scholarly journals Cryo Electron Tomography of Native HIV-1 Budding Sites

2010 ◽  
Vol 6 (11) ◽  
pp. e1001173 ◽  
Author(s):  
Lars-Anders Carlson ◽  
Alex de Marco ◽  
Heike Oberwinkler ◽  
Anja Habermann ◽  
John A. G. Briggs ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Cryo Electron Tomography ◽  
Hiv 1

scholarly journals RNA and Nucleocapsid Are Dispensable for Mature HIV-1 Capsid Assembly

2015 ◽  
Vol 89 (19) ◽  
pp. 9739-9747 ◽  
Author(s):  
Simone Mattei ◽  
Annica Flemming ◽  
Maria Anders-Össwein ◽  
Hans-Georg Kräusslich ◽  
John A. G. Briggs ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Electron Tomography ◽  
Significant Proportion ◽  
Dimensional Structure ◽  
Capsid Assembly ◽  
Interaction Domain ◽  
Cryo Electron Tomography ◽  
Rna Genome ◽  
Hiv 1

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) is released from infected cells in an immature, noninfectious form in which the structural polyprotein Gag is arranged in a hexameric lattice, forming an incomplete spherical shell. Maturation to the infectious form is mediated by the viral protease, which cleaves Gag at five sites, releasing the CA (capsid) protein, which forms a conical capsid encasing the condensed RNA genome. The pathway of this structural rearrangement is currently not understood, and it is unclear how cone assembly is initiated. RNA represents an integral structural component of retroviruses, and the viral nucleoprotein core has previously been proposed to nucleate mature capsid assembly. We addressed this hypothesis by replacing the RNA-binding NC (nucleocapsid) domain of HIV-1 Gag and the adjacent spacer peptide 2 (SP2) by a leucine zipper (LZ) protein-protein interaction domain [Gag(LZ)] in the viral context. We found that Gag(LZ)-carrying virus [HIV(LZ)] was efficiently released and viral polyproteins were proteolytically processed, though with reduced efficiency. Cryo-electron tomography revealed that the particles lacked a condensed nucleoprotein and contained an increased proportion of aberrant core morphologies caused either by the absence of RNA or by altered Gag processing. Nevertheless, a significant proportion of HIV(LZ) particles contained mature capsids with the wild-type morphology. These results clearly demonstrate that the nucleoprotein complex is dispensable as a nucleator for mature HIV-1 capsid assembly in the viral context.IMPORTANCEFormation of a closed conical capsid encasing the viral RNA genome is essential for HIV-1 infectivity. It is currently unclear what viral components initiate and regulate the formation of the capsid during virus morphogenesis, but it has been proposed that the ribonucleoprotein complex plays a role. To test this, we prepared virus-like particles lacking the viral nucleocapsid protein and RNA and analyzed their three-dimensional structure by cryo-electron tomography. While most virions displayed an abnormal morphology under these conditions, some particles showed a normal mature morphology with closed conical capsids. These data demonstrate that the presence of RNA and the nucleocapsid protein is not required for the formation of a mature, cone-shaped HIV-1 capsid.

scholarly journals A flexible framework for multi-particle refinement in cryo-electron tomography

PLoS Biology ◽  
2021 ◽  
Vol 19 (8) ◽  
pp. e3001319
Author(s):  
Alister Burt ◽  
Lorenzo Gaifas ◽  
Tom Dendooven ◽  
Irina Gutsche
Keyword(s):  
Software Package ◽  
Electron Tomography ◽  
Macromolecular Structure ◽  
Computational Tools ◽  
Cryo Electron Tomography ◽  
Flexible Framework ◽  
Subtomogram Averaging ◽  
Particle Refinement ◽  
Hiv 1

