Human Immunodeficiency Virus Type 1 Gag Assembly through Assembly Intermediates

Abstract

Human immunodeficiency virus Gag protein self-assembles into spherical particles, and recent reports suggest the formation of assembly intermediates during the process. To understand the nature of such assembly intermediates along with the mechanism of Gag assembly, we employed expression in Escherichia coli and an in vitro assembly reaction. When E. coli expression was performed at 37°C, Gag predominantly assembled to a high order of multimer, apparently equivalent to the virus-like particles obtained following Gag expression in eukaryotic cells, through the formation of low orders of multimer characterized with a discreet sedimentation value of 60 S. Electron microscopy confirmed the presence of spherical particles in the E. coli cells. In contrast, expression at 30°C resulted in the production of only the 60 S form of Gag multimer, and crescentshaped structures or small patches with double electron-dense layers were accumulated, but no complete particles. In vitro assembly reactions using purified Gag protein, when performed at 37°C, also produced the high order of Gag multimers with some 60 S multimers, whereas the 30°C reaction produced only the 60 S multimers. However, when the 60 S multimers were crosslinked so as not to allow conformational changes, in vitro assembly reactions at 37°C did not produce any higher order of multimers. ATP depletion did not halt Gag assembly in the E. coli cells, and the addition of GroEL-GroES to in vitro reactions did not facilitate Gag assembly, indicating that conformational changes rather than protein refolding by chaperonins, induced at 37°C, were solely responsible for the Gag assembly observed here. We suggest that Gag assembles to a capsid through the formation of the 60 S multimer, possibly a key intermediate of the assembly process, accompanied with conformational changes in Gag.

Analysis of Human Immunodeficiency Virus Type 1 Gag Dimerization-Induced Assembly

“…The 37°C preference for HIV-1 Gag protein in vitro assembly has been observed previously. Morikawa et al demonstrated that bacterially expressed HIV-1 Gag proteins lacking the p6 domain preferentially assembled at 37°C (31). With similar NC-containing HIV-1 Gag proteins, we also observed an assembly temperature dependence, and while assembly levels increased with the addition of RNA at all temperatures, the temperature dependence was not removed (18).…”

“…We speculate that the material in fractions 6 and 7, sedimenting at a somewhat higher rate than bacterial 70S ribosomes (Fig. 5E), may correspond to previously observed assembly intermediates (22,24,31).…”

“…We envision the third step, facilitated by increasing the temperature or by polyanion addition in our systems, to be a conformation change from assembly-restricted dimers or small oligomers to assembly-competent ones. At that stage, final particle assembly can occur, possibly via a set of larger assembly intermediates (22,24,31) (Fig. 5).…”

Intermolecular Interactions between Retroviral Gag Proteins in the Nucleus

“…Higher-order Gag complexes isolated from the cytoplasm represent dimers and multimeric structures that are larger than simple dimers (29,31,56). However, it is not known where Gag-Gag dimers are initially formed within the cell.…”

“…Gag dimers and multimers spontaneously form in vitro when recombinant Gag proteins are mixed with nucleic acids, which promote Gag-Gag multimerization (8,9,12,16,31,32). However, an RNA-independent protein-protein interaction domain can substitute for I domain activity in NC to mediate dimer formation (22).…”