scholarly journals Maturation of the matrix and viral membrane of HIV-1

Science ◽  
2021 ◽  
Vol 373 (6555) ◽  
pp. 700-704 ◽  
Author(s):  
Kun Qu ◽  
Zunlong Ke ◽  
Vojtech Zila ◽  
Maria Anders-Össwein ◽  
Bärbel Glass ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Virus Assembly ◽  
Cell Entry ◽  
Structural Protein ◽  
Partial Removal ◽  
Viral Membrane ◽  
Cryo Electron Tomography ◽  
The Matrix ◽  
Membrane Bound ◽  
Hiv 1

Gag, the primary structural protein of HIV-1, is recruited to the plasma membrane for virus assembly by its matrix (MA) domain. Gag is subsequently cleaved into its component domains, causing structural maturation to repurpose the virion for cell entry. We determined the structure and arrangement of MA within immature and mature HIV-1 through cryo–electron tomography. We found that MA rearranges between two different hexameric lattices upon maturation. In mature HIV-1, a lipid extends out of the membrane to bind with a pocket in MA. Our data suggest that proteolytic maturation of HIV-1 not only assembles the viral capsid surrounding the genome but also repurposes the membrane-bound MA lattice for an entry or postentry function and results in the partial removal of up to 2500 lipids from the viral membrane.

scholarly journals Maturation of the matrix and viral membrane of HIV-1

2020 ◽  
Author(s):  
Kun Qu ◽  
Zunlong Ke ◽  
Vojtech Zila ◽  
Maria Anders-Össwein ◽  
Bärbel Glass ◽  
...  
Keyword(s):  
Virus Assembly ◽  
Acyl Chain ◽  
Cell Entry ◽  
Structural Protein ◽  
Viral Membrane ◽  
The Matrix ◽  
Acyl Chains ◽  
Membrane Bound ◽  
Post Entry ◽  
Hiv 1

AbstractGag – the main structural protein of HIV-1 – is recruited to the plasma membrane for virus assembly by its matrix (MA) domain. Gag is subsequently cleaved into its component domains, causing structural maturation to repurpose the virion for cell entry. We determined the structure and arrangement of MA within immature and mature HIV-1, providing a basis to understand MA’s role in virus assembly. Unexpectedly, we found that MA rearranges during maturation, to form a new, hexameric lattice in which the acyl chain of a phospholipid extends out of the membrane to bind a pocket in MA. Our data suggest that proteolytic maturation of HIV-1 not only achieves assembly of the viral capsid surrounding the genome, but extends to repurpose the membrane-bound MA lattice for an entry or post-entry function, and causes partial removal of 2,500 acyl chains from the viral membrane.

scholarly journals Single-molecule imaging of HIV-1 envelope glycoprotein dynamics and Gag lattice association exposes determinants responsible for virus incorporation

2019 ◽  
Vol 116 (50) ◽  
pp. 25269-25277 ◽  
Author(s):  
Nairi Pezeshkian ◽  
Nicholas S. Groves ◽  
Schuyler B. van Engelenburg
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Envelope Glycoprotein ◽  
Virus Assembly ◽  
Single Molecule Tracking ◽  
Virus Particles ◽  
Cell Plasma ◽  
The Matrix ◽  
Resolution Single ◽  
Hiv 1

The HIV-1 envelope glycoprotein (Env) is sparsely incorporated onto assembling virus particles on the host cell plasma membrane in order for the virus to balance infectivity and evade the immune response. Env becomes trapped in a nascent particle on encounter with the polymeric viral protein Gag, which forms a dense protein lattice on the inner leaflet of the plasma membrane. While Env incorporation efficiency is readily measured biochemically from released particles, very little is known about the spatiotemporal dynamics of Env trapping events. Herein, we demonstrate, via high-resolution single-molecule tracking, that retention of Env trimers within single virus assembly sites requires the Env cytoplasmic tail (CT) and the L12 residue in the matrix (MA) domain of Gag but does not require curvature of the viral lattice. We further demonstrate that Env trimers are confined to subviral regions of a budding Gag lattice, supporting a model where direct interactions and/or steric corralling between the Env-CT and a lattice of MA trimers promote Env trapping and infectious HIV-1 assembly.

