Cryo-electron microscopy in the study of virus entry and infection

Abstract

Viruses have been responsible for many epidemics and pandemics that have impacted human life globally. The COVID-19 pandemic highlighted both our vulnerability to viral outbreaks, as well as the mobilization of the scientific community to come together to combat the unprecedented threat to humanity. Cryo-electron microscopy (cryo-EM) played a central role in our understanding of SARS-CoV-2 during the pandemic and continues to inform about this evolving pathogen. Cryo-EM with its two popular imaging modalities, single particle analysis (SPA) and cryo-electron tomography (cryo-ET), has contributed immensely to understanding the structure of viruses and interactions that define their life cycles and pathogenicity. Here, we review how cryo-EM has informed our understanding of three distinct viruses, of which two - HIV-1 and SARS-CoV-2 infect humans, and the third, bacteriophages, infect bacteria. For HIV-1 and SARS-CoV-2 our focus is on the surface glycoproteins that are responsible for mediating host receptor binding, and host and cell membrane fusion, while for bacteriophages, we review their structure, capsid maturation, attachment to the bacterial cell surface and infection initiation mechanism.

Keywords: 3D reconstructions; HIV-1; SARS-CoV-2; bacteriophage; cryo-EM; cryo-ET; structure-based design; vaccine development.

FIGURE 1

Road map of development of cryo-electron microscopy field. Milestones indicated with blue flags starting from development of electron microscope (1931), visualization of first virus under EM (1939), electron diffraction of frozen, hydrated catalase crystals (1974) and development of electron microscopy of frozen biological samples (1983). Then low resolution cryo-EM maps were reconstructed during early 2000. With the development of direct electron detectors around 2011-2013 there was an upsurge of high resolution cryo-EM structures. In 2017 the Nobel Prize in Chemistry was given to the three pioneers (Jacques Dubochet, Joachim Frank, Richard Henderson) for the development of structure determination by cryo-electron microscopy. By the year 2020 atomic resolution (1.22Å) structure was achieved for apoferritin (EMD-11638) by cryo-EM. Green flags indicate the important milestones of the contributions of cryo-EM in bacteriophages (EMD-1086; 5003), HIV Envelope (EMD-5779; 5019) and SARS-CoV2 spike (EMD-21375; 11494) structure determination, the three topics that are the focus of this paper.

FIGURE 2

Cryo-ET and single particle cryo-EM of HIV-1 Env. (A) From left to right, CD4-binding site antibody VRC01 and CD4-induced antibody 17b (EMD-5461), CD4-binding site antibody b12 (EMD-5018), sCD4 and CD4-induced antibody m36 (EMD-5554), and V3-glycan antibody 10-1074 and CD4-binding site antibody 3BNC117 (EMD-21413). (B) Cryo-ET study of HIV-1 entry mechanism. From left to right, One (EMD-29292), two (EMD-29293) and three (EMD-29294) CD4 molecules sequentially bound to native HIV-1 Env on membrane brings the host and viral membranes close to each other to initiate the fusion process. (C) Single particle cryo-EM of HIV-1 Env conformational states. From left to right, closed (EMD-8714), open-occluded (EMD-25878, bound antibody not shown), open (EMD-8713, bound CD4 and antibody not shown) conformational states are shown with the Env colored sea green, V1V2 loop colored orange, V3 loop colored pink and CD4 binding site colored medium orchid. Each panel includes a schematic representation.

FIGURE 3

High resolution cryo-EM structures of HIV SOSIP Env with antibodies. (A) Naturally-elicited antibodies in complex with HIV SOSIP Env spike: From left to right (first row), CD4 binding site Ab VRC01 (EMD-40281), glycan cluster Ab 2G12 (EMD-20224), V1/V2-apex directed Ab PGT145 (EMD-29288). From left to right (second row), V3-glycan targeted Ab DH270.6 (EMD-20818), gp120-gp41 interface Ab 8ANC195 (EMD-0485), fusion peptide-targeting Ab VRC34 (EMD-28617), membrane proximal external region targeted Ab 4E10 (EMD-25045). (B) Vaccine-elicited antibodies in complex with HIV SOSIP Env spike: From left to right (first row), V3-glycan mouse Ab MU89 (EMD-27706), CD4 binding site macaque Ab DH1285 (EMD-41823), FP-targeting human Ab 2C06 (EMD-29725), MPER targeted human Ab DH1317.4 (EMD-44246). (C) Cryo-EM based polyclonal epitope mapping (cryo-EMPEM): From left to right (first row), polyclonal antibodies were selected from rhesus macaques Rh.32034 (Grp1): pAbC-1(C3/V5 targeted, EMD-23223) and pAbC-2(N241/N289 glycan hole targeted, EMD-23224); From left to right (second row) Rh.33104 (Grp1): pAbC-1(N241/N289 glycan hole targeted, EMD-23227) and pAbC-2(N241/N289 glycan hole targeted, EMD-23228); From left to right (third row) Rh.33311 (Grp2): pAbC-1(FP targeted, EMD-23236), pAbC-2(N611-glycan targeted, EMD-23237), and pAbC-5(C3/V5 targeted, EMD-23240). Antibody heavy chain in medium purple and light chain in pink, Env gp120 in dark gray and gp41 in light steel blue throughout Figure 3. Each panel includes a schematic representation (created with BioRender.com).

