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. 2007 Oct;81(19):10718-28.
doi: 10.1128/JVI.01061-07. Epub 2007 Jul 18.

Overlapping roles of the Rous sarcoma virus Gag p10 domain in nuclear export and virion core morphology

Lisa Z Scheifele  1 , Scott P Kenney, Tina M Cairns, Rebecca C Craven, Leslie J Parent
Affiliations

Overlapping roles of the Rous sarcoma virus Gag p10 domain in nuclear export and virion core morphology

Lisa Z Scheifele et al. J Virol. 2007 Oct.

Abstract

Nucleocytoplasmic shuttling of the Rous sarcoma virus (RSV) Gag polyprotein is an integral step in virus particle assembly. A nuclear export signal (NES) was previously identified within the p10 domain of RSV Gag. Gag mutants containing deletions of the p10 NES or mutations of critical hydrophobic residues at positions 219, 222, 225, or 229 become trapped within the nucleus and exhibit defects in the efficiency of virus particle release. To investigate other potential roles for Gag nuclear trafficking in RSV replication, we created viruses bearing NES mutant Gag proteins. Viruses carrying p10 mutations produced low levels of particles, as anticipated, and those particles that were released were noninfectious. The p10 mutant viruses contained approximately normal amounts of Gag, Gag-Pol, and Env proteins and genomic viral RNA (vRNA), but several major structural defects were found. Thin-section transmission electron microscopy revealed that the mature particles appeared misshapen, while the viral cores were cylindrical, horseshoe-shaped, or fragmented, with some particles containing multiple small, electron-dense aggregates. Immature virus-like particles produced by the expression of Gag proteins bearing p10 mutations were also aberrant, with both spherical and tubular filamentous particles produced. Interestingly, the secondary structure of the encapsidated vRNA was altered; although dimeric vRNA was predominant, there was an additional high-molecular-weight fraction. Together, these results indicate that the p10 NES domain of Gag is critical for virus replication and that it plays overlapping roles required for the nuclear shuttling of Gag and for the maintenance of proper virion core morphology.

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Figures

FIG. 1.
FIG. 1.
Schematic of NES mutant viruses. The wild-type RSV Gag polyprotein is depicted at the top with the MA, p2, p10, CA, SP, NC, and PR domains indicated. Below is the amino acid sequence of the p10 domain, with the four hydrophobic amino acids comprising the NES shown in gray. The p10 mutants are listed below, with horizontal lines depicting unaltered sequence and vertical lines delineating the last amino acid present in each deletion construct. Amino acid sequences for the NES substitutions are indicated. Deletion ΔQM1 removes codons 176 to 200, but preserves the Gag NES. The Δp10.52 and Δp10.31 mutations delete codons 183 to 234 and 193 to 223, respectively, including all or most of the export sequence. The alanines that replace each critical hydrophobic residue are underlined.
FIG. 2.
FIG. 2.
Subcellular localization of Gag-GFP fusion proteins. The Gag-GFP protein is depicted above, illustrating the in-frame fusion of GFP in place of PR, with the additional loss of the C-terminal seven residues of NC during cloning. Below are confocal microscopy images of QT6 cells expressing the indicated Gag-GFP fusion proteins, either with or without the addition of 18 nM LMB 2 h prior to imaging. WT, wild type.
FIG. 3.
FIG. 3.
Virus particle assembly. (A) Quail fibroblasts were transfected with proviral constructs expressing variants of Gag in the context of the RC.V8 plasmid, and budding assays were performed. Budding efficiency was calculated as described in Materials and Methods, and the wild-type level of budding was arbitrarily set at 100%. Error bars represent standard deviations, and each experiment was performed in at least three replicates. (B) The budding assay was performed and analyzed in a fashion identical to that described for panel A except that the gag mutations were introduced into the pRS.V8 background. (C) Gag proteins were immunoprecipitated from lysates or media of transfected cells and analyzed by SDS-PAGE. Molecular mass standards (kDa) are indicated to the left, and the positions of CA-S, CA, and CA-SP are shown to the right.
FIG. 4.
FIG. 4.
Infectivity of viruses with mutations in the Gag p10 NES sequence. Equal amounts of viral particles produced by transfection with the indicated proviral plasmids were added to either QT6 (panels A and B) or TEF cells (panel C). The ability of the viruses to spread throughout the culture was assayed by the RT activity present in cell culture supernatants collected every 3 days (A and B) or by the percentage of cells expressing GFP (C) over time.
FIG. 5.
FIG. 5.
Virion RNA content and analysis of RNA dimer structure. (A) Viral particles were normalized based on RT activity, equivalent numbers of particles were lysed, and vRNA incorporation was quantitatively assessed by RNase protection assay using a labeled probe spanning the 3′ splice site of the vRNA (nucleotides 4998 to 5257). The amount of vRNA detected for each mutant construct was compared to that in the wild type, which was set at 1.0. Each bar represents the average for at least three independent experiments with standard deviations shown. (B) Nondenaturing Northern blotting analysis of vRNA isolated from virus particles. The positions of the HMW species, dimers (D), and monomers (M) are indicated to the left.
FIG. 6.
FIG. 6.
Thin-section electron microscopy analysis of mature viral particles. QT6 cells expressing the wild-type virus (a) or mutants ΔQM1 (b), Δp10.31 (c and d), Δp10.52 (e and f), W222A (g and h), L219A (i, j, and m), V225A (k and n) or L229A (l and o) were fixed, stained, thin sectioned and visualized by transmission electron microscopy. The scale bar represents 80 nm for each image. Black arrows show particles with wild-type morphology (spherical, electron-dense core and a thin lucent region between the core and the viral envelope); arrowheads indicate diffuse, multiple cores; white arrows point to particles with tubular or cylindrical cores; “t” specifies tubular or filamentous particles; and “im” indicates cores with immature morphology.
FIG. 7.
FIG. 7.
Electron microscopy images of cell-associated immature virus-like particles. Quail fibroblasts were processed exactly as described in the Fig. 6 legend. Cells were transfected with plasmids expressing wild-type or mutant Gag proteins (as labeled) with C-terminal truncations that remove the PR sequence. Scale bars are labeled with dimensions. Arrows point to chains of spherical particles and arrowheads indicate elongated tubular particles. Higher magnification insets of individual particles in panels c, f, i, and l are included to show the detailed structure of the immature cores.
FIG. 8.
FIG. 8.
Model of mature virion core morphology. (A) In wild-type virions, the vRNP complex begins as an elongated, uncondensed structure, similar in appearance to influenza virus RNPs (38). The vRNA dimer and NC undergo a conformational change during maturation, and the core condenses into a sphere. (B) With alteration of the p10 domain, maturation of the vRNA and the Gag protein are incomplete. Condensation of the RNP complex is impaired, and viral cores adopt a variety of different appearances, depending on the angle from which they are viewed. This illustration suggests a possible mechanism for how multiple cores, elongated cores, and fragmented or horseshoe-shaped cores might be formed in p10 mutant virus particles.
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