Three-dimensional visualization of gammaherpesvirus life cycle in host cells by electron tomography

Abstract

Gammaherpesviruses are etiologically associated with human tumors. A three-dimensional (3D) examination of their life cycle in the host is lacking, significantly limiting our understanding of the structural and molecular basis of virus-host interactions. Here, we report the first 3D visualization of key stages of the murine gammaherpesvirus 68 life cycle in NIH 3T3 cells, including viral attachment, entry, assembly, and egress, by dual-axis electron tomography. In particular, we revealed the transient processes of incoming capsids injecting viral DNA through nuclear pore complexes and nascent DNA being packaged into progeny capsids in vivo as a spool coaxial with the putative portal vertex. We discovered that intranuclear invagination of both nuclear membranes is involved in nuclear egress of herpesvirus capsids. Taken together, our results provide the structural basis for a detailed mechanistic description of gammaherpesvirus life cycle and also demonstrate the advantage of electron tomography in dissecting complex cellular processes of viral infection.

Figure 1. Major events of viral life cycle and comparison between 2D TEM imaging and 3D visualization by ET

(A) Major events of MHV-68 life cycle illustrated with 2D TEM micrograph. (B) Comparison between 2D micrograph (left) and digital slice from 3D reconstruction (right). (Note: in all figures of this paper, italic abbreviations used for describing cellular compartments: Cy – cytoplasm, Nu – nucleus, Ve – vesicle, Ex – extracellular space)

Figure 2. Virus attachment and entry

(A – C, E & F) 1 nm-thick digital slices from reconstructed tomograms; (A) A cell incubated with MHV-68 virions at 4°C with viral particles attached; (B – D, F) endocytosis of viral particles; (D) a colorized surface rendering of an 11 nm slab of the feature framed in (B); (E) de-enveloped input capsid in the cytoplasm. Color codes in 3D rendering: red – viral DNA; green – capsid; magenta – tegument; orange – envelope; yellow – protrusions on the membrane; light gray – plasma membrane; cyan– membrane coating. Time points (p.i.): (A) – 2 h, (B) – 5 min, (E) – 20 min, (C, D and F) – 5 h Bars: 200nm. Arrows: (E) enveloped virion; (F) L-particle. (Note: For the following figures, grayscale illustrations are 1 nm-think digital slices from reconstructed tomograms, exceptions are otherwise indicated; colorized illustrations [except for Fig. 8] are 3D surface rendering of digital slabs from reconstructed tomograms)

Figure 3. Capsids docking at nuclear pore and viral DNA injection

(A) Incoming capsids close to nuclear pores: one empty capsid docked at a nuclear pore and a capsid with viral DNA inside was located about 200nm away from another nuclear pore; arrow: capsids with DNA core; arrowhead: capsid without DNA; (B) 11 nm-thick slab of the tomogram shown in (A); (C) 3D view of capsid docking at NPC and viral DNA injection (15 nm-thick slab); (D – F) sections from different positions of z-dimension from the 3D volume shown in (C). Filaments emanating from the NPC were indicated by arrows in (D) and the viral DNA was indicated by arrows in (E and F). Color codes in 3D rendering: red – viral DNA; green – capsid; light gray – ribosomes; orange – INM; magenta – ONM; cyan – NPC. Time points (p.i.): (A, B) – 5 h, (C – F) – 24 h. Bars: 200nm.

Figure 4. Maturation of nucleocapsids and encapsidation of viral DNA

(A) A TEM micrograph of a MHV-68 infected NIH3T3 cell; (B, C) various kinds of progeny capsids inside nucleus; (D, E) possible intermediates in capsids assembly (arrows); (F – Q) intermediate stages of viral DNA (arrows) encapsidation process; 3D colored views are rendered from 8nm-thick slabs. Color codes in 3D rendering: red – viral DNA; green – capsid; yellow – scaffolding protein. Time points (p.i.): (A – Q) – 24 h. Bars: (A) 2 μm; others, 100 nm.

Figure 5. Virus-induced nuclear inclusion bodies

(A, D) TEM micrographs of infected cells with inclusion bodies; (B & C) the same inclusion body in (A); (E & F) the inclusion body in (D); (C, F) shaded surface views of entire sections (47 & 135 nm-thick) done in Chimera. Time points (p.i.): (A – F) – 24 h. Bars: (A) 2 μm; (D) 1 μm; (B, E) 200 nm.

Figure 6. Egress from the nucleus

(A) A capsid in the budding process on the INM; (B) a primary enveloped capsid located in the PNS; (C) an 11 nm-thick slab of a fusion event; (D) an 18nm-thick slab of an INM invagination; (E) an inner membrane invagination filled with primary-enveloped capsids; (F) a 13 nm-thick slab of the invagination shown in (E); (G) a 7 nm slab of a double-membrane invagination; (H) a double-membrane invagination; (I) a 100 nm-thick slab of a double membrane invagination; (J – L) 1 nm digital slices from top, middle and bottom of the double structure in (I). Color codes in 3D rendering: red – viral DNA; green – capsid; orange – INM or primary envelope; magenta – ONM or primary envelop in fusion; cyan – NPC; light gray – ribosome. Time points (p.i.): (A, C) – 36 h, (B, D – L) – 24 h. Bars: 200nm.

Figure 7. Tegumentation, secondary envelopment in cytoplasm and extracellular viral particles

(A) Capsids were undergoing tegumentation process in bulk accumulations of tegument proteins in the cytoplasm; (B, C) secondary envelopment in TGNs; (D) a virion and a L-particles in an exocytic vesicle; (E & F) a secondary enveloped particle with large amount of outer layer tegument proteins; (G, H) tegumentation and secondary envelopment of a capsid; (I) a mature virion inside the TGN with spike-like protrusions on the bilayer surface; (J) an exocytic vesicle containing a mature virion in the vicinity of the plasma membrane; (K) an extracellular mature virion; (L) an extracellular enveloped particle with an A-capsid inside; (F, H & K) 11 nm slabs. Color codes in 3D rendering: red – viral DNA; green – capsid; magenta – inner layer of tegument; blue – outer layer of tegument; orange – envelope; yellow – protrusions of glycoprotein. Time points (p.i.): (B & J) – 36 h, (A, C – I, K & L) – 24 h. Bars: 200nm

Figure 8. A 3D illustration of MHV-68 life cycle in the host cell

1) entry via endocytosis; 2) transportation in endosome; 3) viral DNA injection; 4) capsid assembly and DNA encapsidation; 5) intranuclear inclusion bodies; 6) nuclear membrane(s) invaginations; 7) egress from nucleus; 8) tegumentation; 9) secondary envelopment; 10) egress via exocytosis. (Detailed descriptions were presented in the Discussion)