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) are increasingly used for macromolecular structure determination in situ. Here, we introduce a set of computational tools and resources designed to enable flexible approaches to STA through increased automation and simplified metadata handling. We create a bidirectional interface between the Dynamo software package and the Warp-Relion-M pipeline, providing a framework for ab initio and geometrical approaches to multiparticle refinement in M. We illustrate the power of working within this framework by applying it to EMPIAR-10164, a publicly available dataset containing immature HIV-1 virus-like particles (VLPs), and a challenging in situ dataset containing chemosensory arrays in bacterial minicells. Additionally, we provide a comprehensive, step-by-step guide to obtaining a 3.4-Å reconstruction from EMPIAR-10164. The guide is hosted on https://teamtomo.org/, a collaborative online platform we establish for sharing knowledge about cryo-ET.

scholarly journals Cryo-ET of HIV reveals Env positioning on Gag lattice and structural variation among Env trimers

2021 ◽  
Author(s):  
Vidya Mangala Prasad ◽  
Daniel P. Leaman ◽  
Klaus N. Lovendahl ◽  
Mark A. Benhaim ◽  
Edgar A. Hodge ◽  
...  
Keyword(s):  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Structural Variation ◽  
Electron Tomography ◽  
Host Cells ◽  
Detailed Characterization ◽  
Cryo Electron Tomography ◽  
Structural Mass Spectrometry ◽  
Structural Mass ◽  
Hiv 1

SummaryHIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1Å sub-tomogram averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers plus a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.

HIV-1 Biogenesis Studied by Cellular Cryo-Electron Tomography and Biochemical in vitro Reconstitution

2012 ◽  
Vol 18 (S2) ◽  
pp. 50-51
Author(s):  
L. Carlson ◽  
J.H. Hurley ◽  
K. Grünewald ◽  
H. Kräusslich ◽  
J. Briggs
Keyword(s):  
Electron Tomography ◽  
In Vitro Reconstitution ◽  
Cryo Electron Tomography ◽  
Hiv 1

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

scholarly journals Three-Dimensional Structural Characterization of HIV-1 Tethered to Human Cells

2015 ◽  
Vol 90 (3) ◽  
pp. 1507-1521 ◽  
Author(s):  
Joshua D. Strauss ◽  
Jason E. Hammonds ◽  
Hong Yi ◽  
Lingmei Ding ◽  
Paul Spearman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Electron Tomography ◽  
Coiled Coil ◽  
Human Cells ◽  
Three Dimensions ◽  
Distance Measurements ◽  
Linear Fashion ◽  
Cryo Electron Tomography ◽  
Hiv 1

ABSTRACTTetherin (BST2, CD317, or HM1.24) is a host cellular restriction factor that prevents the release of enveloped viruses by mechanically linking virions to the plasma membrane. The precise arrangement of tetherin molecules at the plasma membrane site of HIV-1 assembly, budding, and restriction is not well understood. To gain insight into the biophysical mechanism underlying tetherin-mediated restriction of HIV-1, we utilized cryo-electron tomography (cryo-ET) to directly visualize HIV-1 virus-like particles (VLPs) and virions tethered to human cells in three dimensions (3D). Rod-like densities that we refer to as tethers were seen connecting HIV-1 virions to each other and to the plasma membrane. Native immunogold labeling showed tetherin molecules located on HIV-1 VLPs and virions in positions similar to those of the densities observed by cryo-ET. The location of the tethers with respect to the ordered immature Gag lattice or mature conical core was random. However, tethers were not uniformly distributed on the viral membrane but rather formed clusters at sites of contact with the cell or other virions. Chains of tethered HIV-1 virions often were arranged in a linear fashion, primarily as single chains and, to a lesser degree, as branched chains. Distance measurements support the extended tetherin model, in which the coiled-coil ectodomains are oriented perpendicular with respect to the viral and plasma membranes.IMPORTANCETetherin is a cellular factor that restricts HIV-1 release by directly cross-linking the virus to the host cell plasma membrane. We used cryo-electron tomography to visualize HIV-1 tethered to human cells in 3D. We determined that tetherin-restricted HIV-1 virions were physically connected to each other or to the plasma membrane by filamentous tethers that resembled rods ∼15 nm in length, which is consistent with the extended tetherin model. In addition, we found the position of the tethers to be arbitrary relative to the ordered immature Gag lattice or the mature conical cores. However, when present as multiple copies, the tethers clustered at the interface between virions. Tethered HIV-1 virions were arranged in a linear fashion, with the majority as single chains. This study advances our understanding of tetherin-mediated HIV-1 restriction by defining the spatial arrangement and orientation of tetherin molecules at sites of HIV-1 restriction.

