2 h at 20 °C, and then after washing, shifting to 37 °C for up to 60 min. After cooling to 20 °C, SAR1 was added for 2 h, and incubation continued at 37 °C for 3 days to determine PAN. Fig. 4 shows that SAR1 gave maximum PAN when virus–cell complexes were incubated at 37 °C for 0 or 10 min before adding SAR1, but there was minimal PAN if SAR1 was added after cell–virus complexes were incubated for 30 min or more at 37 °C. Loss of PAN is thus a relatively rapid process, and occurs faster than virus–cell fusion, which takes approximately 90 min at 37 °C for 90 % completion (see Discussion). It appears therefore that the abrogation of SAR1 PAN does not result from the completion of virus–cell fusion, but might reflect the end of an early phase of the fusion process.



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Fig. 4. Kinetics of the loss of SAR1-mediated PAN at 37 °C. Virus was allowed to attach to cells for 2 h at 20 °C, and unattached virus removed by washing. Cell–virus complexes were then incubated at intervals of 0–60 min (as shown on the right) in medium pre-warmed to 37 °C. After cooling to 20 °C, cells were incubated with SAR1 for 2 h. After washing, cultures were incubated at 37 °C for 3 days. Other information is in the legend to Fig. 2. The figure shown is representative of two replicate experiments.

 
SAR1 inhibits fusion of HIV-1-infected cells with non-infected C8166 cells
Because of the suggestion above that SAR1 might inhibit an early stage in the virus–cell fusion process, we examined the ability of SAR1 to directly inhibit HIV-1-mediated cell fusion. In this assay, HIV-1-infected C8166 cells were incubated with non-infected cells for 3·5 h at 37 °C to allow fusion to take place. After fixation and staining, syncytia (defined as cells containing three or more nuclei) were counted. The anti-fusion action of SAR1 was determined by incubating infected cells with the mAb before they were mixed with non-infected cells. Fig. 5 shows that SAR1 was effective in fusion inhibition, with 30 µg ml–1 giving 75 % inhibition. For comparison, mAb b12, which is specific for the gp120 binding site, gave a similar level of fusion inhibition at 3 µg ml–1. However other gp41 Kennedy sequence mAbs (C8, 1575, 1577 and 1583) did not inhibit fusion at concentrations up to 100 µg ml–1, despite the proximity of their epitopes to that of SAR1 (Fig. 1). Only the neutralizing EPES polyclonal antibody inhibited fusion (Cheung et al., 2005). Like SAR1, EPES IgG was made by immunizing mice with CPMV-HIV/29.



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Fig. 5. SAR1 inhibited fusion of HIV-1-infected cells with non-infected cells. C8166 cells were infected and incubated for 3 days at 37 °C. They were then incubated successively with various concentrations of SAR1 (0–100 µg ml–1) for 1 h at 37 °C, and then non-infected cells (3·5 h at 37 °C) for fusion to take place. Other mAbs (C8, 1575, 1577 and 1583) to gp41 tail epitopes adjacent to that of SAR1 gave no inhibition as shown. The gp120 mAb b12 (3 µg ml–1) is the positive control. Cells were fixed and treated with Wright's and Giemsa stains to emphasize the nuclei. Syncytia, defined as having at least three nuclei, were counted using low-power microscopy. The figure shown is representative of two replicate experiments. Error bars are the standard error of the mean.

 
SrCD4 does not enhance binding of SAR1 to HIV-1 particles
As shown above, the SAR1 epitope is optimally exposed on infectious virions only after virus has attached to, and been incubated with, the target cell. To test if this exposure resulted primarily from interaction with the CD4 receptor, we determined if srCD4 enhanced the binding of SAR1 to HIV-1 particles in vitro. mAb ICR 41.1i that reacts with a conformational epitope in the V3 loop of gp120, was the positive control. Fig. 6 shows that srCD4 increased the binding of ICR 41.1i to virions by twofold as others have found (McKeating et al., 1992), but gave no increase in SAR1 binding. Extending the initial virus–srCD4 interaction to 3 h did not enhance SAR1 binding (data not shown). These data show that exposure of and/or conformational changes that affect the SAR1 epitope when virus binds to infected cells are not mimicked by interaction of virus with monomeric srCD4, and thus may result from more complex virus–cell interactions.



