The architecture and chemical stability of the archaeal Sulfolobus turreted icosahedral virus

Abstract

Viruses utilize a diverse array of mechanisms to deliver their genomes into hosts. While great strides have been made in understanding the genome delivery of eukaryotic and prokaryotic viruses, little is known about archaeal virus genome delivery and the associated particle changes. The Sulfolobus turreted icosahedral virus (STIV) is a double-stranded DNA (dsDNA) archaeal virus that contains a host-derived membrane sandwiched between the genome and the proteinaceous capsid shell. Using cryo-electron microscopy (cryo-EM) and different biochemical treatments, we identified three viral morphologies that may correspond to biochemical disassembly states of STIV. One of these morphologies was subtly different from the previously published 27-A-resolution electron density that was interpreted with the crystal structure of the major capsid protein (MCP). However, these particles could be analyzed at 12.5-A resolution by cryo-EM. Comparing these two structures, we identified the location of multiple proteins forming the large turret-like appendages at the icosahedral vertices, observed heterogeneous glycosylation of the capsid shell, and identified mobile MCP C-terminal arms responsible for tethering and releasing the underlying viral membrane to and from the capsid shell. Collectively, our studies allow us to propose a fusogenic mechanism of genome delivery by STIV, in which the dismantled capsid shell allows for the fusion of the viral and host membranes and the internalization of the viral genome.

FIG. 1.

Disassembly states of STIV. (A) Micrographs of frozen hydrated STIV samples collected at ×80,000 magnification, with a 1.5-μm underfocus. Micrograph of particles purified using a CsSO₄ gradient step at room temperature. Roman numerals I and III identify the type I and III (undecorated and lipid-core) particles (top). Micrograph of particles purified using a CsCl₂ gradient step at room temperature. Roman numeral II identifies the type II (disassembled) particles. CsCl₂ is clearly a chaotrope for STIV (bottom). Black arrows identify the double-layered electron density associated with every particle that is perceivably the viral membrane of STIV. The double-ended black arrow highlights portions of the capsid shell associated with type II particles. The scale bar measures 200 nm. (B) SDS-PAGE of the particles purified using the CsSO₄ (left) and CsCl₂ (right) gradients. Labeled are the STIV ORFs corresponding to each protein band. Proteins belonging to ORFs C381 and A223 are absent in the type II (disassembled) particles.

FIG. 2.

Surface representations of the STIV cryo-EM image reconstructions contoured at 100% MCP mass content (1.1 σ). (A) Decorated and undecorated image reconstructions on the left and right, respectively. The turret-like vertices are colored yellow, the MCP shell cyan, the lipid layer red, and the genome blue. The genome in the undecorated image reconstruction cannot be visualized at the rendered contour threshold. (B) The segmented turret-like vertices of the decorated and undecorated image reconstruction on the left and right, respectively. Colored in yellow, magenta, and blue are the densities assigned to proteins from ORFs A223 and C381 (indicated on the figure), C557, and A55/B130, respectively. (C) Segmented densities for the petal- (magenta) and feet-like (blue) regions of the vertices. These densities pertain to substituents with masses of 63 and 19 kDa and are assigned to the proteins from ORFs C557 and A55/B130, respectively.

FIG. 3.

Surface representation of difference maps calculated from the undecorated image reconstruction. Densities for the genome, viral membrane, and vertex turrets are contoured at 1.1 σ, and densities for the MCP region are contoured at 1.7 σ. The higher contour threshold for the MCP region is to visually reduce the noise present in the difference maps. (A) Outline of an icosahedral facet. Colored in red is the viral membrane, in blue are the finger-like densities, in yellow are the vertex turrets, and in gold and outlined in black are the glycosylation sites. Two of the iASUs are outlined with red and cyan polygons. The letters identify the five capsomers in the iASU. (B) Difference map comparison of the decorated and undecorated image reconstructions. (C) Side view of capsomer E overlaid with the undecorated difference map. The MCP C-terminal helix has been modeled into finger-like densities (blue).

FIG. 4.

Analysis of type I to III particles. (A) Averages of translationally centered type I to III particles. Density for the type I particle lipid layer is highlighted by two black arrowheads. (B) Radially averaged density profiles identifying the three different regions of STIV (genome, lipid, and capsid). The profiles have been scaled to one another according to the peaks corresponding to the lipid membrane. (C) Reference-free class averages of type II particles using rotational power spectra with 7-by-7-matrix self-organizing maps. Sixfold class averages are clearly visible in the lower left-hand corner of the matrix. White arrowheads highlight the nodular densities responsible for the symmetry of these class averages.

FIG. 5.

Sequence alignment of the fused STIV A223 and C381 sequences and the STIV2 B631 sequence. The alignment was carried out using the default parameters of the PROMALS server. Cyan boxes are identical residues. The green and yellow boxes at the top identify the STIV A223 and C381 sequences.