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A virus that infects a hyperthermophile encapsidates A-form DNA

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Science  22 May 2015:
Vol. 348, Issue 6237, pp. 914-917
DOI: 10.1126/science.aaa4181

A viral DNA form that survives extremes

The prokaryote Sulfolobus islandicus lives at extreme temperatures (∼80°C) and acidity (pH 3). It is infected by the rudivirus SIRV2. DiMaio et al. determined the structure of the SIRV2 virus using cryo–electron microscopy to understand how the virus survives these brutal conditions. Most DNA in nature assumes a B-form shape. The virion, on the other hand, contains highly unusual A-form DNA that may help it survive adverse conditions. The viral capsid protein forms an extended α-helical structure that wraps around the viral DNA, possibly stabilizing the A-form DNA.

Science, this issue p. 914

Abstract

Extremophiles, microorganisms thriving in extreme environmental conditions, must have proteins and nucleic acids that are stable at extremes of temperature and pH. The nonenveloped, rod-shaped virus SIRV2 (Sulfolobus islandicus rod-shaped virus 2) infects the hyperthermophilic acidophile Sulfolobus islandicus, which lives at 80°C and pH 3. We have used cryo–electron microscopy to generate a three-dimensional reconstruction of the SIRV2 virion at ~4 angstrom resolution, which revealed a previously unknown form of virion organization. Although almost half of the capsid protein is unstructured in solution, this unstructured region folds in the virion into a single extended α helix that wraps around the DNA. The DNA is entirely in the A-form, which suggests a common mechanism with bacterial spores for protecting DNA in the most adverse environments.

Extreme geothermal environments, with temperatures above 80°C, are the habitat of hyperthermophilic DNA viruses that parasitize Archaea (1). These viruses have more than 92% of genes without homologs in databases (2, 3), distinct protein folds (4), and distinct mechanisms of viral egress (5). The high diversity of virion morphotypes may underpin virion morphogenesis and DNA packaging, which could determine the high stability of the virions. Viruses from the family Rudiviridae (6) consist of a nonenveloped, helically arranged nucleoprotein composed of double-stranded DNA (dsDNA) and thousands of copies of a 134-residue protein. To understand the mechanisms stabilizing rudiviral DNA in natural habitats of host cells, which involve high temperatures (~80°C) and low pH values (~pH 3), we used cryo–electron microscopy (cryo-EM) to analyze the rudivirus SIRV2 (Sulfolobus islandicus rod-shaped virus 2) (6), which infects the hyperthermophilic acidophilic archaeon Sulfolobus islandicus (7) (see supplementary materials and methods). Members of the archaeal genus Sulfolobus maintain their cytoplasmic pH neutral at pH 5.6 to 6.5 (8, 9). SIRV2 is therefore exposed to a wide range of pH values: from about pH 6 in the cellular cytoplasm, where it assembles and maturates (10), to pH 2 to 3 in the extracellular environment. We performed our studies at pH 6. SIRV2 is stable over a wide range of temperatures: from –80°C, the temperature at which the virus can be stored for years without loss of infectivity, to 80°C, the temperature of its natural environment. The overall morphology of the virion is maintained, regardless of the use of negative-stain imaging at 75°C (11) or cryo-EM with a sample at 4°C before vitrification (Fig. 1A).

Fig. 1 Cryo-EM and 3D reconstruction of SIRV2.

(A) Micrograph showing SIRV2 virions in vitreous ice. Scale bar, 1000 Å. (B) Side view of the reconstructed virion with a ribbon model for the protein (magenta). The asymmetric unit in the virion contains a protein dimer, and one is shown with one chain in yellow and the other in green. (C) Cutaway view showing the hollow lumen with the all α-helical protein segments that line the lumen. These α helices wrap around the dsDNA (blue) and encapsidate it. (D) Close-up view of the region shown within the rectangle in (C).

Electron cryo-micrographs of SIRV2 (Fig. 1A) showed strong helical striations in most of the virions with a periodicity of 42 Å. We performed three-dimensional (3D) reconstruction using the iterative helical real space reconstruction method (12), after first determining the helical symmetry. Only one solution (with 14.67 subunits per turn of the 42 Å pitch helix) yielded a reconstruction with recognizable secondary structure, almost all α helical, and a resolution of ~3.8 Å in the more-ordered interior, which surrounds the DNA (fig. S2). The asymmetric unit was a symmetrical dimer, the α helices of which were wrapping around a continuous dsDNA. The DNA was in an A-form, in contrast to the B-form DNA (B-DNA) observed in icosahedral bacteriophages (1315).

