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Cryo electron tomography of native HIV-1 budding sites.

Carlson LA, de Marco A, Oberwinkler H, Habermann A, Briggs JA, Kräusslich HG, Grünewald K - PLoS Pathog. (2010)

Bottom Line: Besides the immature lattice, a previously not described Gag lattice was detected in some budding sites and released particles; this lattice was found at high frequencies in a subset of infected T-cells.Buds and released particles carrying this lattice consistently lacked the viral ribonucleoprotein complex, suggesting that they correspond to aberrant products due to premature proteolytic activation.We hypothesize that cellular and/or viral factors normally control the onset of proteolytic maturation during assembly and release, and that this control has been lost in a subset of infected T-cells leading to formation of aberrant particles.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Virology, Universitätsklinikum Heidelberg, Heidelberg, Germany.

ABSTRACT
The structure of immature and mature HIV-1 particles has been analyzed in detail by cryo electron microscopy, while no such studies have been reported for cellular HIV-1 budding sites. Here, we established a system for studying HIV-1 virus-like particle assembly and release by cryo electron tomography of intact human cells. The lattice of the structural Gag protein in budding sites was indistinguishable from that of the released immature virion, suggesting that its organization is determined at the assembly site without major subsequent rearrangements. Besides the immature lattice, a previously not described Gag lattice was detected in some budding sites and released particles; this lattice was found at high frequencies in a subset of infected T-cells. It displays the same hexagonal symmetry and spacing in the MA-CA layer as the immature lattice, but lacks density corresponding to NC-RNA-p6. Buds and released particles carrying this lattice consistently lacked the viral ribonucleoprotein complex, suggesting that they correspond to aberrant products due to premature proteolytic activation. We hypothesize that cellular and/or viral factors normally control the onset of proteolytic maturation during assembly and release, and that this control has been lost in a subset of infected T-cells leading to formation of aberrant particles.

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Filamentous actin at HIV-1 budding sites.The 39 budding sites reconstructed by cryo electron tomography were sorted into five categories, based on the type of filamentous actin structures (if any) present in their vicinity. Top panels are a sketch of the category, showing the Gag lattice in red, the plasma membrane in blue and filamentous actin in black. The lower panels show a computational slice through one budding site from the category, and the numbers below state the number of budding sites (and the number of tomograms they were obtained from) in the category. (A) Budding site at the side of actin-filled filopodium; (B) budding site at the tip of actin-filled filopodium; (C) budding site with cortical actin parallel to the plasma membrane; (D) budding site with cortical actin directed towards or protruding into the budding site; (E) budding site at the plasma membrane, not adjacent to filamentous actin. Scale bar is 100 nm.
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ppat-1001173-g003: Filamentous actin at HIV-1 budding sites.The 39 budding sites reconstructed by cryo electron tomography were sorted into five categories, based on the type of filamentous actin structures (if any) present in their vicinity. Top panels are a sketch of the category, showing the Gag lattice in red, the plasma membrane in blue and filamentous actin in black. The lower panels show a computational slice through one budding site from the category, and the numbers below state the number of budding sites (and the number of tomograms they were obtained from) in the category. (A) Budding site at the side of actin-filled filopodium; (B) budding site at the tip of actin-filled filopodium; (C) budding site with cortical actin parallel to the plasma membrane; (D) budding site with cortical actin directed towards or protruding into the budding site; (E) budding site at the plasma membrane, not adjacent to filamentous actin. Scale bar is 100 nm.

Mentions: In cryo electron tomograms of AdGag or AdGagPol transduced cells (Fig. 2) structural features of the cytoplasm were preserved at the high level previously reported for cellular cET [30], with single ribosomes, actin filaments and microtubules being clearly resolved. Both viral budding sites (Fig. 2A, C–F) and released virions (Fig. 2B–C, E) were present in the recorded tomograms. The budding sites were found at the plasma membrane on lamellipodia–like areas (Fig. 2C, E), occasionally in membrane invaginations (Fig. 2B, D) or at the tips (Fig. 2F, 3B) or sides (Fig. 2A, 3A) of filopodial structures. In the immature Gag lattice of these budding sites and released particles, the two density layers corresponding to CA and NC-RNA could be resolved, and occasionally the hexagonal symmetry in the CA layer was apparent by eye in more tangential tomographic slices. Released immature particles (ip) were found adjacent to cells expressing Gag (Fig. 2B, D) or GagPol (Fig. 2E), whereas released particles with mature morphology (mp) were only found adjacent to cells expressing GagPol (Fig. 2E). No density attributable to components of the ESCRT complex was detected at budding sites at the current resolution.


