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. 2012 Jan;86(2):1145-57.
doi: 10.1128/JVI.06463-11. Epub 2011 Nov 9.

Evidence against extracellular exposure of a highly immunogenic region in the C-terminal domain of the simian immunodeficiency virus gp41 transmembrane protein

Thomas S Postler  1 , José M Martinez-Navio, Eloísa Yuste, Ronald C Desrosiers
Affiliations

Evidence against extracellular exposure of a highly immunogenic region in the C-terminal domain of the simian immunodeficiency virus gp41 transmembrane protein

Thomas S Postler et al. J Virol. 2012 Jan.

Abstract

The generally accepted model for human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein topology includes a single membrane-spanning domain. An alternate model has been proposed which features multiple membrane-spanning domains. Consistent with the alternate model, a high percentage of HIV-1-infected individuals produce unusually robust antibody responses to a region of envelope, the so-called "Kennedy epitope," that in the conventional model should be in the cytoplasm. Here we show analogous, robust antibody responses in simian immunodeficiency virus SIVmac239-infected rhesus macaques to a region of SIVmac239 envelope located in the C-terminal domain, which in the conventional model should be inside the cell. Sera from SIV-infected rhesus macaques consistently reacted with overlapping oligopeptides corresponding to a region located within the cytoplasmic domain of gp41 by the generally accepted model, at intensities comparable to those observed for immunodominant areas of the surface component gp120. Rabbit serum raised against this highly immunogenic region (HIR) reacted with SIV envelope in cell surface-staining experiments, as did monoclonal anti-HIR antibodies isolated from an SIVmac239-infected rhesus macaque. However, control experiments demonstrated that this surface staining could be explained in whole or in part by the release of envelope protein from expressing cells into the supernatant and the subsequent attachment to the surfaces of cells in the culture. Serum and monoclonal antibodies directed against the HIR failed to neutralize even the highly neutralization-sensitive strain SIVmac316. Furthermore, a potential N-linked glycosylation site located close to the HIR and postulated to be outside the cell in the alternate model was not glycosylated. An artificially introduced glycosylation site within the HIR was also not utilized for glycosylation. Together, these data support the conventional model of SIV envelope as a type Ia transmembrane protein with a single membrane-spanning domain and without any extracellular loops.

