Cellular Gag-Containing Complexes and HIV Assembly

Abstract

HIV-1 Gag and Gag-Pol structural proteins are required for intracellular assembly, budding, and maturation to an infectious particle. Previous studies have shown that discrete Gag-containing complexes can be isolated from Gag-expressing cell lysates, using velocity sedimentation. We have further studied these GCCs using a variety of biophysical techniques. Give that many GCCs were found to have high molecular mass, but only contain gag monomer, we speculated that some GCCs could result from monomeric Gag binding to large cellular macromolecular complexes. Careful analyses employing varied lysis and gradient conditions confirmed that Gag co-migrated with ribosomes and its subunits under both native and ribosomal-dissociative conditions. Gag co-migration was observed when purified ribosomes and Gag were incubated together, which strongly implied physical interactions. Through combining stable isotopic labeling (SILAC) and quantitative mass spectrometry methodologies, we found that smaller GCCs were interconverting populations potentially functioning as a “feeder pool” for HIV assembly, whereas larger GCCs, containing Gag oligomerization complexes, support VLP formation. These findings provide new perspectives regarding the roles of GCCs in HIV assembly.