The topology of HIV-1 gp41 continues to be the subject of considerable controversy. Reports have appeared even recently in support of the alternate model (58
) and of the classical model (34
). In our current study, we have expanded this debate for the first time to include gp41 of SIVmac. We have characterized a highly immunogenic region in gp41CTD which elicits strong serum reactivity in SIV-infected rhesus macaques. An analogous reactivity against the corresponding region of HIV-1 gp41CTD has been documented for sera from HIV-infected individuals (6
). The existence of such highly immunogenic regions in the gp41CTD of both HIV-1 and SIVmac, which in the generally accepted model should not be exposed on the surface, underscores the functional conservation between the envelope glycoproteins of these two viruses in the absence of consistently high sequence conservation. This is further emphasized by the correspondence in location of these two highly immunogenic regions within the linear amino acid sequence and the conservation of a hydrophobic stretch with the theoretical potential to span the plasma membrane just downstream of the highly immunogenic region in both cases.
In the serum of an infected individual, the presence of immunoglobulins specific for cytoplasmic or nuclear viral antigens is a common phenomenon. For example, serum of patients infected with human herpesvirus 8 (HHV-8) characteristically reacts with latency-associated nuclear antigen 1 (LANA-1), which is localized to the nucleus (21
). Similarly, HIV-1-infected individuals routinely develop antibodies against Gag, which is either located in the cytoplasm or encapsulated in virions during its normal life cycle (24
). However, the reactivity of patient sera against such intracellular antigens is typically much lower than against extracellular antigens that are naturally and directly exposed to the humoral immune system. The observation that sera from SIV-infected rhesus macaques display reactivity against a specific area of the gp41CTD at levels comparable to the reactivity against the extracellular immunodominant regions of Env suggests that this region of the CTD either is naturally exposed on the cell or virion surface or is unusually immunogenic. The data presented here argue strongly that the natural topology of gp41 does not feature an extracellular loop of gp41CTD and that it is its inherent immunogenicity that must be responsible for the consistently strong antibody responses.
While anti-HIR serum and the monoclonal anti-HIR antibodies 4B2 and 6E6 did indeed bind to the surface of Env-expressing cells ( and C, respectively), we show here that this reactivity is due in whole or in part to free Env released from Env-expressing cells binding to the surface of cells in the culture with remarkable efficiency (). This observation may have important implications for other studies as well, as it indicates that epitopes which appear to be accessible by cell surface staining may actually not be accessible on functional Env trimers as expressed naturally on the surface of cells. For the envelope of HIV, several groups have reported cell surface staining of HIV-infected cells with serum or antibodies directed against the HIR of gp41CTD (12
), whereas others have described the absence of such staining (45
). One recent study reported surface staining of cells expressing HIV-1 Env only under conditions which triggered fusion and at cooled temperature and concluded that gp41CTD was exposed only during the fusion event itself (35
). The data presented here differ from these previous observations, as Env was expressed in HEK293T cells. These cells do not express CD4 and are therefore not able to trigger the fusion mechanism of Env; thus, the observed cell surface presentation of the HIR described here is independent of the fusion event.
A recent publication by Steckbeck et al. (58
) employing a similar method of transfection and detection found that an epitope tag inserted into HIV-1 gp41CTD reacted with its antibody in cell surface staining when located in the immunogenic region but not when inserted closer to the C terminus. This observation is in direct contradiction to our results obtained with a C-terminal FLAG epitope (C). The results of Steckbeck et al. might reflect an inaccessibility of the C-terminal epitope tag at the location of insertion, a possibility that was not experimentally excluded by the authors and that is supported by the fact that the tag was inserted into the lentivirus lytic peptide 2 (LLP2) region, which is believed to form a tight secondary structure and possibly even a direct interaction with the plasma membrane (18
). Interestingly, an Env239 expression construct with a FLAG epitope substitution located only 21 to 29 aa before the C terminus, close to the equivalent of the LLP1 region in HIV-1 gp41, exhibited little or no reactivity with the anti-FLAG antibody in our own surface-staining experiments (data not shown). The same Env mutant reacted with the monoclonal anti-gp120 antibody 1.9C at the same level as wild-type Env. Our data demonstrate unequivocally that surface staining of the HIR can be fully explained by the release of Env protein into the cell-free supernatant and the subsequent attachment of this Env to the surface of cells.
The results of our control experiments for the detection of cell surface staining raised serious concerns about the suitability of this assay for examining the potential extracellular exposure of the HIR. Independent evidence against such a natural HIR exposure on the surface of Env-expressing cells was obtained by other means. We have shown that the HIR is not glycosylated, neither at a naturally occurring nor at an artificially introduced glycosylation motif (). N-linked glycosylation of extracellularly exposed NXS/T motifs is so reliable that so-called “glycosylation scanning,” where artificial glycosylation motifs are introduced at various points within a protein of interest, has been commonly used to determine membrane topology (for examples, see references 11
, and 56
). The naturally occurring 759
sequence within gp41CTD may or may not be too close to the unknown starting location of a hypothetical second membrane-spanning domain to be glycosylated; the artificial site introduced by the Q733N mutation, however, is far removed from any potential steric interference. Neither motif was glycosylated, providing strong evidence against a natural exposure of the HIR outside of the cell.
While the previously discussed methods were aimed at investigating the possibility of an extracellular loop formed by gp41CTD on the surface of Env-expressing cells, we also examined the possibility of HIR exposure on infectious virions by means of neutralization assays. It has been suggested that the ability of an antibody to neutralize viral infectivity relates directly to its ability to bind to the surfaces of target virion (7
). The SIVmac316 strain is closely related to SIVmac239 but is exquisitely sensitive to neutralization by a wide range of antibodies with a wide range of specificities (27
). If the HIR were indeed exposed on the surfaces of virions, anti-HIR antibodies would be expected to neutralize SIVmac316 to one extent or another. However, neither IgG purified from anti-HIR serum (A) nor the monoclonal anti-HIR antibodies 4B2 and 6E6 (B) displayed any ability to neutralize SIVmac316. This observation is consistent with the classical model of gp41CTD conformation without an external loop. It is also consistent with the report by Steckbeck et al. (58
), who did not find any evidence for exposure of the HIV-1 gp41CTD immunogenic region on intact virions.
Taken together, the data presented in this study argue strongly against a natural extracellular exposure of the highly immunogenic region located within the C-terminal domain of SIVmac gp41. We have shown that recognition of the HIR on the surface of Env-expressing cells by anti-HIR serum and monoclonal anti-HIR antibodies does not increase with the level of cell surface expression of Env (C) and is independent of structural elements that would be expected to play a role in the formation of an extracellular loop (A and B). Instead, this surface reactivity can be fully explained by the release of free Env protein from Env-expressing cells into the supernatant and the subsequent binding of this free Env to the surface of cells in the culture (). Further, the region of gp41CTD that is predicted to be exposed extracellularly in the alternative model is not glycosylated to any detectable extent (), providing evidence that this region of gp41 is not presented to the lumen of the endoplasmic reticulum and therefore not located outside of the cell after transport to the cell membrane. Finally, anti-HIR serum and anti-HIR antibodies are not able to neutralize even the highly neutralization-sensitive strain SIVmac316 (), despite efficient binding to the Env protein under nondenaturing and nonreducing conditions.