Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells

Gheysen, Dirk; Jacobs, Eric J.; Foresta, F. de; Thiriart, Clotilde; Francotte, Myriam; Thines, Denise; Wilde, Michel De

doi:10.1016/0092-8674(89)90873-8

Cited by 693 publications

(577 citation statements)

References 42 publications

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“…To a first approximation, all of the information necessary for retroviral particle assembly resides in the Gag polypeptide. For example, Gag alone can form extracellular virus-like particles in the absence of other viral proteins [11] and Gag molecules can spontaneously assemble into spherical, immature viruslike particles in vitro [12][13][14]. Nevertheless, although Gag itself encodes the necessary tertiary and quaternary interactions, it must be emphasized that assembly requires nonspecific RNA interactions both in vivo and in vitro, and is assisted by host factors in vivo, including trafficking factors, assembly chaperones, and the ESCRT budding pathway, as reviewed elsewhere [15][16][17][18].…”

Section: Gag As An Assembly Machinementioning

confidence: 99%

The structural biology of HIV assembly

Ganser‐Pornillos

Yeager

Sundquist

2008

Current Opinion in Structural Biology

409

422

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HIV assembly and replication proceed through formation of morphologically distinct immature and mature viral capsids that are organized by the Gag polyprotein (immature) and by the fully processed CA protein (mature). The Gag polyprotein is composed of three folded polypeptides (MA, CA, and NC) and three smaller peptides (SP1, SP2, and p6) that function together to coordinate membrane binding and Gag-Gag lattice interactions in immature virions. Following budding, HIV maturation is initiated by proteolytic processing of Gag, which induces conformational changes in the CA domain and results in assembly of the distinctive conical capsid. Retroviral capsids are organized following the principles of fullerene cones, and the hexagonal CA lattice is stabilized by three distinct interfaces. Recently identified inhibitors of viral maturation act by disrupting the final stage of Gag processing, or by inhibiting formation of a critical intermolecular CA-CA interface in the mature capsid. Following release into a new host cell, the capsid disassembles and host cell factors can potently restrict this stage of retroviral replication. Here, we review the structures of immature and mature HIV virions, focusing on recent studies that have defined the global organization of the immature Gag lattice, identified sites likely to undergo conformational changes during maturation, revealed the molecular structure of the mature capsid lattice, demonstrated that capsid architectures are conserved, identified the first capsid assembly inhibitors, and begun to uncover the remarkable biology of the mature capsid.

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Section: Gag As An Assembly Machinementioning

confidence: 99%

The structural biology of HIV assembly

Ganser‐Pornillos

Yeager

Sundquist

2008

Current Opinion in Structural Biology

409

422

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“…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.…”

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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“…Whereas expression of the myristylated full-length Gag protein leads to an efficient targeting of the Gag molecules to the inner leaflet of the host cell membrane supporting the assembly of polyvalent lipoprotein aggregates to be released from the plasma membrane by budding [34,35], myristylation deficient Gag proteins and the p24 capsid moiety of Gag have been suggested to remain primarily in the cytoplasm [36,33]. Accordingly, the induction of Gag-specific antibodies provides indirect evidence that full-length Gag as well as the-per definition-cytoplasmic derivatives thereof, GagMyr − and p24, are in vivo released from cells in a way, which allows almost identical engagement of Gag-specific T helper and B cells.…”

Section: Discussionmentioning

confidence: 99%