Structural and functional analysis of a retroviral Gag polyprotein and some of its mammalian homologs

Abstract

Retroviruses cause immunological disorders and cancers in many vertebrate species. Formation of immature retroviral particles is driven by the Gag polyprotein. Subsequent cleavage of Gag by the viral protease, releases the capsid protein (CA), and leads to formation of the mature and infectious virion. High-resolution images of immature particles were lacking at the onset of this project. To understand how Gag packs within the immature virion, we carried out X-ray crystallographic investigations on truncated variants of the Rous Sarcoma Virus (RSV) Gag protein. This structural analysis confirmed existing models, and highlighted the intrinsic flexibility of the sequences tethering Gag subunits together, which may contribute to the observed structural heterogeneity of the immature particle. The physical forces driving assembly of mature RSV CA, were also investigated using in vitro assembly protocols coupled with sitedirected mutagenesis. We found that near-physiological temperatures trigger in vitro assembly of CA hexamer tubes that effectively model the mature capsid surface. Assembly is strongly modulated by charge-charge interactions, and is a nucleated process that remains thermodynamically favored at lower temperatures, but is effectively arrested by the large energy barrier associated with nucleation. The mammalian genome contains a number of Gag homologs believed to be historically domesticated from retroviruses or LTR retroelements. LTR retroelements are transposable genetic sequences anciently related to retroviruses and are thought to have contributed significantly to the evolution of higher eukaryotes. Domesticated Gag-like proteins play critical roles in essential biological processes. Abnormalities in the production of these proteins are linked to a wide range of disorders such as autism spectrum disorders, cancers and embryonic malformations. The properties of these proteins are surprisingly understudied. Therefore, in vitro characterization of 4 human Gag-like proteins was carried out, in attempt to clarify the nature of their relationship with the ancestral Gag polyprotein and to illuminate some fundamental aspects of their neofunctionalization. Biophysical and biochemical experiments established that several of these Gag homologs have retained a multi-domain architecture and has the ability to drive particle assembly, similar to the retroviral Gag protein. CA-like domains from several of the Gag homologs were isolated and crystallized, and X-ray diffraction data collected, although the structures have not yet been resolved.