Membrane proteins are of outstanding importance in biology, drug discovery and vaccination. A common limiting factor in research and applications involving membrane proteins is the ability to solubilize and stabilize membrane proteins. Although detergents represent the major means for solubilizing membrane proteins, they are often associated with protein instability and poor applicability in structural and biophysical studies. Here, we present a novel lipoprotein nanoparticle system that allows for the reconstitution of membrane proteins into a lipid environment that is stabilized by a scaffold of Saposin proteins. We showcase the applicability of the method on two purified membrane protein complexes as well as the direct solubilization and nanoparticle-incorporation of a viral membrane protein complex from the virus membrane. We also demonstrate that this lipid nanoparticle methodology facilitates high-resolution structural studies of membrane proteins in a lipid environment by single-particle electron cryo-microscopy (cryo-EM) and allows for the stabilization of the HIV-envelope glycoprotein in a functional state.

Type 5 adenovirus (Ad5) is a human pathogen that has been widely developed for therapeutic uses, with only limited success to date. We report here the novel finding that human erythrocytes present Coxsackie virus-adenovirus receptor (CAR) providing an Ad5 sequestration mechanism that protects against systemic infection. Interestingly, erythrocytes from neither mice nor rhesus macaques present CAR. Excess Ad5 fiber protein or anti-CAR antibody inhibits the binding of Ad5 to human erythrocytes and cryo-electron microscopy shows attachment via the fiber protein of Ad5, leading to close juxtaposition with the erythrocyte membrane. Human, but not murine, erythrocytes also present complement receptor (CR1), which binds Ad5 in the presence of antibodies and complement. Transplantation of human erythrocytes into nonobese diabetic/severe combined immunodeficiency mice extends blood circulation of intravenous Ad5 but decreases its extravasation into human xenograft tumors. Ad5 also shows extended circulation in transgenic mice presenting CAR on their erythrocytes, although it clears rapidly in transgenic mice presenting erythrocyte CR1. Hepatic infection is inhibited in both transgenic models. Erythrocytes may therefore restrict Ad5 infection (natural and therapeutic) in humans, independent of antibody status, presenting a formidable challenge to Ad5 therapeutics. “Stealthing” of Ad5 using hydrophilic polymers may enable circumvention of these natural virus traps.