Visualizing the virus world inside the cell by cryo-electron tomography

Abstract

Structural studies on purified virus have revealed intricate architectures, but there is little structural information on how viruses interact with host cells in situ. Cryo-focused ion beam (FIB) milling and cryo-electron tomography (cryo-ET) have emerged as revolutionary tools in structural biology to visualize the dynamic conformational of viral particles and their interactions with host factors within infected cells. Here, we review the state-of-the-art cryo-ET technique for in situ viral structure studies and highlight exemplary studies that showcase the remarkable capabilities of cryo-ET in capturing the dynamic virus-host interaction, advancing our understanding of viral infection and pathogenesis.

Keywords: Cryo-ET; in situ structure; infection; virus.

Fig 1

Cryo-ET sample preparation to study in situ virus structures. Infected cells are grown on a cryo-EM grid, which is then plunge-frozen in liquid ethane. Cryo-ET tilt series can be directly collected For thin samples. For thick samples such as cells, sample thinning techniques, such as FIB milling guided by CLEM are required to obtain electron-penetrable thin lamellae (<200 nm) prior to cryo-ET data collection. Figure created with BioRender.com.

Fig 2

Bacteriophage life cycle with representative in situ cryo-ET studies. Phage infection cycle begins with cell binding and genetic material ejection into the cytoplasm. (a) Cryo-ET shows phage ϕCb13 initially binding to the bacterial flagellum before cell pore binding. Panel shows phages (blue and yellow) wrapping around the flagellum (green) [adapted from Guerrero-Ferreira et al. (17) with permission of the publisher]. (b) Genome ejection strategies: Contractile-tailed phage T4 shows tail sheath contraction upon E. coli minicell binding. Panel displays tomogram slices and 3D models of T4 binding to the cell membrane [adapted from Hu et al. (19) with permission of the publisher]. Non-contractile-tailed phage P22 creates a membrane channel for DNA injection. Panel displays the central section of the averaged structure of P22 tail during genome ejection and 3D model [adapted from Wang et al. (20) with permission of the publisher]. Tailless phage ΦX174 extrudes a tube for DNA penetration. Panel shows tomogram slices of ΦX174 attaching to E. coli and the enlarged images with a model [adapted from Sun et al. (21) with permission of the publisher]. (c) Genome replication and phage assembly in the bacterial cytosol: Phage 201φ2–1 forms assembly compartments with capsids docked on its surface. Panel shows a tomogram slice of 201φ2–1 infected Pseudomonas chlororaphis and segmentation with capsid (green), ribosome (yellow), shell (purple), and outer membrane (red) [adapted from Chaikeeratisak et al. (22) with permission of the publisher]. (d) Capsid movement along PhuZ filaments for DNA packaging during assembly. Panel shows a tomogram slice of phage ΦPA3 infection in Pseudomonas aeruginosa and segmentation with phage capsid (green), cell membrane (red), and PhuZ filaments (blue) [adapted from Chaikeeratisak et al. (23) with permission of the publisher]. (e) Late-stage infection exhibits membrane disturbances, likely preparing for phage release. Panel shows a tomogram section of phage Syn5-infected Synechococcus and segmentation with capsid (magenta) and cell membrane (yellow) [adapted from Dai et al. (24) with permission of the publisher]. Figure created with BioRender.com.

Fig 3

HIV life cycle with representative in situ cryo-ET studies. (a) HIV envelope proteins (Env) show mobility on viral surface. Panel shows Env distribution on the mature HIV VLP and the averaged Env structure [adapted from Prasad et al. (42)]. HIV life cycle begins with Env attachment to CD4⁺ cells, fusion with the cell membrane, and core release into the cell. Host factor TRIM5 affects HIV uncoating, while CypA can bind to the HIV capsid and modulate infectivity. (b) HIV capsids are transported through the nuclear pore complex (NPC) into the nucleus. Panel shows a tomogram slice of FIB-milled HIV-infected cells and segmentation with the HIV capsid (magenta), NPC (cyan) and nuclear envelope (yellow), and the core transport model [adapted from Zila et al. (43)]. Inside the nucleus, vRNA is reverse transcribed into cDNA, which integrates into the host genome. (c) Viral particles assemble in the cytosol and bud through the cell membrane, forming immature HIV particles. Host factor IP₆ interacts with CA to stabilize the core. Panel shows a tomogram slice of HIV budding [adapted from Woodward et al. (41)]. (d and e) Gag proteins undergo conformational changes to form the mature matrix (d) and core (e). (d) Panel shows tomogram slices of immature and mature HIV particles and a lattice map [adapted from Qu et al. (39) with permission of the publisher]. (e) Panel shows tomogram slices and lattice maps of mature HIV particles with cone-shaped cores [adapted from Mattei et al. (40) with permission of the publisher]. Figure created with BioRender.com.

Fig 4

SARS-CoV-2 life cycle with representative in situ cryo-ET studies. (a) SARS-CoV-2 life cycle begins with spike (S) protein attaching to ACE2 receptors, leading to membrane fusion. Panel shows tomogram slices of SARS-CoV-2 particles and a model of S protein with flexible hinges (red) allowing large motions during receptor binding [adapted from Turoňová et al. (69)]. (b-d) Virus induces formation of double membrane vesicles (DMV) for replication (b), transports RNA through DMV pores (c), and assembles new virions at the ER–Golgi intermediate compartment (ERGIC) (d). Panel (b) shows a tomogram slice and segmentation of DMV membrane (green) and inner filaments [adapted from Klein et al. (73)]. Panel (c) shows a tomogram slice and segmentation of DMV pore [adapted from Wolff et al. (74)]. Panel (d) shows a tomogram slice and segmentation virion assembly at the ERGIC [adapted from Klein et al. (73)]. (e) Virions bud into membrane vesicles. Panel shows a tomogram slice and segmentation of intracellular virion [adapted from Klein et al. (73)]. (f) Virus exits the cell via exocytosis. Panel shows a tomogram slice of virus exiting through tunnel [adapted from Mendonça et al. (75)]. (g) SARS-CoV-2 virus-virus fusion in six stages. Panel shows tomogram slices of virus–virus fusion and the model [adapted from Song et al. (70)]. Figure created with BioRender.com.

Fig 5

CHIKV life cycle with representative in situ cryo-ET studies. CHIKV infection cycle begins with viral envelope proteins binding to cellular receptors, membrane fusion, RNA release, and translation of non-structural proteins for viral replication. (a and b) Replication happens in spherules on the plasma membrane (PM). Panels (a and b) show a tomogram slice, segmentation and averaged structure of spherules [adapted from Tan et al. (97) and Laurent et al. (98)]. (c) After translation, the nucleocapsid core forms and moves to the PM and interacts with envelope proteins to initiate budding. Panel shows a tomogram slice of CHIKV budding at the PM and the averaged structure of budding intermediates [adapted from Chmielewski et al. (100) with permission of the publisher]. (d) Antibody inhibition of viral budding traps the nucleocapsid in the cytosol. Panel shows a tomogram slice of arrested nucleocapsid particles and the segmentation with capsid (red), spherules (green), and PM (orange) [adapted from Jin et al. (101) with permission of the publisher]. Figure created with BioRender.com.