pubmed.ncbi.nlm.nih.gov

Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells - PubMed

  • ️Thu Jan 01 2015

Review

Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells

Inés Romero-Brey et al. Viruses. 2015.

Abstract

As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications.

Keywords: cell membranes; cryo-EM; electron tomography; focus ion beam-scanning electron microscopy; scanning transmission electron microscopy; serial block face-scanning electron microscopy; serial sectioning; transmission electron microscopy; ultrastructure; virus-host interactions.

PubMed Disclaimer

Figures

Figure 1
Figure 1

Schematic representation of the different methods for preparing virus-infected cells for electron microscopy (EM). Adherent cells can be either chemically fixed with aldehydes (A) or fast frozen by high-pressure freezing (HPF) or plunge/jet freezing (B). (A) After chemical fixation, infected cells can be further processed as a cell monolayer (grown on glass coverslips) or be pelleted prior to further processing for EM. Post-fixation is done with heavy metals (e.g., osmium tetroxide (OsO4) and uranyl acetate (UA)), samples are dehydrated with increasing concentrations of an organic solvent (e.g., ethanol or acetone) and embedded into a plastic resin (plastic-EM; highlighted in green). Coverslips must be removed from the polymerized resin block by successive immersions in liquid nitrogen and hot water; (B) For rapid immobilization of cells by HPF, they must be grown as monolayers on sapphire (or aclar, not shown) discs, which are clamped in-between two aluminium planchettes and then loaded into a HPF machine for rapid freezing. Alternatively, cell pellets are directly placed into the aluminium planchettes or into capillary tubes (not shown) for freezing. Frozen cells can be subjected to freeze substitution (FS), dehydration with an organic solvent and resin embedding (plastic-EM; highlighted in green). Alternatively, high-pressure frozen cell pellets can be further analyzed by CEMOVIS (cryo-electron microscopy of vitreous sections) (cryo-EM, highlighted in violet). Cells growing on EM grids can be also plunge/jet frozen and analyzed directly by cryo-EM (highlighted in violet). Both cryo-EM approaches, which do not require further processing of the cells, allow visualizing cells in their closest-to-native status. Further details about these approaches can be found in the main text.

Figure 2
Figure 2

Impact of different fixation methods on the morphology of double membrane vesicles (DMVs) induced by Hepatitis C Virus (HCV). (A) Huh7 cells grown on sapphire discs were infected with HCV and processed after chemical fixation by high pressure freezing (HPF) and freeze substitution (FS); (B) Huh7 cells were transfected by electroporation with a subgenomic HCV replicon RNA and 48 h later subjected directly to HPF-FS without chemical fixation. DMVs were also found in high abundance in these samples excluding that they are an artifact caused by chemical fixation. Also note the minimal extraction of the cytosol surrounding the DMVs and their content in comparison to chemically fixed cells; (C) HCV-infected Huh7.5 cells grown on coverslips were chemically fixed and then embedded in epon; (D) Huh7.5 cells were infected with HCV and 48 h later cells were fixed, scraped off the culture dish and sedimented by gentle centrifugation prior to embedding of the cell pellet in epon resin. Note the “fried-egg”-like morphology of the DMVs observed in chemically fixed cells (C,D), in comparison to the very well-delineated membranes of cells subjected to HPF (A,B). EM micrographs were taken with permission from Romero-Brey et al. [20].

Figure 3
Figure 3

Schematic representation depicting the post-processing steps prior to the analysis of virus-infected cells by conventional-EM (“plastic-EM”). (A) Virus-infected cells embedded into a plastic resin are sectioned with an ultramicrotome: the resin block containing the embedded virus-infected cells must be trimmed to a trapezium-like shape with an average size of approximately 200 × 250 μm prior to its sectioning with a diamond knife equipped with a water shank; (B) The obtained ultrathin sections (60–80 nm, in yellow) float on the water surface from where the section ribbon can be mounted onto a formvar-coated EM grid; (C) After contrasting with heavy metals, the cell sections can be examined with a conventional transmission electron microscope (TEM) operated at accelerating voltages between 70 and 100 kV. In brief, an electron beam is generated (by a thermionic or a field emission gun) and accelerated under vacuum. The electrons are then transmitted through the cell sections. After passing through the specimen, scattered electrons are focused by electromagnetic lenses and magnified onto an imaging device such as a fluorescent screen or recorded with a digital camera (CCD, charged-coupled device, or a CMOS, complementary metal-oxide semiconductor). The diagram of the TEM-working principle shown on the right is adapted from Briggman and Bock [78].

Figure 4
Figure 4

Overview of the 3D-EM methods that can be used to analyze the architecture of virus-infected cells by means of plastic-EM (A-E, on the left) or cryo-EM (F-K, on the right). (A,B) Thicker sections of approximately 300 nm and 500–1000 nm of a region of interest (ROI, highlighted in green) can be analyzed by ET and Scanning Transmission Electron Microscopy (STEM) tomography, respectively; (C) Several sequential tomograms can be also joined together to reconstruct a larger volume of the ROI; (D) Manually obtained serial ultrathin sections of a ROI can be imaged by conventional TEM and the information of these 2D images combined to create a 3D map of this particular area. Alternatively, sectioned cells can be analyzed by Scanning Electron Microscopy (SEM); (E) With serial block face (SBF) or focus ion beam (FIB)-SEM 3D information of a whole virus-infected cell (framed in orange) can be obtained; (F) Alternatively, ~200 nm sections of high-pressure frozen cells can be further analyzed at very low temperatures by CETOVIS (cryo-electron tomography of vitreous sections); (G) A FIB can be also used to slice 200–500 nm thick lamellae from vitrified (high pressure or plunge frozen) cells that can be then analyzed by cryo-ET; (H) Cryo-ET (CET) can be applied to entire plunge frozen cells. However, it only allows getting information from where the cell thickness is ≤1 µm (at the cell periphery); (I) Cryo-STEM tomography can be used to study ~600 nm thick sections of vitrified cells (at 200 kV); (J) With soft X-ray cryo-tomography 3D information of up to 10 µm can be retrieved from frozen hydrated specimens; (K) A FIB can be also used to slice 30 nm sections from the block face of vitrified cells that can be then recorded in a sequential fashion by SEM. Note that techniques allowing for wider fields of view are highlighted in orange, whereas in green are those allowing the imaging of smaller fields of view.

