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

Strauss, J; Hammonds, J.; Yi, Hong; Ding, Ling; Spearman, Paul; Wright, Elizabeth

doi:10.1128/jvi.01880-15

Cited by 32 publications

(52 citation statements)

References 71 publications

(140 reference statements)

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“…We previously used live-cell fluorescence imaging before vitrification to identify fluorescently tagged ROIs within the cell 14 . Although this approach is useful for targeting cells expressing the labeled protein, it is difficult to obtain exact coordinates for CLEM because cells can grow and move on the grid between the live-cell imaging and freezing steps.…”

Section: Introductionmentioning

confidence: 99%

“…It should be noted that within the field of cryo-EM the procedures and instrumentation used for CLEM and cryo-CLEM may vary and are dependent on the biological questions to be addressed 22,23 . In this protocol, we describe the CLEM and cryo-CLEM approaches that we have adapted for use on specific biological targets of interest to us, and with the hardware and software available to us 14,15 . However, the streamlined procedures for sample preparation and generation of multiscale image maps for correlation should be applicable to a broad range of cryo-CLEM experiments.…”

Section: Introductionmentioning

confidence: 99%

“…In addition, (cryo-)CLEM procedures were used to determine the arrangement of HIV-1 particles anchored to cell plasma membranes via tetherin, a host cellular restriction factor that inhibits enveloped virus release 14 . Cryo-CLEM of prokaryotes has also been described 22,27–29 and sections of this protocol that describe imaging and analysis parameters are also relevant to studies of these systems.…”

Section: Introductionmentioning

confidence: 99%

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Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

et al. 2016

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Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5–15 d for an individual experienced in cryo-EM.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

et al. 2016

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“…10A). HIV-1 VLPs, however, have a Gag lattice electron density that exclusively follows the curvature of the lipid bilayer (26,39,40,41). We next characterized the morphologies of the chimeric Gag-based VLPs that were shown to produce particles: HIV/HTLV CA, HTLV/HIV CA, HIV/HIV-C, and HTLV/HIV-C.…”

mentioning

confidence: 99%

Disparate Contributions of Human Retrovirus Capsid Subdomains to Gag-Gag Oligomerization, Virus Morphology, and Particle Biogenesis

Martin

Mendonça

Angert

et al. 2017

J Virol

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The capsid domain (CA) of the retroviral Gag protein is a primary determinant of Gag oligomerization, which is a critical step for immature Gag lattice formation and virus particle budding. Although the human immunodeficiency virus type 1 (HIV-1) CA carboxy-terminal domain (CTD) is essential for CA-CA interactions, the CA CTD has been suggested to be largely dispensable for human T-cell leukemia virus type 1 (HTLV-1) particle biogenesis. To more clearly define the roles of the HTLV-1 CA amino-terminal domain (NTD) and CA CTD in particle biogenesis, we generated and analyzed a panel of Gag proteins with chimeric HIV-1/HTLV-1 CA domains. Subcellular distribution and protein expression levels indicated that Gag proteins with a chimeric HIV-1 CA NTD/HTLV-1 CA CTD did not result in Gag oligomerization regardless of the parent Gag background. Furthermore, chimeric Gag proteins with the HTLV-1 CA NTD produced particles phenotypically similar to HTLV-1 immature particles, highlighting the importance of the HTLV-1 CA NTD in HTLV-1 immature particle morphology. Taken together, these observations support the conclusion that the HTLV-1 CA NTD can functionally replace the HIV-1 CA CTD, but the HIV-1 CA NTD cannot replace the HTLV-1 CA CTD, indicating that the HTLV-1 CA subdomains provide distinct contributions to Gag-Gag oligomerization, particle morphology, and biogenesis. Furthermore, we have shown for the first time that HIV-1 and HTLV-1 Gag domains outside the CA (e.g., matrix and nucleocapsid) impact Gag oligomerization as well as immature particle size and morphology. IMPORTANCE A key aspect in virus replication is virus particle assembly, which is a poorly understood process for most viruses. For retroviruses, the Gag structural protein is the primary driver of virus particle biogenesis, and the CA CTD is the primary determinant of Gag-Gag interactions for HIV-1. In this study, the HTLV-1 capsid amino-terminal domain was found to provide distinct contributions to Gag-Gag oligomerization, particle morphology, and biogenesis. This study provides information that will aid efforts for discovery of therapeutic targets for intervention.

