Nucleic acid-independent retrovirus assembly can be driven by dimerization - PubMed (original) (raw)

Nucleic acid-independent retrovirus assembly can be driven by dimerization

Marc C Johnson et al. J Virol. 2002 Nov.

Abstract

The Gag protein of retroviruses alone can polymerize into regular virus-like particles (VLPs) both in vitro and in vivo. In most circumstances the capsid (CA) and nucleocapsid (NC) domains of Gag as well as some form of nucleic acid are required for this process. The mechanism by which NC-nucleic acid interaction promotes assembly has remained obscure. We show here that while deletion of the NC domain of Rous sarcoma virus Gag abolishes formation and budding of VLPs at the plasma membranes of baculovirus-infected insect cells, replacement of NC with a dimer-forming leucine zipper domain restores budding of spherical particles morphologically similar to wild-type VLPs. The positioning of the dimerization domain appears to be critical for proper assembly, as the insertion of a 5-amino-acid flexible linker upstream of the zipper domain leads to budding of tubular rather than spherical particles. Similar tubular particles are formed when the same linker is inserted upstream of NC. The tubes are morphologically distinct from tubes formed when the p10 domain upstream of CA is deleted. The fact that a foreign dimerization domain can functionally mimic NC suggests that the role of nucleic acid in retroviral assembly is not to serve as a scaffold but rather to promote the formation of Gag dimers, which are critical intermediates in the polymerization of the Gag shell.

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Figures

FIG. 1.

FIG. 1.

Schematic representation of expressed proteins. MA-NC corresponds to the RSV Gag protein lacking its PR domain at the C terminus.

FIG. 2.

FIG. 2.

EM images of budding structures. Cells were infected with baculovirus vectors expressing either MA-NC (A and B), MA-CA (C and D), MA-CA-Zip (E and F), or MA-NC-fZip (G and H). Panels A, C, E, and G are SEM images (insets are low-magnification images), and panels B, D, F, and H are thin-section TEM images. Bars, 500 nm.

FIG. 3.

FIG. 3.

Size distribution of MA-NC, MA-CA-Zip, and MA-CA-fZip. Diameters of spherical (MA-NC and MA-CA-Zip) or tubular (MA-CA-fZip) particles were measured in TEM thin sections.

FIG. 4.

FIG. 4.

EM images of budding structures of MA-CA-fNC. (Top) SEM; (Bottom) TEM. Bar, 500 nm.

FIG. 5.

FIG. 5.

EM images of p10 budding structures. Cells were infected with baculovirus vectors expressing either MA-NC dp10 (A and B) or MA-NC dp10 + 25 AA (C and D). (A and C) SEM images; (B and D) thin-section TEM images. Bars, 500 nm.

FIG. 6.

FIG. 6.

Size distribution of MA-NC, MA-NC dp10, and MA-NC dp10 + 25 AA particles. Diameters of spherical (MA-NC and MA-NC dp10 + 25 AA) or tubular (MA-NC dp10) particles were measured in TEM thin sections.

FIG. 7.

FIG. 7.

Detergent stability of particles. Particles were pelleted after incubation with or without Triton X-100 (Tx-100) as described in Materials and Methods. Pellets were dissolved in SDS, and the proteins were separated by SDS-PAGE and then either stained with Coomassie blue, probed with an antibody against RSV CA, or probed with an antibody against baculovirus gp64.

FIG. 8.

FIG. 8.

Density of particles. Pelleted particles from each expression construct were resuspended and then centrifuged to equilibrium in 10-to-60% (wt/wt) sucrose gradients. A portion of each fraction was separated by SDS-PAGE and probed with an antibody against RSV CA. The density of each fraction was determined by refractometry. Fractions from the boxed lanes were pooled, diluted to reduce the sucrose concentration, and then centrifuged again to collect the particles. The pellets were then viewed by thin-section TEM and are shown on the right.

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