Atomic structure of the 75 MDa extremophile Sulfolobus turreted icosahedral virus determined by CryoEM and X-ray crystallography

Abstract

Sulfolobus turreted icosahedral virus (STIV) was isolated in acidic hot springs where it infects the archeon Sulfolobus solfataricus. We determined the STIV structure using near-atomic resolution electron microscopy and X-ray crystallography allowing tracing of structural polypeptide chains and visualization of transmembrane proteins embedded in the viral membrane. We propose that the vertex complexes orchestrate virion assembly by coordinating interactions of the membrane and various protein components involved. STIV shares the same coat subunit and penton base protein folds as some eukaryotic and bacterial viruses, suggesting that they derive from a common ancestor predating the divergence of the three kingdoms of life. One architectural motif (β-jelly roll fold) forms virtually the entire capsid (distributed in three different gene products), indicating that a single ancestral protein module may have been at the origin of its evolution.

Fig. 1.

Near-atomic resolution electron cryomicroscopy reconstruction of STIV. (A) The overall virus reconstruction is displayed with the different protein components individually colored (B345, light blue; A223, light pink; C381, purple) and with an icosahedral cage overlaid onto it. (B) Blow-up view of an icosahedral face with one capsid icosahedral asymmetric unit colored as in A and labeled (1–5 for the trimeric B345 capsomers and P for the A223 penton base). (C) Cross-section of the reconstruction revealing the presence of the viral membrane (gold) and the internal genome (red). The cement protein (dark gray) has been removed from the two vertex complexes located on the right-hand side to allow a better visibility of the β-pore.

Fig. 2.

The capsid shell. (A) CryoEM density of a single B345 coat subunit at 3.9 Å resolution. (B) Fit of the B345 crystal structure into the corresponding density. The crystal structure stops at residue 324 whereas the cryoEM density is continuous until the C-terminal residue and folds as an α-helix (delineated by the two red arrows). The red star indicates the position of the E-F loop belonging to the smaller jelly-roll of the B345 subunit. (C) The B345 C-terminal α-helix (residues 325–345) was modeled de novo in the cryoEM density. The red arrows are matching the region shown in B. (D) The electrostatic surface potential of the B345 C-terminal helix reveals that its most C-terminal moiety is strongly positively charged due to the presence of many basic residues. Electrostatics calculations were carried out at pH 3.0 and 80 °C, and the result is displayed colored from red (−5 kT/e) to blue (+5 kT/e).

Fig. 3.

The penton base structure. (A) The pentameric A223 structure was built by combining de novo modeling in the cryoEM density with X-ray crystallography of its C-terminal domain jelly-roll. (B) The A223 pentamer is formed of three layers from the N to the C terminus: (i) a 10-stranded β-pore formed with the A55 membrane protein (steel blue), (ii) the penton base jelly-roll ring, and (iii) the third layer, also forming a ring of jelly-rolls, which is connected to the rest of the structure via an additional strand added to the penton base jelly-roll domain. (C) A single A223 monomer. The A223 N-terminal β-strand is associated in an anti-parallel manner to the A55 N-terminal β-strand (steel blue). (D) Each A223 jelly-roll interacts via its B-C face with the larger jelly-roll of B345 belonging to the same icosahedral asymmetric unit whereas its prominent E-F loop fits in the concave surface defined by the two jelly-rolls of B345 from the next asymmetric unit (indicated by a red star). B345 subunits are shown in light blue. The β-strands B and C from the A223 penton base domain rendered in light pink are labeled for clarity.

Fig. 4.

Architecture of the turret. (A) The pentameric C381 turret protein X-ray structure is shown fit into the corresponding region of the cryoEM density. (B) Each C381 monomer is formed of three layers of jelly-rolls with markedly different orientations. (C) The interior of the C381 pentamer exhibits a positive electrostatic surface potential and several constrictions (indicated by black arrows). These characteristics are unfavorable to allow dsDNA genome transit during packaging or infection. Electrostatics calculations were carried out at pH 3.0 and 80 °C, and the result is displayed colored from red (−5 kT/e) to blue (+5 kT/e). One C381 monomer has been removed to allow visualization of the pentamer central cavities.

Fig. 5.

Organization of the viral membrane. (A) The A55 protein (steel blue) accounts for the unique transmembrane helix present within each icosahedral asymmetric unit as well as for the 38-Å-high cylindrically shaped extension contacting the A223 penton base protein (light pink) and for the additional β-strand contributing to the formation of the β-pore structure. The lipid headgroups belonging to the monolayered viral membrane are visualized as two layers rendered in gold whereas the most external layer of the genome is displayed in red. Four out of five of the B345 capsomers surrounding A223 at the icosahedral fivefold axis are also shown (light blue). (B) The A55 pentameric bundle of transmembrane helices is shown within the corresponding cryoEM density. (C) Tilted view of the A223/A55 complex showing the N-terminal β-strand of each A55 monomer connecting to the density appended below the β-pore and reaching the transmembrane domain. Only the extramembranous region of the A55 membrane protein is represented.

Fig. 6.

Assembly of the STIV virion from a single protein module. The B345 coat subunit, A223 penton base, and C381 turret protein are all formed of various numbers of jelly-roll domains fused on a single polypeptide chain. We therefore propose that a unique ancestral protein module gave rise to many of the STIV structural proteins by duplication and sequence diversification events.