PR Domain of Rous Sarcoma Virus Gag Causes an Assembly/Budding Defect in Insect Cells

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100

115

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.

Section: Resultsmentioning

confidence: 99%

“…2). Because the PR domain of RSV inhibits proper VLP assembly in insect cells (17), all of the constructs described here lack a PR domain.…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Nucleic Acid-Independent Retrovirus Assembly Can Be Driven by Dimerization

Johnson

Scobie

May

et al. 2002

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100

115

“…Although both MA and NC demonstrate functional conservation, few detailed comparisons of virion size and morphology across genera have been carried out to address whether functional equivalence leads to the same physical characteristics of the VLPs. For instance, although cryo-EM and thin-section transmission EM (TEM) measurements are available for RSV and HIV-1 (39,65), only two reports directly compared wild-type and domain-exchanged VLPs in the same experiment (34,36).…”

mentioning

confidence: 99%

The Retroviral Capsid Domain Dictates Virion Size, Morphology, and Coassembly of Gag into Virus-Like Particles

Ako-adjei

Johnson

Vogt

2005

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The retroviral structural protein, Gag, is capable of independently assembling into virus-like particles (VLPs) in living cells and in vitro. Immature VLPs of human immunodeficiency virus type 1 (HIV-1) and of Rous sarcoma virus (RSV) are morphologically distinct when viewed by transmission electron microscopy (TEM). To better understand the nature of the Gag-Gag interactions leading to these distinctions, we constructed vectors encoding several RSV/HIV-1 chimeric Gag proteins for expression in either insect cells or vertebrate cells. We used TEM, confocal fluorescence microscopy, and a novel correlative scanning EM (SEM)-confocal microscopy technique to study the assembly properties of these proteins. Most chimeric proteins assembled into regular VLPs, with the capsid (CA) domain being the primary determinant of overall particle diameter and morphology. The presence of domains between matrix and CA also influenced particle morphology by increasing the spacing between the inner electron-dense ring and the VLP membrane. Fluorescently tagged versions of wild-type RSV, HIV-1, or murine leukemia virus Gag did not colocalize in cells. However, wild-type Gag proteins colocalized extensively with chimeric Gag proteins bearing the same CA domain, implying that Gag interactions are mediated by CA. A dramatic example of this phenomenon was provided by a nuclear export-deficient chimera of RSV Gag carrying the HIV-1 CA domain, which by itself localized to the nucleus but relocalized to the cytoplasm in the presence of wild type HIV-1 Gag. Wild-type and chimeric Gag proteins were capable of coassembly into a single VLP as viewed by correlative fluorescence SEM if, and only if, the CA domain was derived from the same virus. These results imply that the primary selectivity of Gag-Gag interactions is determined by the CA domain.

“…By this convention, within the immature virus, CA is in the form ␤ Ϫ /SP ϩ , and within the mature virus, it is in the form ␤ ϩ /SP Ϫ . In vitro, the HIV-1 ⌬MACANC and RSV ⌬PRGag proteins, where ␤-hairpin formation is prevented by downstream N-terminal sequences of CA but SP is present (␤ Ϫ /SP ϩ ), assembled into immature spherical particles (21,27). In vitro and in vivo assembly studies on HIV CANC and RSV CANC show that the presence of both N-and C-terminal deter- minants (␤ ϩ /SP ϩ ) of CA results in mature-like tubular (and, in the case of HIV-1, also conical) particle assembly (8,18,22).…”

Section: Discussionmentioning

confidence: 99%

In Vitro Assembly of Virus-Like Particles of a Gammaretrovirus, the Murine Leukemia Virus XMRV

Hadravová

Marco

Ulbrich

et al. 2012

Immature retroviral particles are assembled by self-association of the structural polyprotein precursor Gag. During maturation the Gag polyprotein is proteolytically cleaved, yielding mature structural proteins, matrix (MA), capsid (CA), and nucleocapsid (NC), that reassemble into a mature viral particle. Proteolytic cleavage causes the N terminus of CA to fold back to form a ␤-hairpin, anchored by an internal salt bridge between the N-terminal proline and the inner aspartate. Using an in vitro assembly system of capsid-nucleocapsid protein (CANC), we studied the formation of virus-like particles (VLP) of a gammaretrovirus, the xenotropic murine leukemia virus (MLV)-related virus (XMRV). We show here that, unlike other retroviruses, XMRV CA and CANC do not assemble tubular particles characteristic of mature assembly. The prevention of ␤-hairpin formation by the deletion of either the N-terminal proline or 10 initial amino acids enabled the assembly of ⌬ProCANC or ⌬10CANC into immature-like spherical particles. Detailed three-dimensional (3D) structural analysis of these particles revealed that below a disordered N-terminal CA layer, the C terminus of CA assembles a typical immature lattice, which is linked by rod-like densities with the RNP.