scholarly journals Hierarchical assembly governs TRIM5α recognition of HIV-1 and retroviral capsids

2019 ◽  
Vol 5 (11) ◽  
pp. eaaw3631 ◽  
Author(s):  
Katarzyna A. Skorupka ◽  
Marcin D. Roganowicz ◽  
Devin E. Christensen ◽  
Yueping Wan ◽  
Owen Pornillos ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Hexagonal Lattice ◽  
Hierarchical Assembly ◽  
Virus Core ◽  
Capsid Shell ◽  
Cryo Electron Tomography ◽  
Nonself Recognition ◽  
Subtomogram Averaging ◽  
Dependent Processes ◽  
Hiv 1

TRIM5α is a restriction factor that senses incoming retrovirus cores through an unprecedented mechanism of nonself recognition. TRIM5α assembles a hexagonal lattice that avidly binds the capsid shell, which surrounds and protects the virus core. The extent to which the TRIM lattice can cover the capsid and how TRIM5α directly contacts the capsid surface have not been established. Here, we apply cryo–electron tomography and subtomogram averaging to determine structures of TRIM5α bound to recombinant HIV-1 capsid assemblies. Our data support a mechanism of hierarchical assembly, in which a limited number of basal interaction modes are successively organized in increasingly higher-order structures that culminate in a TRIM5α cage surrounding a retroviral capsid. We further propose that cage formation explains the mechanism of restriction and provides the structural context that links capsid recognition to ubiquitin-dependent processes that disable the retrovirus.

scholarly journals Structural Characterization of Tethered HIV-1 VLPs by Light Microscopy and Cryo-Electron Tomography

2014 ◽  
Vol 20 (S3) ◽  
pp. 1256-1257 ◽  
Author(s):  
Joshua D. Strauss ◽  
Jason E. Hammonds ◽  
Paul W. Spearman ◽  
Elizabeth R. Wright
Keyword(s):  
Light Microscopy ◽  
Structural Characterization ◽  
Electron Tomography ◽  
Cryo Electron Tomography ◽  
Hiv 1

scholarly journals Cryo-Electron Tomography and Nucleocapsid Protein Labeling by Tomo-Bubblegram Imaging Reveal a Role for HIV-1 Integrase in Viral Maturation

2016 ◽  
Vol 110 (3) ◽  
pp. 155a
Author(s):  
Juan Fontana ◽  
Kellie A. Jurado ◽  
Naiqian Cheng ◽  
Alan N. Engelman ◽  
Alasdair C. Steven
Keyword(s):  
Nucleocapsid Protein ◽  
Electron Tomography ◽  
Protein Labeling ◽  
Cryo Electron Tomography ◽  
Viral Maturation ◽  
Hiv 1

scholarly journals Structural Studies of Trimeric SIV and HIV-1 Envelope Glycoproteins Using Cryo-Electron Tomography

2010 ◽  
Vol 16 (S2) ◽  
pp. 882-883
Author(s):  
TA White ◽  
A Bartesaghi ◽  
MJ Borgnia ◽  
MJV de la Cruz ◽  
JLS Milne ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Envelope Glycoproteins ◽  
Structural Studies ◽  
Cryo Electron Tomography ◽  
Hiv 1

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

scholarly journals Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Martin Obr ◽  
Clifton L. Ricana ◽  
Nadia Nikulin ◽  
Jon-Philip R. Feathers ◽  
Marco Klanschnig ◽  
...  
Keyword(s):  
Rous Sarcoma Virus ◽  
Electron Tomography ◽  
Structural Mechanism ◽  
Rous Sarcoma ◽  
Cryo Electron Tomography ◽  
Sarcoma Virus ◽  
Subtomogram Averaging ◽  
Opposing Effects ◽  
Hiv 1

AbstractInositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.

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