scholarly journals Membrane Binding and Subcellular Localization of Retroviral Gag Proteins Are Differentially Regulated by MA Interactions with Phosphatidylinositol-(4,5)-Bisphosphate and RNA

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Jingga Inlora ◽  
David R. Collins ◽  
Marc E. Trubin ◽  
Ji Yeon J. Chung ◽  
Akira Ono
Keyword(s):  
Virus Assembly ◽  
Membrane Binding ◽  
Endogenous Retrovirus ◽  
Single Amino Acid ◽  
Human Endogenous Retrovirus ◽  
Structural Protein ◽  
Acidic Phospholipid ◽  
Specific Localization ◽  
Hiv 1

ABSTRACTThe matrix (MA) domain of HIV-1 mediates proper Gag localization and membrane binding via interaction with a plasma-membrane (PM)-specific acidic phospholipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. HIV-1 MA also interacts with RNA, which prevents Gag from binding to membranes containing phosphatidylserine, a prevalent cellular acidic phospholipid. These results suggest that the MA-bound RNA promotes PM-specific localization of HIV-1 Gag by blocking nonspecific interactions with cellular membranes that do not contain PI(4,5)P2. To examine whether PI(4,5)P2dependence and RNA-mediated inhibition collectively determine MA phenotypes across a broad range of retroviruses and elucidate the significance of their interrelationships, we compared a panel of Gag-leucine zipper constructs (GagLZ) containing MA of different retroviruses. We found thatin vitromembrane binding of GagLZ via HIV-1 MA and Rous sarcoma virus (RSV) MA is both PI(4,5)P2dependent and susceptible to RNA-mediated inhibition. The PM-specific localization and virus-like particle (VLP) release of these GagLZ proteins are severely impaired by overexpression of a PI(4,5)P2-depleting enzyme, polyphosphoinositide 5-phosphatase IV (5ptaseIV). In contrast, membrane binding of GagLZ constructs that contain human T-lymphotropic virus type 1 (HTLV-1) MA, murine leukemia virus (MLV) MA, and human endogenous retrovirus K (HERV-K) MA is PI(4,5)P2independent and not blocked by RNA. The PM localization and VLP release of these GagLZ chimeras were much less sensitive to 5ptaseIV expression. Notably, single amino acid substitutions that confer a large basic patch rendered HTLV-1 MA susceptible to the RNA-mediated block, suggesting that RNA readily blocks MA containing a large basic patch, such as HIV-1 and RSV MA. Further analyses of these MA mutants suggest a possibility that HIV-1 and RSV MA acquired PI(4,5)P2dependence to alleviate the membrane binding block imposed by RNA.IMPORTANCEMA basic residues in the HIV-1 structural protein Gag interact with phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and RNA. RNA inhibits HIV-1 MA binding to non-PI(4,5)P2acidic lipids. This inhibition may promote PM specificity of Gag membrane binding, an early essential step in virus assembly. However, whether and how relationships between these interactions have developed among retroviruses are poorly understood. In this study, by comparing diverse retroviral MA domains, we elucidated a strong correlation among PI(4,5)P2dependence, susceptibility to RNA-mediated inhibition, and cellular behaviors of Gag. Mutagenesis analyses suggest that a large basic patch on MA is sufficient to confer susceptibility to RNA-mediated inhibition but not for PI(4,5)P2-dependent membrane binding. Our findings highlight RNA’s role as a general blocker of large basic patches and suggest a possibility that some retroviruses, including HIV-1, have evolved to bind PI(4,5)P2, while others have adopted smaller basic patches on their MA domains, to overcome the RNA-mediated restriction of membrane binding.

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.

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