Topology of the C-Terminal Tail of HIV-1 gp41: Differential Exposure of the Kennedy Epitope on Cell and Viral Membranes

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.

Evidence against Extracellular Exposure of a Highly Immunogenic Region in the C-Terminal Domain of the Simian Immunodeficiency Virus gp41 Transmembrane Protein

“…The HIR within gp41CTD of SIVmac239 is highly similar in location, if not in sequence, to the immunogenic region of the HIV-1 gp41CTD, the so-called "Kennedy epitope" (13,30,58). It was this high immunogenicity in HIV-1 which led to the proposal of an alternative topology featuring an extracellular loop (14,17,26).…”

“…Proponents of the alternate model have addressed this inconsistency by suggesting that only a minority of Env molecules assume the conformation with an extracellular loop or that the immunogenic region is only exposed during or after fusion. This alternate model remains controversial; while Steckbeck et al (58) recently reported reactivity of antibodies against the immunogenic region on the surface of Env-expressing cells but not on intact virions, another recent study by Liu et al (34) found no conclusive evidence supporting the formation of an extracellular loop on Env-expressing cells.…”

Neutralizing Epitopes in the Membrane-Proximal External Region of HIV-1 gp41 Are Influenced by the Transmembrane Domain and the Plasma Membrane

“…Further, based upon a model initially proposed in studies by Cleveland et al (11,52), the TM of MPER-TM1 construct could span the membrane twice; conflicting views exist regarding the degree of membrane spanning of the gp41 TM (28). The TM also appears to play a role in the overall exposure of the MPER, as selected amino-acid substitutions in this region of the TM affect binding by all three Nt MAbs (Fig.…”

Unique Functional Properties of Conserved Arginine Residues in the Lentivirus Lytic Peptide Domains of the C-terminal Tail of HIV-1 gp41

“…Predictive Modeling of LLP Peptides Interaction with the Plasma Membrane-The three LLP motifs of the CTT of gp41 have been suggested to interact with the plasma membrane (9,(22)(23)(24)(25)(26)(27)(28), although the depth and orientation of their insertion inside the phospholipid bilayer has not been described. Recently the computational modeling of the E z potential has been described as the ability of peptides to insert into lipid membranes (15,16).…”