Familial relationships in hyperthermo- and acidophilic archaeal viruses

Abstract

Archaea often live in extreme, harsh environments such as acidic hot springs and hypersaline waters. To date, only two icosahedrally symmetric, membrane-containing archaeal viruses, SH1 and Sulfolobus turreted icosahedral virus (STIV), have been described in detail. We report the sequence and three-dimensional structure of a third such virus isolated from a hyperthermoacidophilic crenarchaeon, Sulfolobus strain G4ST-2. Characterization of this new isolate revealed it to be similar to STIV on the levels of genome and structural organization. The genome organization indicates that these two viruses have diverged from a common ancestor. Interestingly, the prominent surface turrets of the two viruses are strikingly different. By sequencing and mass spectrometry, we mapped several large insertions and deletions in the known structural proteins that could account for these differences and showed that both viruses can infect the same host. A combination of genomic and proteomic analyses revealed important new insights into the structural organization of these viruses and added to our limited knowledge of archaeal virus life cycles and host-cell interactions.

FIG. 1.

Electron micrographs of the archaeal virus STIV2. Shown is a cryo-electron micrograph taken at 2.9-μm underfocus showing STIV2 viral particles (black arrow). Big, turreted spikes (white arrows), which might be used in host cell infection, are visible protruding from the vertices. The diameter of the particles is 71 nm facet to facet and 93 nm with the turrets included. Flagellar impurities in the virus preparation are indicated by the asterisk. The turrets are shown more prominently in an electron micrograph of the negatively stained STIV2 (inset A) (a turret is indicated by a white arrow). Very rarely, empty capsid shells lacking DNA were observed (inset B; underfocus, 4.1 μm). The membrane is indicated by an open arrow. Bar, 100 nm.

FIG. 2.

Genome comparison of the STIVs. The genomes are represented by solid black bars, and the predicted ORFs are indicated by arrows showing their relative orientations. Black arrows, structural proteins detected by mass spectrometry and labeled with their gene names; gray arrows, other ORFs. The gray ORFs that are labeled have a corresponding protein structure in STIV (27, 32-34) or we have determined a homology model (STIV2). The parallelograms between the two genomes indicate regions of more than 70% nucleotide sequence identity.

FIG. 3.

Structure of STIV2 and its turrets. (A) A radially depth-cued isosurface representation (at 2 σ above the mean) of the 20-Å-resolution STIV2 reconstruction viewed down a 2-fold symmetry axis. Symmetry axes are indicated by a white ellipse (2-fold), triangle (3-fold), and pentagon (5-fold). (B) A 0.44-nm-thick central section through the STIV2 virion (left). The right side shows the capsid protein density generated by fitting the homology-modeled coat protein into the reconstruction. Symmetry axes are indicated as in panel A. The viral capsid (C) and the underlying membrane (M) are indicated. Bar, 20 nm. (C) A close-up (at 1 σ above the mean) of the STIV2 turrets. (D) Comparison of the central sections of the manually segmented STIV2 (red outline) and STIV (blue outline) turrets. The dimensions are indicated for the STIV2 turret. The open arrow points to the channel in the middle of the turrets through which DNA may travel (27). (E) A close-up (at 1 σ above the mean) of the STIV turrets clearly showing the central barrel and the attached ear-like structures (55). (F) A radially depth-cued isosurface representation of the additional turret density present in STIV compared to STIV2 generated by difference imaging of the virion reconstructions. (G) Collage showing the major capsid protein trimers (C-α backbone; green), the segmented density of the STIV2 turret (red surface), and the remaining density of the difference map of STIV2 minus the major capsid protein density (yellow mesh). (H) Schematic diagram of STIV2 structural-protein organization. The major capsid protein A345 is drawn in blue surrounding the pentameric turret (red), which is proposed to contain B631 and C510. B72 is shown binding to the DNA genome. Based on transmembrane prediction using hidden Markov models (TMHMM), proteins A259, C141, E132, A55, and A103 have transmembrane regions and are thus drawn in contact with the membrane (30). (B, D, and G) The connection of the turret to the membrane underneath the capsid is marked with an asterisk.