In Vitro Assembly of Human Immunodeficiency Virus Type 1 Gag Protein

Abstract

Retroviral Gag protein is sufficient to produce Gag virus-like particles when expressed in higher eukaryotic cells. Here we describe the in vitro assembly reaction of human immunodeficiency virus Gag protein, which consists of two sequential steps showing the optimal conditions for each reaction. Following expression and purification, Gag protein lacking only the C-terminal p6 domain was present as a monomer (50 kDa) by velocity sedimentation analysis. Initial assembly of the Gag protein to 60 S intermediates occurred by dialysis at 4°C in low salt at neutral to alkaline pH. However, higher order of assembly required incubation at 37°C and was facilitated by the addition of Mg 2؉ . Prolonged incubation under these conditions produced complete assembly (600 S), equivalent to Gag virus-like particles obtained from Gag-expressing cells. Neither form disassembled by treatment with nonionic detergent, suggesting that correct assembly might occur in vitro. Electron microscopic observation confirmed that the 600 S assembly products were spherical particles similar to authentic immature human immunodeficiency virus particles. The latter assembly stage but not the former was accelerated by the addition of RNA although not inhibited by RNaseA treatment. These results suggest that Gag protein alone assembles in vitro, but that additional RNA facilitates the assembly reaction.

Human Immunodeficiency Virus Type 1 Gag Assembly through Assembly Intermediates

“…Following extensive wash with binding buffer and subsequently with wash buffer (20 mM Tris (pH 7.9), 150 mM NaCl, and 60 mM imidazole), bound protein was eluted with elute buffer (20 mM Tris (pH 7.9), 150 mM NaCl, and 1 M imidazole). The details of the purification protocol have been described previously (26).…”

“…In Vitro Assembly Reaction-In vitro assembly reaction of purified Gag protein was performed as described previously (26). In brief, following metal chelate chromatography, eluted fractions were desalted using Sephadex G-25 (PD-10) equilibrated with buffer A (20 mM Tris (pH 8.6 adjusted at room temperature), 100 mM NaCl, 0.2 mM EDTA, 5 mM MgCl 2 , and 1 mM dithiothreitol).…”

“…DNA Construction and Protein Expression-The HIV-1 gag gene encoding the Gag region essential for virus particle formation (MA-CAp2-NC) with the additional 6 histidine residues at the C terminus was cloned into E. coli expression vector pTrcHisA, as described previously (26). Transformed E. coli cells were cultured at 37 and 30°C.…”

“…The particles are a hollow sphere surrounded by an electron-dense ring structure, similar to the immature form of HIV VLPs produced by Gag expression in eukaryotic cells (24). Interestingly, spherical particles with similar morphologies are also produced when purified soluble Gag protein is subjected to in vitro assembly reactions (25,26), although the particle sizes are heterogeneous when compared with those of VLPs produced by eukaryotic cells. Similar findings have been reported for other retroviral Gags (27,28).…”

“…We have shown previously, using purified Gag protein lacking the C-terminal p6 domain, that the in vitro assembly reaction is composed of two sequential steps: the formation of a 60 S complex, possibly an assembly intermediate, and complete assembly to 600 S, equivalent to the immature form of HIV capsids (26). Although the ideal for study of Gag assembly would be in higher eukaryotic cell expression systems, the level of Gag expression in such systems is often insufficient for biochemical and structural analyses, and neither expression nor assembly of Gag is synchronized.…”