Investigation of membrane protein-protein interactions using correlative FRET-PLA.

Fluorescence resonance energy transfer (FRET) analysis and the recently developed proximity ligation assay (PLA) are widely used to study protein-protein interactions in situ. We have developed correlative FRET-PLA to monitor interactions between membrane proteins that frequently cause problems in confirmatory co-immunoprecipitation assays. Correlative FRET-PLA is particularly aimed at delivering robust and reliable results and is useful for investigating protein-protein interactions.

Immunization with hybrid proteins containing the membrane proximal external region of HIV-1.

The transmembrane envelope (TM) protein gp41 of HIV-1 is an attractive target when designing a vaccine to induce neutralizing antibodies. A few broadly neutralizing antibodies (2F5, 4E10, and 10E8) that target conserved epitopes in the membrane proximal external region (MPER) of gp41 have been isolated from infected individuals. However, attempts to induce such antibodies by immunizations with gp41 and Env derivatives containing the MPER were successful only to some extent.

Improved split-ubiquitin screening technique to identify surface membrane protein-protein interactions.

Yeast-based methods are still the workhorse for the detection of protein-protein interactions (PPIs) in vivo. Yeast two-hybrid (Y2H) systems, however, are limited to screening for a specific group of molecules that interact in a particular cell compartment. For this reason, the split-ubiquitin system (SUS) was developed to allow screening of cDNA libraries of full-length membrane proteins for protein-protein interactions in Saccharomyces cerevisiae.

Modulation of cytokine release and gene expression by the immunosuppressive domain of gp41 of HIV-1.

The transmembrane envelope protein gp41 of the human immunodeficiency virus HIV-1 plays an important role during infection allowing fusion of the viral and cellular membrane. In addition, there is increasing evidence that gp41 may contribute to the immunodeficiency induced by HIV-1. Recombinant gp41 and a synthetic peptide corresponding to a highly conserved domain in gp41, the immunosuppressive (isu) domain, have been shown to inhibit mitogen-induced activation of human peripheral blood mononuclear cells (PBMCs) and to increase release of IL-6 and IL-10 from these cells.

Neutralization of porcine endogenous retrovirus by antibodies against the membrane-proximal external region of the transmembrane envelope protein.

Immunization of different species including goats, rats, hamsters and guinea pigs with the recombinant ectodomain of the transmembrane envelope (TM) protein p15E of porcine endogenous retrovirus (PERV) has been shown to result in the production of virus-neutralizing antibodies. The sera recognize two groups of epitopes, one located in the fusion peptide-proximal region (FPPR) and the second in the membrane-proximal external region (MPER) of p15E. Most interestingly, the epitopes in the MPER are similar to epitopes in the TM protein gp41 of human immunodeficiency virus type 1 (HIV-1) recognized by mAbs 2F5 and 4E10, which broadly neutralize HIV-1.

Characterization of a monoclonal anti-capsid antibody that cross-reacts with three major primate lentivirus lineages.

Mouse monoclonal antibodies with varying specificities against the Gag capsid of simian and human immunodeficiency virus (SIV/HIV) were generated by immunizing mice with whole inactivated SIVagmTYO-1. Monoclonal antibody AG3.0 showed the broadest reactivity recognizing the Gag capsid protein (p24-27) and Gag precursors p38, p55, and p150 of HIV-1, HIV-2, SIVmac, and SIVagm.

Generation of neutralising antibodies against porcine endogenous retroviruses (PERVs).

Antibodies neutralising porcine endogenous retroviruses (PERVs) were induced in different animal species by immunisation with the transmembrane envelope protein p15E. These antibodies recognised epitopes, designated E1, in the fusion peptide proximal region (FPPR) of p15E, and E2 in the membrane proximal external region (MPER). E2 is localised in a position similar to that of an epitope in the transmembrane envelope protein gp41 of the human immunodeficiency virus-1 (HIV-1), recognised by the monoclonal antibody 4E10 that is broadly neutralising.

Mode of interaction between the HIV-1-neutralizing monoclonal antibody 2F5 and its epitope.

Objective: To determine the mechanism of interaction between the HIV-1 gp41-specific broadly neutralizing monoclonal antibody (mAb) 2F5, its epitope in the membrane proximal external region and a domain located in the fusion peptide proximal region in the N-terminal region of gp41. Knowledge of these interactions would be useful for the design of antigens used to induce 2F5-like antibodies.

Methods: The binding and avidity of the mAb 2F5 were analyzed using enzyme-linked immunosorbent assays, epitope mapping and surface plasmon resonance analysis.