Cryo-electron microscopy reveals ordered domains in the immature HIV-1 particle

Fuller, Stephen D.; Wilk, Thomas; Gowen, Brent; Kräusslich, Hans‐Georg; Vogt, Volker M.

doi:10.1016/s0960-9822(06)00331-9

Cited by 223 publications

(280 citation statements)

References 63 publications

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“…Fluorescence imaging has been used to follow the time-dependent increase in the intensity of Gag clusters in living cells 3,4 , revealing the time scale of virion formation. Electron microscopy (EM) has elucidated the spatial arrangement of Gag in fully formed virions [5][6][7][8] . However, studying the complete assembly process requires nanoscale resolution over a large dynamic range, since the size of a cluster ranges from a few molecules to several thousand.…”

Section: Abstract: Super-resolution Imaging; Protein Assembly; Proteimentioning

confidence: 99%

Quantitative Super-Resolution Imaging Reveals Protein Stoichiometry and Nanoscale Morphology of Assembling HIV-Gag Virions

et al. 2012

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The HIV structural protein Gag assembles to form spherical particles of radius ~70 nm. During the assembly process, the number of Gag proteins increases over several orders of magnitude, from a few at nucleation to thousands at completion. The challenge in studying protein assembly lies in the fact that current methods such as standard fluorescence or electron microscopy techniques cannot access all stages of the assembly process in a cellular context. Here, we demonstrate an approach using super-resolution fluorescence imaging that permits quantitative morphological and molecular counting analysis over a wide range of protein cluster sizes. We applied this technique to the analysis of hundreds of HIV-Gag clusters at the cellular plasma membrane, thus elucidating how different fluorescent labels can change the assembly of virions. Keywords: Super-resolution imaging; protein assembly; protein counting; HIV-GagPage 2 of 12 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 3 Viruses represent a major class of pathogens, whose assembly in the cellular context contains important information about the complex processes governing viral infection. Viruses are nanoscale objects that assemble from small nucleation complexes to ensembles containing thousands of molecules. In the case of human immunodeficiency virus (HIV), the viral components are targeted to the plasma membrane of infected cells where they assemble and eventually form spheres ~70 nm in radius. Viral assembly is widely studied using HIV-Gag, the main structural protein of HIV, which is sufficient to drive the assembly of virus-like particles (VLPs) in the absence of other viral components 1,2 . Fluorescence imaging has been used to follow the time-dependent increase in the intensity of Gag clusters in living cells 3,4 , revealing the time scale of virion formation. Electron microscopy (EM) has elucidated the spatial arrangement of Gag in fully formed virions [5][6][7][8] . However, studying the complete assembly process requires nanoscale resolution over a large dynamic range, since the size of a cluster ranges from a few molecules to several thousand. This cannot be achieved with standard fluorescence imaging methods since they lack both the necessary resolution to determine cluster morphology, and the sensitivity to detect smaller clusters. EM-based methods in turn lack information on protein identity; thus, complexes composed of small numbers of Gag proteins are difficult to identify, precluding the first step toward quantitative analysis. As an alternative approach, super-resolution fluorescence imaging based on single molecule localization (SR) 9-11 offers nanoscale resolution of structures formed by specific proteins. Here, we use an SR-based approach to quantitatively image hundreds of forming HIV-Gag virions in different stages of cluster formation. With this inf...

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Section: Abstract: Super-resolution Imaging; Protein Assembly; Proteimentioning

confidence: 99%

Quantitative Super-Resolution Imaging Reveals Protein Stoichiometry and Nanoscale Morphology of Assembling HIV-Gag Virions

et al. 2012

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“…The morphology of this particle is quite distinct from that of FFV. The FLV core is round rather than angular and displays a radial arrangement of rod-like features (small arrows) characteristic of Gag in other retroviruses (Fuller et al, 1997;Wilk et al, , 2001b. The membrane lacks prominent surface spikes and appears broader (large arrow) due to the presence of a submembrane protein layer.…”

Section: A Suggestive Imagementioning

confidence: 99%

Do lipid rafts mediate virus assembly and pseudotyping?

Briggs

Wilk

Fuller

2003

Journal of General Virology

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113

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“…In fact, studies by high resolution electron microscopy have revealed that Gag molecules are arranged in a fullerene-or cage-like network consisting of hexagonal and trigonal units (13,14), suggesting the ordered multimerization of Gag in a virus particle. However, little is known about the mechanisms involved in the formation of Gag capsid.…”

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confidence: 99%

Human Immunodeficiency Virus Type 1 Gag Assembly through Assembly Intermediates

Morikawa

Goto

Momose

2004

Journal of Biological Chemistry

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Human immunodeficiency virus Gag protein self-assembles into spherical particles, and recent reports suggest the formation of assembly intermediates during the process. To understand the nature of such assembly intermediates along with the mechanism of Gag assembly, we employed expression in Escherichia coli and an in vitro assembly reaction. When E. coli expression was performed at 37°C, Gag predominantly assembled to a high order of multimer, apparently equivalent to the virus-like particles obtained following Gag expression in eukaryotic cells, through the formation of low orders of multimer characterized with a discreet sedimentation value of 60 S. Electron microscopy confirmed the presence of spherical particles in the E. coli cells. In contrast, expression at 30°C resulted in the production of only the 60 S form of Gag multimer, and crescentshaped structures or small patches with double electron-dense layers were accumulated, but no complete particles. In vitro assembly reactions using purified Gag protein, when performed at 37°C, also produced the high order of Gag multimers with some 60 S multimers, whereas the 30°C reaction produced only the 60 S multimers. However, when the 60 S multimers were crosslinked so as not to allow conformational changes, in vitro assembly reactions at 37°C did not produce any higher order of multimers. ATP depletion did not halt Gag assembly in the E. coli cells, and the addition of GroEL-GroES to in vitro reactions did not facilitate Gag assembly, indicating that conformational changes rather than protein refolding by chaperonins, induced at 37°C, were solely responsible for the Gag assembly observed here. We suggest that Gag assembles to a capsid through the formation of the 60 S multimer, possibly a key intermediate of the assembly process, accompanied with conformational changes in Gag.

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Cryo-electron microscopy reveals ordered domains in the immature HIV-1 particle

Cited by 223 publications

References 63 publications

Quantitative Super-Resolution Imaging Reveals Protein Stoichiometry and Nanoscale Morphology of Assembling HIV-Gag Virions

Quantitative Super-Resolution Imaging Reveals Protein Stoichiometry and Nanoscale Morphology of Assembling HIV-Gag Virions

Do lipid rafts mediate virus assembly and pseudotyping?

Human Immunodeficiency Virus Type 1 Gag Assembly through Assembly Intermediates

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