Association of Nef with the human immunodeficiency virus type 1 core - PubMed (original) (raw)
Association of Nef with the human immunodeficiency virus type 1 core
A Kotov et al. J Virol. 1999 Oct.
Abstract
Highly conserved among primate lentiviruses, the human immunodeficiency virus type 1 (HIV-1) Nef protein enhances viral infectivity by an unknown mechanism. Nef-defective virions are blocked at a stage of the HIV-1 life cycle between entry and reverse transcription, possibly virus uncoating. Nef is present in purified HIV-1 particles; however, it has not been determined whether Nef is specifically recruited into HIV-1 particles or whether virion-associated Nef plays a functional role in HIV-1 replication. To address the specificity and potential functionality of virion-associated Nef, we determined the subviral localization of Nef. HIV-1 cores were isolated by detergent treatment of concentrated virions followed by equilibrium density gradient sedimentation. Relative to HIV-1 virions, HIV-1 cores contained equivalent amounts of reverse transcriptase and integrase, decreased amounts of the viral matrix protein, and trace quantities of the viral transmembrane glycoprotein gp41. Examination of the particles by electron microscopy revealed cone-shaped structures characteristic of lentiviral cores. Similar quantities of proteolytically processed Nef protein were detected in gradient fractions of HIV-1 cores and intact virions. In addition, detergent-resistant subviral complexes isolated from immature HIV-1 particles contained similar quantities of Nef as untreated virions. These results demonstrate that Nef stably associates with the HIV-1 core and suggest that virion-associated Nef plays a functional role in accelerating HIV-1 replication.
Figures
FIG. 1
Isolation of HIV-1 core structures by equilibrium sedimentation through a detergent layer. HIV-1 supernatants were harvested from 293T cells transiently transfected with the R9 wild-type HIV-1 proviral plasmid and filtered to remove cellular debris. Virions were concentrated by ultracentrifugation, resuspended in STE buffer, and layered onto 30 to 70% linear sucrose gradients containing a layer of 1% Triton X-100 at the top. The virions were sedimented through the detergent layer and into the sucrose gradient. Fractions (1 ml) were collected from the bottom of the gradient and assayed for CA concentration by ELISA and density by refractometry. The lane numbers correspond to gradient fractions. (A) Detergent-treated virions; (B) virions subjected to ultracentrifugation on a control gradient lacking detergent.
FIG. 2
Immunoblot analysis of isolated HIV-1 cores. Fractions (1 ml) from sucrose gradients were diluted to 1.5 ml with STE buffer to reduce the density of the solutions, and the particulate material was pelleted by ultracentrifugation. Pellets were dissolved in sample buffer and subjected to immunoblot analysis using various HIV-1 protein-specific antisera. Separate gels containing fractions from detergent-treated virions and untreated virions were transferred onto a single polyvinylidene difluoride filter. Blots were developed by chemiluminescence after probing with the appropriate horseradish peroxidase-conjugated secondary antiserum. In each panel, the upper half of the image shows the material isolated from detergent-treated virions, and the lower part shows control virions isolated in the absence of detergent. (A) Anti-CA plus anti-MA; (B) anti-RT plus anti-IN; (C) anti-gp41; (D) anti-Nef. The molecular masses of protein standards run on the same gel are shown in kilodaltons to the right of each panel.
FIG. 3
Electron microscopic analysis of HIV-1 cores. HIV-1 cores isolated from _env_-defective virions were pelleted, resuspended in 20 μl of STE buffer, and applied to carbon-coated grids that were glow discharged within 30 min of sample application. The grids were subsequently rinsed with 5 to 7 drops of STE buffer and stained with 1% uranyl acetate. Samples were examined in a Philips CM12 transmission electron microscope operating at 120 kV. Electron micrographs were recorded at a nominal magnification of ×35,000. Cone-shaped particles are clearly visible. The size and shape of the particles are identical to those observed in sectioned HIV-1 virions. Bar, 100 nm.
FIG. 4
Nef is associated with immature subviral complexes isolated by detergent treatment of protease-defective virions. Concentrated supernatants from cells from the protease-defective proviral clone R9.PR− were subjected to equilibrium sedimentation through a layer of 1% Triton X-100 into a linear sucrose density gradient. Fractions (1 ml) were collected from the bottom of the tube and assayed for relative Pr55_gag_ content by a CA-specific ELISA. (A) Fractions from the detergent-containing gradient. (B) fractions from a parallel gradient lacking detergent. Particles in each fraction were pelleted by ultracentrifugation and assayed for Nef and Pr55_gag_ by immunoblotting. In each blot, the top panel contains samples from the detergent-containing gradient, while the bottom panel contains samples from a parallel gradient lacking detergent. (C) Analysis of the peak fractions of detergent-treated and untreated virions for Nef. (D) The blot shown in panel C reprobed with antiserum specific for HIV-1 CA.
References
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- Aiken, C. Unpublished data.
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