Characterization of the archaeal thermophile Sulfolobus turreted icosahedral virus validates an evolutionary link among double-stranded DNA viruses from all domains of life

Abstract

Icosahedral nontailed double-stranded DNA (dsDNA) viruses are present in all three domains of life, leading to speculation about a common viral ancestor that predates the divergence of Eukarya, Bacteria, and Archaea. This suggestion is supported by the shared general architecture of this group of viruses and the common fold of their major capsid protein. However, limited information on the diversity and replication of archaeal viruses, in general, has hampered further analysis. Sulfolobus turreted icosahedral virus (STIV), isolated from a hot spring in Yellowstone National Park, was the first icosahedral virus with an archaeal host to be described. Here we present a detailed characterization of the components forming this unusual virus. Using a proteomics-based approach, we identified nine viral and two host proteins from purified STIV particles. Interestingly, one of the viral proteins originates from a reading frame lacking a consensus start site. The major capsid protein (B345) was found to be glycosylated, implying a strong similarity to proteins from other dsDNA viruses. Sequence analysis and structural predication of virion-associated viral proteins suggest that they may have roles in DNA packaging, penton formation, and protein-protein interaction. The presence of an internal lipid layer containing acidic tetraether lipids has also been confirmed. The previously presented structural models in conjunction with the protein, lipid, and carbohydrate information reported here reveal that STIV is strikingly similar to viruses associated with the Bacteria and Eukarya domains of life, further strengthening the hypothesis for a common ancestor of this group of dsDNA viruses from all domains of life.

FIG. 1.

Model of an STIV particle. Shown is a cutaway view of the T=31 icosahedral capsid of STIV based on cryo-EM reconstruction (30). Extending from each of the fivefold vertices are turret-like projections. The protein shell is blue and has been removed from one quarter of the particle to reveal the inner lipid layers (yellow). The capsid diameter is ∼70 nm, and the turrets extend 13 nm above the surface.

FIG. 2.

Analysis of STIV capsid proteins by SDS-PAGE. Purified virus was denatured and separated (A) on a 4 to 20% SDS-PAGE gel or (B) on isoelectric focusing (3 to 10 nonlinear) gel strips and then separated in the second dimension on an 8 to 18% SDS-PAGE gel. Gels stained with Coomassie brilliant blue and individual protein spots and bands were then excised and digested with thermolysin and/or trypsin, and the peptides were analyzed using mass spectrometry. The unmodified major capsid protein expressed in E. coli runs similarly to 14 (*).

FIG. 3.

Location of capsid proteins on the STIV genome map. ORFs are named according to frame (A to F) and number of predicted amino acids. Proteins identified by protease mass mapping (solid black arrows), in general, cluster together. There are eight proteins from annotated ORFs and one additional protein, A78. This protein lacks a start methionine and was not included in the original annotation. The map was created using Vector NTI Advance 10.1.1.

FIG. 4.

B164 alignment with several proteins from the family Poxviridae. SwissProt accession numbers are separated by an underscore from species abbreviations (FOWPV, Fowlpox virus; 9POXV, Vultur gryphus poxvirus; POXVV, Vaccinia virus; MCV1, Molluscum contagiosum virus subtype 1; SWPV, Swinepox virus; YMTV, Yaba monkey tumor virus; RPOXV, Rabbit fibroma virus). Residues are shaded according to 90% consensus, with white letters on black background signifying perfect conservation. Abbreviations on the consensus line are as follows: h, hydrophobic; s, small; l, aliphatic, b, big; a, aromatic; c, charged.

FIG. 5.

Glycosylation of STIV proteins. Purified virus and whole-cell lysates of S. solfataricus P2 were digested with O- or N-deglycosidase enzymes and loaded onto a 4 to 20% SDS-PAGE gel. The proteins were analyzed for glycoproteins and visualized using a Pro-Q glycoprotein stain. CC Mr, CandyCane glycoprotein marker. The glycosylated major capsid protein ran at ∼41 kDa (lanes 3, 4, and 5). The band at ∼35 kDa is the N-link-specific glycosidase and not a deglycosylated product (lane 5). In contrast to the viral proteins, total Ssp2 proteins show a significant reduction in glycosylation after treatment (lanes 7 and 8).

FIG. 6.

Total lipid extracts from host and viral membranes. Lipids isolated from host cells (upper) and STIV (lower) were analyzed by negative-mode electrospray mass spectrometry. Viral lipids are a subset of those found in the host.

FIG. 7.

Experimental and theoretical isotope distributions for the lipid at m/z ∼1,858. A) Close-up of peak at m/z 1,858 in the STIV sample. The minor component present at m/z 1,856 has an additional cyclopentane group in the iospranyl chain. Based on the unit molecular weight, this could be PGL1 or PGL2 with four cyclopentane groups (B). However, a closely related neutral molecule that has become ionized by chloride differs by only 0.0362 amu (C). Theoretical isotope distributions for the acidic (B) and neutral (C) lipids clearly show the effect chloride has on the pattern. The influence is largest 2 mass units above the monoisotopic peak. A comparison of the STIV sample and the two theoretical spectra reveals that STIV has the acidic lipid. The measured m/z of 1,858.3745 ± 0.009 also matches that for the acidic species (1,858.3703m/z) but not that for the neutral molecule plus Cl⁻ (1,858.3341m/z).

FIG. 8.

Isotope pattern for the ion at m/z 1,534. Shown are the experimental (A) and theoretical (B) patterns for the STIV predominant lipid molecule. The acidic lipid has a measured mass that is consistent with PL1. Approximately 30% of this lipid is present with an additional cyclopentane unit (m/z 1,532), and a small amount has a second cyclopentane unit (m/z 1,530). (C) Lipids found in STIV. The cyclic tetraether lipids found in Sulfolobus have the general backbone shown above. The number of cyclopentane groups per chain is variable.