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. 2010 Dec 7;5(12):e15261.
doi: 10.1371/journal.pone.0015261.

Topology of the C-terminal tail of HIV-1 gp41: differential exposure of the Kennedy epitope on cell and viral membranes

Jonathan D Steckbeck  1 , Chengqun Sun, Timothy J Sturgeon, Ronald C Montelaro
Affiliations

Topology of the C-terminal tail of HIV-1 gp41: differential exposure of the Kennedy epitope on cell and viral membranes

Jonathan D Steckbeck et al. PLoS One. .

Abstract

The C-terminal tail (CTT) of the HIV-1 gp41 envelope (Env) protein is increasingly recognized as an important determinant of Env structure and functional properties, including fusogenicity and antigenicity. While the CTT has been commonly referred to as the "intracytoplasmic domain" based on the assumption of an exclusive localization inside the membrane lipid bilayer, early antigenicity studies and recent biochemical analyses have produced a credible case for surface exposure of specific CTT sequences, including the classical "Kennedy epitope" (KE) of gp41, leading to an alternative model of gp41 topology with multiple membrane-spanning domains. The current study was designed to test these conflicting models of CTT topology by characterizing the exposure of native CTT sequences and substituted VSV-G epitope tags in cell- and virion-associated Env to reference monoclonal antibodies (MAbs). Surface staining and FACS analysis of intact, Env-expressing cells demonstrated that the KE is accessible to binding by MAbs directed to both an inserted VSV-G epitope tag and the native KE sequence. Importantly, the VSV-G tag was only reactive when inserted into the KE; no reactivity was observed in cells expressing Env with the VSV-G tag inserted into the LLP2 domain. In contrast to cell-surface expressed Env, no binding of KE-directed MAbs was observed to Env on the surface of intact virions using either immune precipitation or surface plasmon resonance spectroscopy. These data indicate apparently distinct CTT topologies for virion- and cell-associated Env species and add to the case for a reconsideration of CTT topology that is more complex than currently envisioned.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Schematic models of the HIV-1 CTT.
A.) Traditional CTT model with one membrane-spanning α-helix and a completely intracytoplasmic localization of the remaining CTT sequence. LLP domains have been placed at their presumed membrane-localized position. B.) Alternative CTT model with multiple MSD segments as proposed by Hollier and Dimmock . This model proposes three membrane-spanning β-sheets and an extracellular localization of the KE.
Figure 2
Figure 2. Sequence alignment of variant Env CTT.
Sequence alignment of the membrane-spanning domain (MSD) and CTT of the Env proteins used in this study. All Env proteins were full-length gp160, however, only the sequences from K665 to the C-terminus are shown for simplicity. Structural and sequence domains are indicated in boxes above the corresponding sequence. VSV-G epitope tag indicated in bold; SAR1 and 1577 epitope indicated in gray box. KE – Kennedy epitope; LLP – lentivirus lytic peptide, MSD – membrane spanning domain.
Figure 3
Figure 3. FACS analysis of intact Env-expressing cells demonstrates extracellular exposure of the KE sequence.
Cells transfected with HIV-1 89.6 WT, VSV-G-KE, or VSV-G-LLP2 were analyzed by FACS to determine VSV-G epitope accessibility to anti-VSV-G MAb. A.) Intact, non-permeabilized Env-expressing cells were stained with α-VSV-G (AlexaFluor (AF) 700) and α-actin (AF 488). B.) Intact cells expressing Env were stained for surface exposure of the KE using a native KE antibody, SAR1. C.) Same as (B) except cells were permeabilized prior to staining.
Figure 4
Figure 4. VSV-G epitope insertions do not disrupt Env association with detergent-resistant membranes.
Env association with detergent resistant membranes was determined by sucrose gradient floatation followed by western blot analysis. Lane 1 represents the top of the gradient and lane 12 the bottom of the gradient. Blots were stained with anti-gp41 MAb Chessie 8 to determine the localization of gp41 in the bands shown.
Figure 5
Figure 5. Anti-KE MAbs do not bind to intact virions.
The indicated proteins and viral particles were immunoprecipitated using reference MAbs coupled to protein G-coated paramagnetic beads. (Top) Open bars represent % of target antigen precipitated when incubated with solubilized virus, while closed bars represent the % of input p24 precipitated by the corresponding MAb under native (intact virus) conditions. #  = p<0.05 for MAbs compared to IgG control with solubilized virus; *  = p<0.05 for MAbs with intact virus compared to IgG control. (Bottom) Representative p24 bands immunoprecipitated using the MAbs indicated in top panel with intact virus (Intact) or the bands of the target antigen from each MAb in detergent-disrupted virus (Solublized).
Figure 6
Figure 6. Comparison of MPER and KE MAb binding using SPR spectroscopy.
MAbs 2F5 (MPER) and SAR1 (KE) were compared for relative binding rates and affinity using SPR spectroscopy to monitor antibody binding to purified intact HIV-1 virions. RU – resonance units.
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References

    1. Evans DT, Desrosiers RC. Immune evasion strategies of the primate lentiviruses. Immunol Rev. 2001;183:141–158. - PubMed
    1. Luciw PA. Human immunodeficiency viruses and their replication. In: Fields BN, Knipe DM, Howley PM, editors. Fields' Virology. Third ed. Philadelphia: Lippincott-Raven Publishers; 2002. pp. 1881–1952.
    1. Craigo JK, Zhang B, Barnes S, Tagmyer TL, Cook SJ, et al. Envelope variation as a primary determinant of lentiviral vaccine efficacy. Proc Natl Acad Sci U S A. 2007;104:15105–15110. - PMC - PubMed
    1. Jiang J, Aiken C. Maturation-dependent human immunodeficiency virus type 1 particle fusion requires a carboxyl-terminal region of the gp41 cytoplasmic tail. J Virol. 2007;81:9999–10008. - PMC - PubMed
    1. Freed EO, Martin MA. 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. 1995;69:1984–1989. - PMC - PubMed

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