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Figures

Fig 1
Fig 1
Reactivity of sera from SIV-infected rhesus macaques with overlapping peptides corresponding to the full-length SIVmac239 envelope protein. (A) Average of 10 ELOISAs with sera taken between 16 and 22 weeks after infection. Sample sets included individual sera from five rhesus macaques infected with SIVmac239, two rhesus macaques infected with SIVmac239 g23, which has two N-linked glycosylation sites deleted in the extracellular portion of gp41, and two rhesus macaques infected with SIVmac239 g123, which has three N-linked glycosylation sites deleted in the extracellular portion of gp41 (64); also included was one serum pool from eight rhesus macaques infected with SIVmac239. V1/V2, V1/V2 loop region; V3, V3 loop region; V4, V4 region; V5, V5 region; MSD, membrane-spanning domain; A450, absorbance at 450 nm. (B) Sequence correlates of high reactivity within gp41CTD. The HIR is underlined in the wild-type sequence. A450 > 0.5, number of sera with absorbance values of >0.5 at 450 nm; aa, amino acid.
Fig 2
Fig 2
Hydrophilicity distribution of SIVmac239 envelope protein. (A) Kyte-Doolittle hydrophilicity plot of the entire envelope protein. (B) Kyte-Doolittle hydrophilicity plot of the classical membrane-spanning domain and gp41CTD. MSD1, classical membrane-spanning domain; MSD2, hypothetical second membrane-spanning domain. Green and red, area forming an extracellular loop in a hypothetical alternative structure. Gray, area of gp41CTD unambiguously located within the cytoplasm. (C) Classical and alternative models of gp41CTD structure. Color coding corresponds to that in panel B. The black T shape in the classical model indicates the palmitoyl residue attached to C787. See text for details.
Fig 3
Fig 3
Rabbit serum raised against an oligopeptide corresponding to the highly immunogenic region within gp41CTD (α-HIR serum) reacts with Env in cell surface staining. HEK293T cells were transiently transfected with Env239 and derivatives and then analyzed by flow cytometry. (A) Staining of Env239 with rabbit serum drawn before immunization (preimmune serum) (left), α-HIR serum (middle), or IgG purified from this serum (right). (B) Staining of Env239 and Env316 with α-HIR serum (top) or monoclonal α-gp120 antibody 1.9C (bottom). (C) Staining of progressively truncated Env239 with α-HIR serum (top) or monoclonal α-gp120 antibody 1.9C (bottom). PE, phycoerythrin; APC, allophycocyanin. Mock-transfected samples are depicted in gray shade, env-transfected cells as the black line.
Fig 4
Fig 4
The monoclonal α-HIR antibodies 4B2 and 6E6 react specifically with the HIR of Env of SIVmac239 and SIVmac316 and stain the surface of Env-expressing cells. (A) Reactivity by ELISA of 4B2 and 6E6 with gp120, gp140, an oligopeptide representing the full HIR, and an oligopeptide representing the core of the HIR. A450, absorbance at 450 nm. (B) Reactivity of 4B2 and 6E6 in ELOISAs with oligopeptides representing the full length of Env239. 4B2 reacted only with peptides 6711 and 6712, which correspond to the respective indicated sequences. (C) Cell surface staining of HEK293T cells transiently transfected with Env239, Env239 F725stop, or Env316 with 4B2 (top), 6E6 (middle), or 1.9C (bottom), by flow cytometry. Mock-transfected samples are depicted in gray shade, env-transfected cells as the black line.
Fig 5
Fig 5
Neither the palmitoylated C787, nor R705, which is located within the membrane-spanning domain of gp41, is relevant for cell surface staining with α-HIR serum, while a C-terminal FLAG tag is recognized without permeabilization. HEK293T cells were transiently transfected with Env239 with point mutations or C-terminal FLAG tag and then analyzed by flow cytometry. (A) Staining of envelope with the palmitoylated cysteine mutated to alanine (C787A), with α-HIR serum (top) or 1.9C (bottom). (B) Staining of full-length or truncated Env239 with the arginine of the membrane-spanning domain mutated to leucine (R705L), with α-HIR serum (top) or 1.9C (bottom). (C) Staining of Env239 with a C-terminal FLAG tag (Env239 C-FLAG), with α-HIR serum (top) or monoclonal α-FLAG antibody M2 (bottom). Mock-transfected samples are depicted in gray shade, env-transfected cells as the black line.
Fig 6
Fig 6
The supernatant of Env-expressing cells contains free Env that binds to the surfaces of cells which do not express Env. HEK293T cells were transiently transfected with DNA encoding Env239 or Env239 with a C-terminal FLAG tag (Env239 C-FLAG) or with unrelated empty vector DNA. Thirty-six hours after transfection, medium from these cells was removed, large cellular debris and floating cells were removed by centrifugation, and the supernatant was transferred onto cells that had been transfected with unrelated empty vector DNA. After 2.5 h of incubation at 37°C, cells were washed and processed for cell surface staining by flow cytometry. Cells that received supernatant from mock-transfected cells are depicted with gray shading; cells that received supernatant from Env-expressing cells are depicted as the black line. Top, staining with monoclonal α-HIR antibody 4B2; bottom, staining with monoclonal α-FLAG antibody M2.
Fig 7
Fig 7
gp41CTD is not glycosylated. Immunoblot of cell lysates from HEK293T cells transiently transfected with DNA encoding Env239 and derivates deficient in a known N-linked glycosylation site located in the extracellular part of gp41 (N634Q, N650Q), with a newly introduced N-linked glycosylation consensus motif within the HIR (Q733N) and with an N-linked glycosylation consensus motif within gp41CTD deleted (N759Q). wt, wild type; mock, lysate of mock-transfected cells.
Fig 8
Fig 8
IgG purified from α-HIR serum and monoclonal α-HIR antibodies do not neutralize SIVmac316. Neutralization assays with SIVmac316. (A) Neutralization by IgG purified from α-HIR serum. (B) Neutralization by monoclonal α-HIR antibodies 4B2 and 6E6. The bottom abscissa indicates concentrations of 4B2 and 6E6 in μg/ml; the top abscissa indicates reciprocal serum dilutions of the SIV316+ serum pool and SPF serum. SEAP, secreted alkaline phosphatase; SIVmac316+ serum pool, pooled sera from SIV-infected rhesus macaques; SPF serum, serum from specific-pathogen-free rhesus macaque; KK41 mAb, hybridoma supernatant containing monoclonal α-gp41 antibody KK41; mAb, monoclonal antibody.
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