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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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A previously undescribed Gag lattice in budding sites and released particles.(A) Computational slice through cryo electron tomograms of U-373 MG cells transduced with AdGagPol. Budding sites (b) and released particles (p) exhibit a Gag lattice different from both the immature and the mature lattice. (B) Enlarged slice through the top budding site from (A). Density is observed at the expected position of the CA domain, but no prominent density corresponding to NC-RNA is detected. (C) For comparison, a slice through a budding site with the immature Gag lattice showing the characteristic two density layers underneath the plasma membrane. Thickness of computational slices through cryo electron tomograms is 1.6 nm. pm: plasma membrane. Scale bars are 100 nm.
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ppat-1001173-g006: A previously undescribed Gag lattice in budding sites and released particles.(A) Computational slice through cryo electron tomograms of U-373 MG cells transduced with AdGagPol. Budding sites (b) and released particles (p) exhibit a Gag lattice different from both the immature and the mature lattice. (B) Enlarged slice through the top budding site from (A). Density is observed at the expected position of the CA domain, but no prominent density corresponding to NC-RNA is detected. (C) For comparison, a slice through a budding site with the immature Gag lattice showing the characteristic two density layers underneath the plasma membrane. Thickness of computational slices through cryo electron tomograms is 1.6 nm. pm: plasma membrane. Scale bars are 100 nm.

Mentions: Immunoblot analysis of AdGagPol-transduced cells and the particle fraction recovered from the culture medium showed proteolytic processing of Gag (Fig. 1C). Furthermore, particles containing mature-looking cores were found in cryo electron tomograms in immediate proximity of cellular budding sites and next to immature particles (Fig. 2E). Of note, structures resembling the mature capsid were never found in budding sites still connected to the plasma membrane. Besides the well-described mature and immature particles, we observed a third, previously not described form of particles in the tomograms of AdGagPol-transduced cells (Fig. 6A). This apparently “intermediate morphology” exhibiting a thinner Gag lattice than the immature structure was observed both in extracellular particles (Fig. 6A, Fig. S2 in Supporting Information S1) and in HIV-1 budding sites connected to the cell (Fig. 6A–B). Budding sites and extracellular particles with the previously unrecognized Gag lattice were characterized by a single density layer bound to the inner face of the membrane (Figure 6B), as opposed to the two layers (CA and NC-RNA) seen in the immature lattice (Fig. 6C). Density plots orthogonal to the cell membrane confirmed that the density layer found in these novel structures was located at the same position relative to the plasma membrane (peak at −12 nm) as the CA layer of the complete immature lattice (Fig. S1 in Supporting Information S1).


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)

A previously undescribed Gag lattice in budding sites and released particles.(A) Computational slice through cryo electron tomograms of U-373 MG cells transduced with AdGagPol. Budding sites (b) and released particles (p) exhibit a Gag lattice different from both the immature and the mature lattice. (B) Enlarged slice through the top budding site from (A). Density is observed at the expected position of the CA domain, but no prominent density corresponding to NC-RNA is detected. (C) For comparison, a slice through a budding site with the immature Gag lattice showing the characteristic two density layers underneath the plasma membrane. Thickness of computational slices through cryo electron tomograms is 1.6 nm. pm: plasma membrane. Scale bars are 100 nm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991257&req=5

ppat-1001173-g006: A previously undescribed Gag lattice in budding sites and released particles.(A) Computational slice through cryo electron tomograms of U-373 MG cells transduced with AdGagPol. Budding sites (b) and released particles (p) exhibit a Gag lattice different from both the immature and the mature lattice. (B) Enlarged slice through the top budding site from (A). Density is observed at the expected position of the CA domain, but no prominent density corresponding to NC-RNA is detected. (C) For comparison, a slice through a budding site with the immature Gag lattice showing the characteristic two density layers underneath the plasma membrane. Thickness of computational slices through cryo electron tomograms is 1.6 nm. pm: plasma membrane. Scale bars are 100 nm.
Mentions: Immunoblot analysis of AdGagPol-transduced cells and the particle fraction recovered from the culture medium showed proteolytic processing of Gag (Fig. 1C). Furthermore, particles containing mature-looking cores were found in cryo electron tomograms in immediate proximity of cellular budding sites and next to immature particles (Fig. 2E). Of note, structures resembling the mature capsid were never found in budding sites still connected to the plasma membrane. Besides the well-described mature and immature particles, we observed a third, previously not described form of particles in the tomograms of AdGagPol-transduced cells (Fig. 6A). This apparently “intermediate morphology” exhibiting a thinner Gag lattice than the immature structure was observed both in extracellular particles (Fig. 6A, Fig. S2 in Supporting Information S1) and in HIV-1 budding sites connected to the cell (Fig. 6A–B). Budding sites and extracellular particles with the previously unrecognized Gag lattice were characterized by a single density layer bound to the inner face of the membrane (Figure 6B), as opposed to the two layers (CA and NC-RNA) seen in the immature lattice (Fig. 6C). Density plots orthogonal to the cell membrane confirmed that the density layer found in these novel structures was located at the same position relative to the plasma membrane (peak at −12 nm) as the CA layer of the complete immature lattice (Fig. S1 in Supporting Information S1).

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
Related in: MedlinePlus