OA30.06 LB
Background: HIV-1 has 7–15 envelope spike glycoproteins (Env) per virion consisting of a trimer of non-covalently linked gp120-gp41 heterodimers. These “functional” spikes are essential to virion binding and fusion, however it is unclear as to number required to mediate efficient fusion. Indeed other non-functional Env conformations, including uncleaved precursors (gp160), aberrant oligmers, monomers and gp41 stumps devoid of gp120 are also thought to be displayed on virions. The extent to which functional and non-function forms of Env are co-displayed on individual virions is currently a matter of debate. Recently, we developed a new technique, Flow Virometry that allows the study of proteins on the surface of individual virions. We have applied this technique to probe the conformation of Envs on the surface of individual particles using a panel of anti-env antibodies that discriminate between different conformations of these molecules.
Methods: HIV-1 virions of BaL strain grown in PM1 CD4 T cells were captured with 15 nm magnetic nanoparticles (MNPs) coupled to one of several monoclonal antibodies recognizing particular conformations of Env. Captured virions were then stained with fluorescent anti-Env antibodies different from the capture antibody and separated from free antibodies on a magnetic column. A range of neutralizing and non-neutralizing antibodies targeting functional and non-function Env forms were used to stain virions and their representation on individual viral particles assessed by flow virometry.
Results: Our data indicate a non-uniform distribution of functional and non-functional Env within a viral population, where differential patterns of antibody staining revealed virion populations that were homogenous or mosaic with respect to functional and non-functional forms of Env.
Conclusions: These finding have important implications for understanding antibody binding and neutralization, as well as other antibody effector functions.