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Fig. 6. Incubation of srCD4 with HIV-1 virions increased the binding of the gp120 V3 loop-specific mAb ICR41.1i, but not SAR1. Purified virus (5x105 s.f.u.) was incubated with 0 (–) or 0·5 µg ml–1 (+) srCD4 for 1 h at 20 °C as indicated, and the mix then transferred to wells of a microtitre plate and incubation continued overnight at the same temperature. The plate was washed and blocked and virus reacted again overnight with 10 µg ml–1 of mAbs SAR1 or ICR41.1i. After fixation with paraformaldehyde, bound antibody was detected by ELISA. The value for an irrelevant IgG binding to virus (<0·2 OD405 units) has been subtracted. The figures shown are representative of three replicate experiments. Error bars are the standard error of the mean.

 

   DISCUSSION
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Even though SAR1 gives no significant STAN, it causes PAN, fusion inhibition and NIP (Table 1). A key observation in explaining SAR1 PAN is the finding that SAR1 inhibits the fusion of HIV-1-infected cells to non-infected cells (Fig. 5). Although cell–cell fusion is not necessarily the same phenomenon as virion–cell fusion (Cao et al., 1993; Konopka et al., 1995; Simmons et al., 1995), inhibition of cell–cell fusion by SAR1 suggests that SAR1 effects PAN by abrogating virion–cell fusion. This could mean that the gp41 C-terminal tail is directly implicated in the lentivirus-mediated fusion process. Others have reported that modifications to the tail can affect lentivirus fusogenicity (Mulligan et al., 1992; Ritter et al., 1993; Sodroski et al., 1986; Spies & Compans, 1994; Wilk et al., 1992; Zingler & Littman, 1993). Alternatively, the SAR1 epitope (745GERDRDR751) may be positioned close enough for the SAR1 IgG to sterically interfere with the fusion process mediated by the gp41 ectodomain (e.g. Follis et al., 2002; He et al., 2003; Jones et al., 1998; Lu et al., 2001; Weissenhorn et al., 1997). However, while the neutralizing EPES IgG also inhibited fusion (Cheung et al., 2005), mAbs to the other 746ERDRD751 conformers (mAbs 1577 and 1583; Vella et al., 1993) did not. Nor did mAbs to the adjacent epitopes 734PDRPEG739 (C8) and 740IEEE743 (1575) inhibit fusion, and IEEE is only two glycine residues upstream of the SAR1 epitope. Thus, certain neutralizing conformations of the ERDRD epitope correlate with fusion-inhibition, while mAbs 1577 and 1583 (also specific to ERDRD) are both non-neutralizing and non-fusion-inhibiting. How such a short sequence can manifest several different epitopes will be discussed separately (N. J. Dimmock, in preparation). Finally it remains to be determined if SAR1 inhibits fusion specifically, or binds with a different topology to other Kennedy sequence mAbs and interferes with fusion sterically.


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Table 1. Summary of some HIV-1-neutralizing properties of mAb SAR1

STAN, Standard neutralization; PAN, post-attachment neutralization; FI, fusion-inhibition of infected and non-infected cells; NIP, neutralization of infectious progeny virus; IC50, 50 % inhibitory concentration. NA, Not applicable; ND, not done.

 
Temperature requirements for PAN by SAR1 are summarized in Table 1. PAN increased and reached a maximum when virus and target cells were incubated together for 3 h at 20 °C before the addition of antibody. Thus, the SAR1 epitope appears to slowly attain its cognate conformation or become more accessible to antibody after virus reacts with cell receptors. Similar levels of PAN were achieved after infection at 4 °C (Fig. 2a; Reading et al., 2003). Epitope activation appears to be indirect, as there is no known cellular receptor for the gp41 external tail loop. Once the epitope becomes available, SAR1 achieves maximum PAN in 30 min at 20 °C (Fig. 3). After incubation of virus–cell complexes at 20 °C and shifting the temperature to 37 °C, PAN was maximal after 10 min, but was largely abrogated after 30 min incubation (Fig. 4). Thus, the PAN process is complex with an epitope-maturation phase that does not require physiological temperatures, and a loss of epitope phase that takes place at 37 °C (Table 1). Overall PAN is transient and temperature-dependent. Loss of PAN occurs more rapidly than the loss of membrane fusion, which in our system and those of others takes at least 90 min at 37 °C (Armstrong & Dimmock, 1996; Cleveland et al., 2000b; Jackson et al., 1999; Lu et al., 1992; McInerney & Dimmock, 2001; Pelchen-Matthews et al., 1995; Rieber et al., 1992; Srivastava et al., 1991). Loss of PAN is more reminiscent of the rapid changes in gp120–gp41 conformation induced by binding CD4 (Doranz et al., 1999; Jones et al., 1998), or of inhibition of cell–cell fusion mediated by the gp41 T-20 peptide (Munoz-Barroso et al., 1998).

