The retroviral capsid domain dictates virion size, morphology, and coassembly of gag into virus-like particles - PubMed (original) (raw)

The retroviral capsid domain dictates virion size, morphology, and coassembly of gag into virus-like particles

Danso Ako-Adjei et al. J Virol. 2005 Nov.

Abstract

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.

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Figures

FIG. 1.

FIG. 1.

Schematic diagram and descriptions of wild-type and chimeric RSV/HIV-1 Gag proteins and summary of phenotype. RSV domains are in white; HIV-1 domains are in gray.

FIG. 2.

FIG. 2.

Thin-section TEM of wild-type and chimeric RSV/HIV-1 VLPs. (A) Pellet from mixed medium collected from SF9 cells expressing RSV and HIV-1 Gag. (B and C) Pellets from medium collected from DF1 cells expressing Gag proteins by transient transfection. (D to M) SF9 cells expressing Gag proteins by baculovirus infection. Wild-type RSV Gag (B); wild-type HIV-1 Gag (C); HR (D); RH (E); RHR (F); HRH2 (G); pellet from medium collected from SF9 cells expressing RHR2 (H); HRH (I); RnHc (J); HnRc (K); high magnification of RnHc (L); high magnification of HnRc (M). Scale bars, 100 nm.

FIG. 3.

FIG. 3.

Fluorescence visualization of CFP or YFP labeled RSV, HIV-1, and MLV Gag in DF1 cells. HIV-1 Gag-CFP and HIV-1 Gag-YFP (A); RSV Gag-CFP and HIV-1 Gag-YFP (B); HIV-1 Gag-CFP and MLV Gag-YFP (C); RSV Gag-CFP and MLV Gag-YFP (D). Images are pseudocolored (CFP = green; YFP = red) and represent a single focal plane.

FIG. 4.

FIG. 4.

Fluorescence visualization of chimeric and wild type Gag proteins. Samples were prepared as in the results shown in Fig. 3. HRH2-CFP and RSV Gag-YFP (A); RSV Gag-CFP and RHR-YFP (B); HIV-1 Gag-CFP and RHR-YFP (C); HRH2-CFP and HIV-1 Gag-YFP (D); RHR2 alone (40× objective) (E); RHR2-YFP and nonlabeled HIV-1 Gag (40× objective) (F); RSV Gag-CFP and RHR2-YFP (G); RHR2-YFP and HIV-1 Gag-CFP (H). Images are pseudocolored (CFP = green, YFP = red) and represent a single focal plane.

FIG. 5.

FIG. 5.

Correlative SEM and confocal visualization of VLPs from fluorescent wild-type and chimeric Gag proteins from DF1 cells. (A) RSV Gag-YFP and RSV Gag-CFP. Black arrows point to VLPs in SEM images (B) HIV-1 Gag-CFP and RHR-YFP. (C) HIV-1 Gag-YFP and HRH2-CFP particles. The last panel in each row represents an overlay of the SEM and confocal images. Fluorescent images are pseudocolored (CFP = green, YFP = red) and represent a single focal plane.

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References

    1. Amarasinghe, G. K., R. N. De Guzman, R. B. Turner, K. J. Chancellor, Z. R. Wu, and M. F. Summers. 2000. NMR structure of the HIV-1 nucleocapsid protein bound to stem-loop SL2 of the psi-RNA packaging signal. Implications for genome recognition. J. Mol. Biol. 301:491-511. - PubMed
    1. Andrawiss, M., Y. Takeuchi, L. Hewlett, and M. Collins. 2003. Murine leukemia virus particle assembly quantitated by fluorescence microscopy: role of Gag-Gag interactions and membrane association. J. Virol. 77: 11651-11660. - PMC - PubMed
    1. Bennett, R. P., T. D. Nelle, and J. W. Wills. 1993. Functional chimeras of the Rous sarcoma virus and human immunodeficiency virus gag proteins. J. Virol. 67:6487-6498. - PMC - PubMed
    1. Bennett, R. P., and J. W. Wills. 1999. Conditions for copackaging Rous sarcoma virus and murine leukemia virus Gag proteins during retroviral budding. J. Virol. 73:2045-2051. - PMC - PubMed
    1. Bowzard, J. B., R. P. Bennett, N. K. Krishna, S. M. Ernst, A. Rein, and J. W. Wills. 1998. Importance of basic residues in the nucleocapsid sequence for retrovirus Gag assembly and complementation rescue. J. Virol. 72:9034-9044. - PMC - PubMed

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