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“…Subsequently, cells were infected with MeV strains, including wild type and recombinant strains at a multiplicity of infection of 1, 6 or 10. TEM grids were plunge frozen in liquid ethane using a Cryoplunge 3 system (Gatan, Pleasanton, CA) as previously described [4,5]. Cryo-grids were imaged with a JEOL JEM-2200FS TEM (JEOL, Ltd., Japan) equipped with an in-column Omega energy filter, operated with a slit with of 20 eV.…”

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confidence: 99%

The Near-to-Native-State Architecture of Measles Virus Assembly Sites and Isolated Measles Virus Particles

Wright

Strauss

et al. 2017

Microsc Microanal

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Measles virus (MeV) is a pleomorphic, enveloped, single-stranded RNA virus that belongs to the Paramyxovirus family. Details regarding the coordination and native arrangement of the Measles virus macromolecular complexes at sites of virus assembly and in released virus particles are poorly understood. Three-dimensional structural analyses of Measles viruses by cryo-electron tomography (cryo-ET) have been of purified viruses [1,2]. To further investigate the structure of assembled, released Measles virus particles and the processes of virus assembly under native-state conditions, we utilized whole cellular cryo-ET.HeLa and MRC-5 cells were grown directly on gold Quantfoil TEM grids (Quantifoil Micro Tools GmbH, Jena, Germany). Subsequently, cells were infected with MeV strains, including wild type and recombinant strains at a multiplicity of infection of 1, 6 or 10. TEM grids were plunge frozen in liquid ethane using a Cryoplunge 3 system (Gatan, Pleasanton, CA) as previously described [4,5]. Cryo-grids were imaged with a JEOL JEM-2200FS TEM (JEOL, Ltd., Japan) equipped with an in-column Omega energy filter, operated with a slit with of 20 eV. Image montages were acquired at a magnification of 10,000×, equivalent to a pixel size of 6.14 Å on a DE-20 direct electron detector camera (Direct Electron, LP, San Diego, CA). Bidirectional tilt-series were acquired at 2º increments over an angular range of -62º to +62º using the SerialEM software package [6]. Images were recorded at a magnification of 20,000×, corresponding to a pixel size of 2.94 Å on a DE-20 direct electron detector camera at a rate of 12 frames per second. Data was processed as previously described [3] and tomograms were reconstructed using the IMOD software package [7].Thin areas along the periphery of Measles virus-infected cells were surveyed to locate sites of virus assembly and to identify released virus particles. Regions of interest were identified and cryo-ET data was acquired. Sites of assembly were identified by the presence of the matrix protein along the plasma membrane (PM). Short and long-range arrays of the matrix protein were present and consisted of subunits in which the spacing was ~8 nm. In regions where the matrix protein was present along the interior of the plasma membrane, well-ordered arrays of the fusion (F) and hemagglutinin (H) glycoproteins were present on the surface. In addition, the ribonucleoprotein complex (RNP) appeared to associate directly with the ordered arrays of the matrix protein, indicating a coordinated recruitment to sites of virus assembly. Direct visualization of MeV-infected cells by cryo-ET has proved valuable for determining the native organization of the M protein, glycoproteins, and ribonucleoprotein (RNP) at both sites of virus assembly and in released virus particles.

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

Cited by 32 publications

References 71 publications

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

Disparate Contributions of Human Retrovirus Capsid Subdomains to Gag-Gag Oligomerization, Virus Morphology, and Particle Biogenesis

The Near-to-Native-State Architecture of Measles Virus Assembly Sites and Isolated Measles Virus Particles

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