FIGURE 4

Structural determination of SARS-CoV-2 variants of concern (VoC) (built with nextstrain/ncov). 2019–2024 evolution of SARS-CoV-2 spikes of key emerging variants of concern (VoC): 2019-nCoV (EMD-21375), D614G (EMD-22825), Alpha (B.1.1.7) (EMD-14229), Beta (B.1.351) (EMD-27505), Gamma (P.1) (EMD-24987), Delta (B.1.617.2) (EMD-24981), Omicron BA.1 (B.1.1.529) (EMD-25896), Omicron BA.2 (EMD-26433), Omicron (XBB.1.5) (EMD-43320), Omicron (XBB.1.16) (EMD-42860), Omicron (EG.5.1) (EMD-44000). All cryo-EM structures (side and top view) of spike show the three monomers in dark gray, light steel blue and thistle colors and mutations in spike are shown in red color.

FIGURE 5

Cryo-EM studies of SARS-CoV-2 spikes and its interaction with antibodies. (A) Schematic representation of SARS-CoV2 spike with its different parts color labeled with S1 (antique white), S2 (yellow), N-terminal domain (NTD, dark sea green), receptor binding domain (RBD, sea green), SD1 (misty rose), fusion peptide (FP, orange) and stem helix region (pink violet).The same color scheme was maintained throughout Figure 5 except 5G. Antibody heavy chain is shown in purple and light chain is in thistle color. (B) ACE-2 bound (human) SARS-CoV-2 XBB.1.5 spike (EMD-43324). (C) Binding patterns of different NAbs (class 1, EMD-13869), (class 2, EMD-23156), (class 3, EMD-22732), (class 4, EMD-24504) to RBD. (D) Binding of NTD-targeted Ab DH1050.1 (EMD-23277). (E) Binding of SD1-targeted Ab S3H3 (EMD-32564). (F) Binding of glycan-targeted Ab 2G12 (EMD-23094). (G) Binding of stem-helix specific Ab S2P6 along with RBM-bound S2M11 (EMD-24533).

FIGURE 6

Cryo-electron tomography of membrane bound SARS-CoV-2 spikes. (A) From left to right, SARS-CoV-2 virus with spikes from subtomogram averaging (12Å) put back on membrane surface and glimpse of inside, EMD-30430, a representative tomogram slice of SARS-CoV-2 infected and cryo-FIB milled VeroE6 cells (black arrows indicate virions), EMD-11865, and in situ assembly of the ribonucleoprotein inside SARS-CoV-2 virus, EMD-30429. (B) From left to right, In situ spike structures reveal flexibility mediated by three hinges, EMD-11223, prefusion spike viewed at a 40° angle from the normal axis on membrane in a 3 RBD-down conformation, EMD-30426, and post-fusion conformation, EMD-30428.

FIGURE 7

Structural studies on bacteriophages using cryo-EM. (A) Schematic diagram of a long-tailed bacteriophage with its parts labeled. Structure of different parts of bacteriophages were solved either individually or partially together and a representative cryo-EM map is shown for each part: capsid (EMD-40228), portal protein (EMD-43145), collar and whiskers (EMD-5528), tail tube (EMD-29354), tail sheath (EMD-36127), base plate (EMD-8064), tail fiber (EMD-34968), tail pin (EMD-35824) in figure A (created with BioRender.com). (B) Phage infection initiation mechanism is shown with a schematic model. (C) DNA pre injection and post injection states are shown in short tailed (left, EMD-31315, 31318) and long-tailed phages (right, EMD-2774, 6082). (D) Close view of detailed structural changes during intermediate stages of long tail contraction (EMD-1089, 1086). (E) Cryo-EM map of trans envelope channel formation during short-tailed phage infection (EMD-9006). (F) Schematic diagram of various application of phages (created with BioRender.com).