Figure 5
Figure 5

Principle of electron tomography (ET) and 3D reconstruction. (A) Thicker sections (250–1000 nm, in pink) of virus-infected cells can be analyzed by means of electron tomography (ET) or scanning transmission electron microscopy (STEM) tomography using microscopes operated with voltages >100 kV. The sections are tilted relative to the incident electron beam in an automatic fashion with help of a goniometer. 2D projections of the same field of view are acquired every tilted angle, from which the cell volume is subsequently reconstructed in silico; (B) Following the same working principle, cryo-ET, cryo-STEM tomography or soft X-ray cryo-tomography can be applied to vitrified cells grown on EM grids. For this purpose, the microscope must be operated at very low temperatures and with low electron doses (in the case of cryo-ET and cryo-STEM tomography). Alternatively thick cryo-sections collected on an EM grid as in (A) can be analyzed by CETOVIS (see text for further details). The diagrams of the ET-working principle are adapted from Baumeister et al. [147].

Figure 6
Figure 6

Examples of applications of 3D-EM methods to the study of human-relevant viral infections. (A) Electron tomography (ET) of Hepatitis C Virus (HCV)-infected cells [20]. On the left: slice of a tomogram showing HCV-induced double membrane vesicles (DMVs); on the right: 3D surface rendering of the whole tomogram, showing DMVs in close proximity to ER; (B) STEM tomography of Mimivirus-infected cells [124]. On the left: digital slice derived from STEM tomography of 280 nm thick section; on the right: 3D surface rendering revealing that the membrane assembly region consists of an elaborate membrane network; (C) Serial sectioning of cytomegalovirus (CMV)-infected cells [50]. On the left: single micrograph (in X–Y direction) and image stack (slice through the Z-axis) of the assembly complex; on the right: cellular and viral structures were segmented on all 28 sections and superimposed on a single micrograph; (D) FIB-SEM of HIV-infected cells [133]. On the left: 2D FIB-SEM image of an HIV-1 chronically infected H9 cell (left) and an uninfected astrocyte (right), co-cultured for 24 h; on the right: 3D rendering of the FIB-SEM image stack containing the contact zone between the HIV-infected H9 cell and the astrocyte. The target cell extends long filopodial bridges towards the infected cell across the intercellular gap. HIV-1 virions are detected adjacent to the filopodial bridges; (E) Cryo-ET of Herpes Simplex Virus (HSV)-infected cells [138]. On the left: slice of the tomogram, showing secondary envelopment of capsids; on the right: 3D surface rendering of the whole tomogram, showing a capsid in close proximity to an enveloping vesicle. Asterisks: capsids; white arrow: enveloping vesicle; arrowhead: glycoproteins; black arrows: tegument; pm: plasma membrane. All these pictures are reproduced from the original publications with permission (see Acknowledgments).

Figure 7
Figure 7

Serial sectioning workflow. A ribbon of consecutive ultrathin sections (60–80 nm) of a cell can be either obtained manually (A) or automatically on a tape (ATUM, automatic tape-collecting ultramicrotome) (B) and subsequently analyzed by TEM (C) or SEM (D), respectively. Generated micrographs are aligned to obtain a z-stack of this cell. Note also that the same principle is used to join tomograms obtained by serial-ET.

Figure 8
Figure 8

Principle of Focus Ion Beam (FIB)-Scanning Electron Microscopy (SEM). Epon blocks containing the embedded cells are mounted on SEM stubs with silver paint. First a trench is milled with the FIB, generating a cross-section into the epon block. Subsequently the newly generated block face is milled (with the FIB, blue), resulting in the removal of a thin layer (as small as 3 nm; [181]) and imaged (with the SEM, pink) in a sequential manner. This process is repeated automatically as long as needed to obtain a tomographic dataset. Further details about this method can be found in the main text.

Similar articles

Cited by

References

    1. Zhou Z.H. Towards atomic resolution structural determination by single-particle cryo-electron microscopy. Curr. Opin. Struct. Biol. 2008;18:218–228. doi: 10.1016/j.sbi.2008.03.004. - DOI - PMC - PubMed
    1. Cheng Y., Walz T. The advent of near-atomic resolution in single-particle electron microscopy. Annu. Rev. Biochem. 2009;78:723–742. doi: 10.1146/annurev.biochem.78.070507.140543. - DOI - PubMed
    1. Wolf M., Garcea R.L., Grigorieff N., Harrison S.C. Subunit interactions in bovine papillomavirus. Proc. Natl. Acad. Sci. USA. 2010;107:6298–6303. doi: 10.1073/pnas.0914604107. - DOI - PMC - PubMed
    1. Bharat T.A., Davey N.E., Ulbrich P., Riches J.D., de Marco A., Rumlova M., Sachse C., Ruml T., Briggs J.A. Structure of the immature retroviral capsid at 8 A resolution by cryo-electron microscopy. Nature. 2012;487:385–389. doi: 10.1038/nature11169. - DOI - PubMed
    1. Ruska E. Nobel lecture. The development of the electron microscope and of electron microscopy. Biosci. Rep. 1987;7:607–629. doi: 10.1007/BF01127674. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources