Cryo-EM reconstruction of the Cafeteria roenbergensis virus capsid suggests novel assembly pathway for giant viruses

Abstract

Whereas the protein composition and overall shape of several giant virus capsids have been described, the mechanism by which these large capsids assemble remains enigmatic. Here, we present a reconstruction of the capsid of Cafeteria roenbergensis virus (CroV), one of the largest viruses analyzed by cryo-electron microscopy (cryo-EM) to date. The CroV capsid has a diameter of 3,000 Å and a Triangulation number of 499. Unlike related mimiviruses, the CroV capsid is not decorated with glycosylated surface fibers, but features 30 Å-long surface protrusions that are formed by loops of the major capsid protein. Based on the orientation of capsomers in the cryo-EM reconstruction, we propose that the capsids of CroV and related giant viruses are assembled by a newly conceived assembly pathway that initiates at a five-fold vertex and continuously proceeds outwards in a spiraling fashion.

Figure 1

Cryo-EM images of CroV compared to APMV. (A) Cryo-electron micrograph of four CroV particles. (B) Single CroV particle with concave core depression (white arrow). (C) Single APMV particle. Scale bars in (A–C) represent 2,000 Å.

Figure 2

Cryo-EM reconstruction of the CroV virion and capsomer arrangements of other giant icosahedral viruses. (A) Reconstruction of the CroV capsid. The isosurface of the map was colored by pentasymmetrons (purple) and trisymmetrons (blue, red, green, cyan and orange). One of the 30 edges of the icosahedron is marked by a cyan line. Two surface areas (a,b) are magnified and selected capsomers are labeled by yellow triangles to show their orientations. (B–E) Isolated icosahedral faces of CroV, PBCV-1, CIV and PpV01 capsids are shown schematically. Their T-numbers, asymmetric unit capsomer numbers, and trisymmetron capsomer numbers are listed. 5-fold, 3-fold, and 2-fold symbols are indicated in red and ASUs are outlined in blue.

Figure 3

The double jelly-roll major capsid protein (MCP) of large icosahedral viruses. (A) Multiple sequence alignment of the MCPs of CroV, PBCV-1, PpV01 and APMV. Both CroV and APMV have large DE2 loops. (B) The CroV MCP (red) was homologously modeled and superposed with the X-ray structure of PBCV-1 (cyan) to show the longer DE2 loop of CroV. (C) Ribbon diagram of the trimeric homologously modeled CroV double jelly-roll MCP capsomer displaying a pseudo-hexagonal shape. Individual double jelly-roll MCPs are color-coded. (D) Central cross-section of the cryo-EM map of CroV superposed with that of PBCV-1. CroV not only has a larger virion diameter (3000 Å vs 1800 Å), its capsid layer is also thicker (105 Å vs 75 Å), which is consistent with the extended DE2 loop shown in panel (B).

Figure 4

The proposed spiral assembly pathway of large icosahedral capsids. Isosurfaces of CroV (A), PBCV-1 (B), and CIV (C) cryo-EM maps were centered on the 5-fold axis. Capsomers in all three panels are colored based on their orientation in red, blue, green, cyan, and orange. The pentameric capsomers are depicted as purple stars. (D) Schematic diagram showing the right-handed spiral assembly pathway following arrows (1) to (6), with multiple extensions (7) of the trisymmetrons to form similar patterns as shown in (A–C). One set of capsomers (red) is labeled by Roman numerals if they are part of the pentasymmetron and by Arabic numerals if they are part of a trisymmetron.