Characterization of Rous sarcoma virus Gag particles assembled in vitro

Purified retrovirus Gag proteins or Gag protein fragments are able to assem ble into virus-like particles (VLPs) in vitro in the presence of RNA. We ha ve examined the role of nucleic acid and of the NC domain in assembly of VL Ps from a Rous sarcoma virus (RSV) Gag protein and have characterized these VLPs using transmission electron microscopy (TEM), scanning TEM (STEM), an d cryoelectron microscopy (cryo-EM). RNAs of diverse sizes, single-stranded DNA oligonucleotides as small as 22 nucleotides, double-stranded DNA, and heparin all promoted efficient assembly. The percentages of nucleic acid by mass, in the VLPs varied from 5 to 8%. The mean mass of VLPs, as determine d by STEM, was 6.5 x 10(7) Da for both RNA-containing and DNA oligonucleoti de-containing particles, corresponding to a stoichiometry of about 1,200 pr otein molecules per VLP, slightly lower than the 1,500 Gag molecules estima ted previously for infectious RSV. By cryo-EM, the VLPs showed the characte ristic morphology of immature retroviruses, with discernible regions of hig h density corresponding to the two domains of the CA protein. In sphericall y averaged density distributions, the mean radial distance to the density c orresponding to the C-terminal domain of CA was 33 nm, considerably smaller than that of equivalent human immunodeficiency virus type 1 particles. Del etions of the distal portion of NC, including the second Zn-binding motif, had little effect on assembly, but deletions including the charged residues between the two Zn-binding motifs abrogated assembly. Mutation of the cyst eine and histidine residues in the first Zn-binding motif to alanine did no t affect assembly, but mutation of the basic residues between the two Zn-bi nding motifs, or of the basic residues in the N-terminal portion of NC, abr ogated assembly. Together, these findings establish VLPs as a good model fo r immature virions and establish a foundation for dissection of the interac tions that lead to assembly.