Three-Dimensional Analysis of Budding Sites and Released Virus Suggests a Revised Model for HIV-1 Morphogenesis

Carlson, Lars A.; Briggs, John; Glass, Bärbel; Riches, James D.; Simon, Martha N.; Johnson, Marc C.; Müller, Bárbara; Grünewald, Kay; Kräusslich, Hans‐Georg

doi:10.1016/j.chom.2008.10.013

Cited by 213 publications

(267 citation statements)

References 28 publications

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“…HIV-Gag assembles into a hexagonal lattice as observed with cryoEM 20,21 . It is proposed that this hexameric lattice grows from a nucleation point by incorporating more Gag proteins during assembly, with an inherent curvature set by protein-protein interactions.…”

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confidence: 84%

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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confidence: 84%

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

et al. 2012

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“…Concentric circles, each consisting of a unique subunit composition, would also meet this criterion, but a single spiral would not. Because HIV-1 assembly sites in cells will, on average, contain approximately 2,400 Gag molecules at the time of release (52), even the small subset of Gag molecules which expose their p6 domains at the rim of the Gag lattice could provide enough opportunity for the ESCRT-I-mediated and the ALIX-mediated pathways to both contribute to ESCRT-III assembly at the same assembly site.…”

Section: Methodsmentioning

confidence: 99%

In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

Carlson

Hurley

2012

Proc. Natl. Acad. Sci. U.S.A.

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Most membrane-enveloped viruses depend on host proteins of the endosomal sorting complex required for transport (ESCRT) machinery for their release. HIV-1 is the prototypic ESCRT-dependent virus. The direct interactions between HIV-1 and the early ESCRT factors TSG101 and ALIX have been mapped in detail. However, the full pathway of ESCRT recruitment to HIV-1 budding sites, which culminates with the assembly of the late-acting CHMP4, CHMP3, CHMP2, and CHMP1 subunits, is less completely understood. Here, we report the biochemical reconstitution of ESCRT recruitment to viral assembly sites, using purified proteins and giant unilamellar vesicles. The myristylated full-length Gag protein of HIV-1 was purified to monodispersity. Myr-Gag forms clusters on giant unilamellar vesicle membranes containing the plasma membrane lipid PIð4,5ÞP 2 . These Gag clusters package a fluorescent oligonucleotide, and recruit early ESCRT complexes ESCRT-I or ALIX with the appropriate dependence on the Gag PTAP and LYPðXÞ n L motifs. ALIX directly recruits the key ESCRT-III subunit CHMP4. ESCRT-I can only recruit CHMP4 when ESCRT-II and CHMP6 are present as intermediary factors. Downstream of CHMP4, CHMP3 and CHMP2 assemble synergistically, with the presence of both subunits required for efficient recruitment. The very late-acting factor CHMP1 is not recruited unless the pathway is completed through CHMP3 and CHMP2. These findings define the minimal sets of components needed to complete ESCRT assembly at HIV-1 budding sites, and provide a starting point for in vitro structural and biophysical dissection of the system. confocal microscopy | host-pathogen interaction | membrane traffic | virus budding M ost membrane-enveloped viruses utilize host proteins of the endosomal sorting complex required for transport (ESCRT) machinery for their release (1-3). This dependence was first described for HIV-1, where mutation of an ESCRTbinding motif (late domain) in the viral protein Gag was shown to stall virus bud release at a late stage in virus assembly (4, 5). Late domains have subsequently been found in other retroviruses and numerous other virus families including, e.g., flavi-, filo-and rhabdoviruses (6). Indeed, the only well-characterized case of ESCRT-independent release of a membrane-enveloped virus is influenza virus (7). In normal cell physiology, ESCRTs function in cytokinesis, formation of intralumenal vesicles in multivesicular endosomes, and vesicle release from the plasma membrane (8-10). These seemingly disparate processes all involve membrane budding away from the cytoplasm, and ESCRTs are the only described protein machinery to perform vesicle formation with this topology, which is analogous to virus release.The initial events in HIV-1 ESCRT recruitment are well characterized. The structural protein of HIV-1, Gag, binds earlyacting ESCRT factors through two late-domain motifs in its C-terminal p6 domain: a PTAP sequence that interacts with the TSG101 subunit of ESCRT-I (11-16) and a LYPðXÞ n L motif that interacts with the ESCRT...

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“…Previous biochemical analyses of mature HIV-1 particles estimated 1,000 to 10,000 copies of viral RNA for 1 pg of capsid (p24) or ϳ25 million capsid molecules (5). The number of Gag molecules in immature HIV-1 particles was estimated to be 1,400 to 2,400 (6,7). Because each Gag polyprotein yields 1 processed, mature capsid protein; taken together, these numbers imply that each virion contains, on average, somewhere between 0.05 and 1 genomes.…”

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confidence: 99%

High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis

Chen

Nikolaitchik

Singh

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

181

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A long-standing question in retrovirus biology is how RNA genomes are distributed among virions. In the studies presented in this report, we addressed this issue by directly examining HIV-1 RNAs in virions using a modified HIV-1 genome that contained recognition sites for BglG, an antitermination protein in the Escherichia coli bgl operon, which was coexpressed with a fragment of BglG RNA binding protein fused to a fluorescent protein. Our results demonstrate that the majority of virions (>90%) contain viral RNAs. We also coexpressed HIV-1 genomes containing binding sites for BglG or the bacteriophage MS2 coat protein along with 2 fluorescent protein-tagged RNA binding proteins. This method allows simultaneously labeling and discrimination of 2 different RNAs at single-RNA-detection sensitivity. Using this strategy, we obtained physical evidence that virions contain RNAs derived from different parental viruses (heterozygous virion) at ratios expected from a random distribution, and we found that this ratio can be altered by changing the dimerization sequences. Our studies of heterozygous virions also support a generally accepted but unproven assumption that most particles contain 1 dimer. This study provides answers to long-standing questions in HIV-1 biology and illustrates the power and sensitivity of the 2-RNA labeling method, which can also be adapted to analyze various issues of RNA biogenesis including the detection of different RNAs in live cell imaging.Bgl ͉ MS2 ͉ dimerization ͉ DIS ͉ fluorescent protein

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Three-Dimensional Analysis of Budding Sites and Released Virus Suggests a Revised Model for HIV-1 Morphogenesis

Cited by 213 publications

References 28 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

In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis

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