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 degrees C in low salt at neutral to alkaline pH. However, higher order of assembly required incubation at 37 degrees 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.
Highlights
The main structural component of human immunodeficiency virus (HIV)1particle, Gag, is encoded by the gag gene and is the sole protein required for formation of Gag virus-like particles (VLPs), analogous to the immature form of authentic HIV
This process is thought to consist of several steps: N-terminal myristoylation of Gag protein followed by targeting to the plasma membrane and self-assembly of Gag protein underneath the plasma membrane to form Gag VLPs and budding [1, 5, 6]
We described the in vitro assembly of nearly full-length HIV Gag protein (MA-CA-p2-NC), devoid of only the C-terminal p6 domain, showing the optimal condition for formation of a spherical particle with a double-ring structure, similar to authentic immature HIV particles
Summary
Materials—E. coli expression vector pTrcHisA was purchased from Invitrogen and metal chelate resin (HisBind Resin) from Novagen. A high molecular weight calibration kit was purchased from Amersham Pharmacia Biotech, and prestained protein molecular weight markers (low range) were from Bio-Rad. Calf liver RNA and anti-polyhistidine mouse monoclonal antibody were obtained from Sigma. In Vitro Assembly Reaction—Following chromatography, eluted protein solution was initially adjusted with EDTA to a final concentration of 2 mM (to chelate Ni2ϩ) and dialyzed overnight at 4 °C against 20 mM Tris (pH 8.6 adjusted at room temperature), 100 mM NaCl, 0.2 mM EDTA, and 1 mM dithiothreitol (DTT) unless otherwise indicated. Protein in a gel was either directly detected by Coomassie Brilliant Blue or silver staining or was subjected to Western blotting [35] using anti-HIV-1 CA or anti-polyhistidine monoclonal antibody (Sigma) and anti-mouse IgG alkaline phosphatase conjugate (Cappel). Ultrathin sections were stained with uranyl acetate and lead citrate and examined with an electron microscope (Hitachi H-800)
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