Cryo electron tomography of native HIV-1 budding sites.

Carlson LA, de Marco A, Oberwinkler H, Habermann A, Briggs JA, Kräusslich HG, Grünewald K - PLoS Pathog. (2010)

Filamentous actin at HIV-1 budding sites.The 39 budding sites reconstructed by cryo electron tomography were sorted into five categories, based on the type of filamentous actin structures (if any) present in their vicinity. Top panels are a sketch of the category, showing the Gag lattice in red, the plasma membrane in blue and filamentous actin in black. The lower panels show a computational slice through one budding site from the category, and the numbers below state the number of budding sites (and the number of tomograms they were obtained from) in the category. (A) Budding site at the side of actin-filled filopodium; (B) budding site at the tip of actin-filled filopodium; (C) budding site with cortical actin parallel to the plasma membrane; (D) budding site with cortical actin directed towards or protruding into the budding site; (E) budding site at the plasma membrane, not adjacent to filamentous actin. Scale bar is 100 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991257&req=5

ppat-1001173-g003: Filamentous actin at HIV-1 budding sites.The 39 budding sites reconstructed by cryo electron tomography were sorted into five categories, based on the type of filamentous actin structures (if any) present in their vicinity. Top panels are a sketch of the category, showing the Gag lattice in red, the plasma membrane in blue and filamentous actin in black. The lower panels show a computational slice through one budding site from the category, and the numbers below state the number of budding sites (and the number of tomograms they were obtained from) in the category. (A) Budding site at the side of actin-filled filopodium; (B) budding site at the tip of actin-filled filopodium; (C) budding site with cortical actin parallel to the plasma membrane; (D) budding site with cortical actin directed towards or protruding into the budding site; (E) budding site at the plasma membrane, not adjacent to filamentous actin. Scale bar is 100 nm.
Mentions: In cryo electron tomograms of AdGag or AdGagPol transduced cells (Fig. 2) structural features of the cytoplasm were preserved at the high level previously reported for cellular cET [30], with single ribosomes, actin filaments and microtubules being clearly resolved. Both viral budding sites (Fig. 2A, C–F) and released virions (Fig. 2B–C, E) were present in the recorded tomograms. The budding sites were found at the plasma membrane on lamellipodia–like areas (Fig. 2C, E), occasionally in membrane invaginations (Fig. 2B, D) or at the tips (Fig. 2F, 3B) or sides (Fig. 2A, 3A) of filopodial structures. In the immature Gag lattice of these budding sites and released particles, the two density layers corresponding to CA and NC-RNA could be resolved, and occasionally the hexagonal symmetry in the CA layer was apparent by eye in more tangential tomographic slices. Released immature particles (ip) were found adjacent to cells expressing Gag (Fig. 2B, D) or GagPol (Fig. 2E), whereas released particles with mature morphology (mp) were only found adjacent to cells expressing GagPol (Fig. 2E). No density attributable to components of the ESCRT complex was detected at budding sites at the current resolution.

Bottom Line: Besides the immature lattice, a previously not described Gag lattice was detected in some budding sites and released particles; this lattice was found at high frequencies in a subset of infected T-cells.Buds and released particles carrying this lattice consistently lacked the viral ribonucleoprotein complex, suggesting that they correspond to aberrant products due to premature proteolytic activation.We hypothesize that cellular and/or viral factors normally control the onset of proteolytic maturation during assembly and release, and that this control has been lost in a subset of infected T-cells leading to formation of aberrant particles.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Virology, Universitätsklinikum Heidelberg, Heidelberg, Germany.

ABSTRACT
The structure of immature and mature HIV-1 particles has been analyzed in detail by cryo electron microscopy, while no such studies have been reported for cellular HIV-1 budding sites. Here, we established a system for studying HIV-1 virus-like particle assembly and release by cryo electron tomography of intact human cells. The lattice of the structural Gag protein in budding sites was indistinguishable from that of the released immature virion, suggesting that its organization is determined at the assembly site without major subsequent rearrangements. Besides the immature lattice, a previously not described Gag lattice was detected in some budding sites and released particles; this lattice was found at high frequencies in a subset of infected T-cells. It displays the same hexagonal symmetry and spacing in the MA-CA layer as the immature lattice, but lacks density corresponding to NC-RNA-p6. Buds and released particles carrying this lattice consistently lacked the viral ribonucleoprotein complex, suggesting that they correspond to aberrant products due to premature proteolytic activation. We hypothesize that cellular and/or viral factors normally control the onset of proteolytic maturation during assembly and release, and that this control has been lost in a subset of infected T-cells leading to formation of aberrant particles.

Show MeSH