Structural analysis of membrane-bound retrovirus capsid proteins

Abstract

We have developed a system for analysis of histidine-tagged (His-tagged) retrovirus core (Gag) proteins, assembled in vitro on lipid monolayers consisting of egg phosphatidylcholine (PC) plus the novel lipid DHGN. DHGN was shown to chelate nickel by atomic absorption spectrometry, and DHGN-containing monolayers specifically bound gold conjugates of His-tagged proteins. Using PC + DHGN monolayers, we examined membrane-bound arrays of an N-terminal His-tagged Moloney murine leukemia virus (M-MuLV) capsid (CA) protein, His-MoCA, and in vivo studies suggest that in vitro-derived His-MoCA arrays reflect some of the Gag protein interactions which occur in assembling virus particles. The His-MoCA proteins formed extensive two-dimensional (2D) protein crystals, with reflections out to 9.5 A resolution. The image-analyzed 2D projection of His-MoCA arrays revealed a distinct cage-like network. The asymmetry of the individual building blocks of the network led to the formation of two types of hexamer rings, surrounding protein-free cage holes. These results predict that Gag hexamers constitute a retrovirus core substructure, and that cage hole sizes define an exclusion limit for entry of retrovirus envelope proteins, or other plasma membrane proteins, into virus particles. We believe that the 2D crystallization method will permit the detailed analysis of retroviral Gag proteins and other His-tagged proteins.

Assembly of Retrovirus Capsid-Nucleocapsid Proteins in the Presence of Membranes or RNA

“…1,2-Di-O-hexadecyl-sn-glycero-3-(1Ј-(2Љ-R-hydroxy-3Ј-N-(5-amino-1-carboxypentyl)iminodiacetic acid) propyl ester (DHGN), which was prepared by D. Thompson, and 1,2-dioleoyl-sn-glycero-3-((N-(5-amino-1-carboxypentyl)iminodiacetic acid) succinimide) (DOGS) (Avanti; kindly supplied by L. WilsonKubalek [48]) were charged with nickel as described previously (4). Lacey and carbon EM grids (300 mesh) were from Ted Pella.…”

“…For M-MuLV, the major PrGag protein interaction (I) domain essential for virus particle assembly (42) appears to be composed of a C-terminal portion of CA plus a portion of NC. Like human immunodeficiency virus type 1 (HIV-1), deletion of the amino-terminal MA domain has proven compatible with M-MuLV particle assembly (4,31,37,47), as long as an amino-terminal membrane-anchoring (M) sequence (42) is retained. Similarly, studies have shown that p12-deleted M-MuLV gag genes can direct the assembly of virus-like particles (4,27,50).…”

“…Like human immunodeficiency virus type 1 (HIV-1), deletion of the amino-terminal MA domain has proven compatible with M-MuLV particle assembly (4,31,37,47), as long as an amino-terminal membrane-anchoring (M) sequence (42) is retained. Similarly, studies have shown that p12-deleted M-MuLV gag genes can direct the assembly of virus-like particles (4,27,50). By comparison, investigations have shown that an intact NC domain is essential for efficient M-MuLV particle assembly (4,27), while major M-MuLV CA deletions compatible with virus formation have yet to be identified.…”

“…Similarly, studies have shown that p12-deleted M-MuLV gag genes can direct the assembly of virus-like particles (4,27,50). By comparison, investigations have shown that an intact NC domain is essential for efficient M-MuLV particle assembly (4,27), while major M-MuLV CA deletions compatible with virus formation have yet to be identified.…”

“…This is of interest because HIV, M-MuLV, RSV, and a number of other avian and mammalian retroviruses assemble on membranes (42). Our approach to the in vitro analysis of retrovirus Gag protein interactions has been to assemble N-terminally histidine-tagged (His-tagged) Gag proteins onto membrane monolayers containing nickel-chelating lipids (4,5,53). Thus, we have shown that His-tagged HIV-1 CA proteins assemble on lipid monolayers in a fashion similar to PrGag proteins in immature HIV-1 particles (5,35,36).…”