It is interesting how closely our data parallel those in which peptide inhibitors and antibodies to the gp41 main ectodomain were used to probe the temperature requirements for cell–cell fusion directed by vaccinia virus-expressed gp160 (Golding et al., 2002). Here, it was found that peptides (including T-20) block the formation of fusion intermediates (coiled-coil and six-helix bundle formation) more effectively if incubated with Env-expressing cells at a subfusion temperature before shifting to 37 °C. Antibodies to the N-heptad region and the six-helix bundle behaved similarly, and inhibited only if they were pre-incubated with Env-expressing cells at the lower temperature. The half-time at 37 °C transition from the six-helix bundle intermediate to fusion was 3·5–9·5 min, and bearing in mind differences in Env expression systems [vaccinia-expressed Env (Golding et al., 2002) and HIV-1 virus infection] and cells used, were of the same order as we report above for complete transition to fusion (10–30 min). The authors conclude that the coiled-coil and six-helix bundle form prior to fusion, and that the lag time is needed to accumulate sufficient six-helix bundles at the fusion site. Our data are consistent with SAR1 inhibiting an early event in the fusion pathway, and we now need to determine what role the gp41 tail loop has in fusion, and which fusion intermediate is inhibited.

The increase in PAN, discussed above, that arises when virus and cells are incubated at 20 °C reflects greater reactivity of the SAR1 epitope. This recalls the induction of gp120 and gp41 ectodomain epitopes and the co-receptor-binding site on incubation with srCD4 (Allaway et al., 1993; Doranz et al., 1999; Mbah et al., 2001; McKeating et al., 1992; Sattentau & Moore, 1991; Sattentau et al., 1993; Sullivan et al., 1998; Thali et al., 1993; Xiang et al., 2002). However, we found no increase in binding of SAR1 to purified virions complexed with srCD4, even though this doubled the binding of a gp120 V3 loop-specific mAb (Fig. 6; McKeating et al., 1992). Activation of the SAR1 epitope may require multiple contacts between viral envelope proteins and CD4 molecules inserted in the cell membrane, or binding to other virus receptors.

Work with SAR1 has revealed previously unknown properties of the gp41 C-terminal tail, which adds to the case that part of the C-terminal tail is exposed on both the external surface of the virion (Buratti et al., 1998; Cleveland et al., 2000a, b, 2003; McLain et al., 2001), and the infected cell (Cheung et al., 2005; Reading et al., 2003), rather than being entirely inside the virion. Further study of the structure and function of the gp41 external tail loop is required to elucidate its significance for the virus life-cycle. If antibodies targeted to the gp41 tail loop prove to be of clinical benefit, the well defined, cost-effective plant virus chimeras that stimulate SAR1-like antibodies (Reading et al., 2003) and standard neutralizing antibodies (Cheung, 2002; Cheung et al., 2005; Cleveland et al., 2000a, b, 2003; Durrani et al., 1998; McLain et al., 1995, 1996a, b, 2001) could be a useful vaccine component.


   ACKNOWLEDGEMENTS
 
C. J. H was supported by a studentship from the Medical Research Council, UK. We thank C. Vella (NIBSC, Potters Bar, UK) for mAbs 1575, 1577 and 1583, J. Cordell (Institute for Cancer Research, Sutton, UK) for mAb ICR41.1i, D. R. Burton (Scripps Research Institute, La Jolla, CA) for mAb b12, and Y. H. Abacioglou and G. K. Lewis (University of Maryland, Baltimore, MD) for mAb C8. Soluble recombinant CD4 was provided by Werner Meier (Biogen Inc., Cambridge, MA). We are also grateful to H. Holmes of the AIDS Reagent Project, NIBSC, UK and the NIH AIDS Research and Reference Reagent Program for reagents.


   REFERENCES
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DISCUSSION
REFERENCES
 
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Received 2 July 2004; accepted 2 February 2005.



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