Self-assembly in vitro of purified CA-NC proteins from Rous sarcoma virus and human immunodeficiency virus type 1

Abstract

The internal structural proteins of retroviruses are proteolytically processed from the Gag polyprotein, which alone is able to assemble into virus-like particles when expressed in cells. All Gag proteins contain domains corresponding to the three structural proteins MA, CA, and NC. We have expressed the CA and NC domains together as a unit in Escherichia coli, both for Rous sarcoma virus (RSV) and for human immunodeficiency virus type 1 (HIV-1). We also expressed a similar HIV-1 protein carrying the C-terminal p6 domain. RSV CA-NC, HIV-1 CA-NC, and HIV-1 CA-NC-p6 were purified in native form by classic methods. After adjustment of the pH and salt concentration, each of these proteins was found to assemble at a low level of efficiency into structures that resembled circular sheets and roughly spherical particles. The presence of RNA dramatically increased the efficiency of assembly, and in this case all three proteins formed hollow, cylindrical particles whose lengths were determined by the size of the RNA. The optimal pH at which assembly occurred was 5.5 for the RSV protein and 8.0 for the HIV-1 proteins. The treatment of the RSV CA-NC cylindrical particles with nonionic detergent, with ribonuclease, or with viral protease caused disassembly. These results suggest that RNA plays an important structural role in the virion and that it may initiate and organize the assembly process. The in vitro system described should facilitate the dissection of assembly pathways in retroviruses.

Supramolecular Architecture of Severe Acute Respiratory Syndrome Coronavirus Revealed by Electron Cryomicroscopy

“…However, a subsequent cryoelectron tomography study did not detect ordered packing of Moloney murine leukemia virus Env (28). In retrovirus particles, Env distribution may be constrained by interaction with underlying hexagonal lattices of Gag that have been observed for several retroviruses (5,32,33,41,50,60,90). Cryo-EM and image analysis of arenavirus particles revealed ribonucleoprotein densities that appeared to be organized in a two-dimensional, membrane-proximal lattice (62).…”

Proteolytic refolding of the HIV-1 capsid protein amino-terminus facilitates viral core assembly

“…We have demonstrated recently that the cylinders formed in vitro by the HIV-1 capsid protein are helical (J.Finch, S.Li, V.Klishko, C.P. Hill and W.I.Sundquist, in preparation) and speculate that all viral cores will exhibit helical arrays of capsid, perhaps differing in how the helices distort to accommodate the viral RNA genome (Campbell and Vogt, 1995).…”

Virus assembly, allostery and antivirals

“…The elements of Gag that are most important for this discussion are the twodomain capsid (CA) segment (the primary mediator of Gag-Gag interactions), a spacer peptide that follows CA, and the RNA-binding nucleocapsid (NC) segment. The earliest suggestion of allostery in retrovirus assembly came from in vitro assembly studies using DNA oligomers and Gag [22,23]. Gag dimerization was crucial for assembly: HIV Gag would not assemble with very short oligos nor if the oligo had high-affinity sequences separated by a low-affinity sequence, suggesting that protein-protein interaction was required to activate assembly [24].…”