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

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

An engineered poliovirus chimaera elicits broadly reactive HIV-1 neutralizing antibodies

The Sabin type 1 vaccine strain of poliovirus is probably the safest and most successful live-attenuated vaccine virus used in humans. Its widespread use since the early 1960s has contributed significantly to the virtual eradication of poliomyelitis in developed countries. We have reported previously the construction of an intertypic antigen chimaera of poliovirus, based on the Sabin 1 strain, and proposed that this virus could be modified to express on its surface antigenic determinants from other pathogens. We describe here the construction and characterization of a poliovirus antigen chimaera containing an epitope from the transmembrane glycoprotein (gp41) of human immunodeficiency virus type 1 (HIV-1). In antibody absorption experiments, the virus chimaera inhibited neutralization of HIV-1 by antipeptide monoclonal antibodies specific for the gp41 epitope and significantly reduced the group specific neutralizing activity of HIV-1-positive human sera. Rabbit antisera raised by subcutaneous injection of the polio/HIV chimaera in adjuvant was shown to be specific for HIV-1 gp41 in peptide-binding assays and by western blotting. Moreover, the antisera neutralized a wide range of American and African HIV-1 isolates and also inhibited virus-induced cell fusion. Monoclonal antibodies against the HIV-1 derived regions of the chimaera also neutralized HIV-1. These results establish the potential of using poliovirus for the presentation of foreign antigens and suggest that Sabin 1 poliovirus/HIV chimaeras could offer an approach to the development of an HIV vaccine