We used Rosetta guided by restraints from the EM data to determine and refine the atomic structure of SIRV2 (16). We began by docking a model of A-form DNA (A-DNA) into the map. The resulting model showed good agreement with the experimental data, where the rigid phosphate groups were well defined (Fig. 2, A and C). A dimeric crystal structure from a close homolog with 88% identity [Protein Data Bank identifier (PDB ID) 3F2E] was docked into the map. However, this model lacked 51 N-terminal residues, the first 46 of which were shown by nuclear magnetic resonance (NMR) to be unstructured in the monomer in solution and not part of the fragment crystallized (17). In the cryo-EM reconstruction, these residues formed helices wrapping around the DNA. Because the NMR studies were done at pH 6, the same pH used for our cryo-EM studies, the gain in structure of these residues is associated with assembly rather than a change in pH. We used RosettaCM (18) to build these N-terminal residues into the density map. A representative model was chosen from a well-converged, low-energy ensemble (fig. S4); this model shows good agreement with the side-chain density in the map at both protein-protein interfaces (Fig. 2B) and protein-DNA interfaces (Fig. 2C). Seven N-terminal residues could not be placed in the density.

Fig. 2 The refined atomic model in the 3D reconstruction.

(A) The density allows accurate tracing of the nucleotide chain. (B) The interface between helices within the asymmetric unit features large numbers of aromatic residues, which aid in correct registration of the sequence. (C) Side chains at the protein-DNA interface are well defined in density.

The final structure reveals that the N-terminal residues form a helix-turn-helix motif encapsulating the A-DNA, with helices from each subunit in the asymmetric unit packing in an antiparallel configuration (Fig. 3, A and B). Proline residues in this region (Pro27, Pro39, and Pro50) allow some helical deformation to tightly wrap the DNA. These interdigitated helices form a solvent-inaccessible surface surrounding the DNA (Fig. 3C). The DNA was confirmed as A-form (19), where the parameters [including an average base pair inclination of 25°, a negative slide (average = –1.6), and a negative x-displacement (average = –4.8)] match A-DNA, whereas the slide and x-displacement are positive for B-DNA. The average phosphate-phosphate distance along the DNA backbone is 5.9 Å, as opposed to ~7.0 Å for B-DNA. The diameter of the DNA is ~24 Å. At this resolution, the sugar pucker is not discernable. A slight bulge in the DNA occurs near the dimer interface, where a buried arginine side chain (Arg73) interacts with one of the DNA phosphate groups, leading to some local deviations from A-DNA. Model bias was tested by starting with the B-form, which converged to the same final model (fig. S5). The DNA (Fig. 3D), whose axis is at a helical radius of ~60 Å, has three right-handed superhelical turns every 44 (= 3 × 14.67) repeats (turns) of the DNA. So there are 528 base pairs (= 44 × 12) per 47 right-handed turns, which yields an overall twist of 11.2 base pairs per turn (Fig. 3D).

Fig. 3 The SIRV2 protein dimer helices fully encapsulate the DNA.

(A) Three asymmetric units of the virion are shown, illustrating how the N-terminal helices wrap around the DNA, forming antiparallel helix-helix packing. (B) Side view. (C) Surface view of the protein (using a 1.4 Å probe radius). (D) The right-handed solenoidal supercoiling of the DNA, with three turns shown.

DNA-protein contacts (Fig. 4, A to C) are largely polar, with nine conserved side chains (Arg8, Lys16, Lys20, Asn44, Asn48, Arg73, Lys82, Arg85, and Arg89) directly interacting with the DNA phosphate groups. Additionally, the backbone of Ser9 makes contact with the phosphate backbone of the DNA. There are also several hydrophobic contacts with the DNA, most notably the aromatic residues Trp17, Phe21, Phe24, and Phe52, as well as Val37. All of these residues are conserved in related rudiviruses (Fig. 4D), suggesting a similar method of DNA stabilization and protection. Extensive protein-DNA interactions in SIRV2 virions alleviate the necessity to package charge-neutralizing counterions (13, 14). Within the dimer, protein-protein interfaces are largely hydrophobic. The interface is extensive, comprising ~17% of each monomer’s surface area, with a total interface area of 1491 Å2. Five aromatic residues (Tyr10, Tyr14, Trp17, Phe21, and Phe45) form a well-packed barrier separating DNA from solvent. The protein-protein interface between adjacent dimers also forms a largely hydrophobic interface, with 1706 Å2 of contact area between dimers on both sides. Interactions between dimers across the groove of the helix are weak and largely polar.

Fig. 4 Overview of protein-DNA contacts in the virion.

(A) The protein-DNA interface is mainly polar, with largely Arg and Lys side chains contacting phosphate groups in the DNA. (B) Side view. (C) Schematic indicating all of the polar protein-DNA contacts. The coloring of each subunit is the same as in (A) and (B). (D) A multiple sequence alignment with related archaeal rod-shaped viruses indicates that all of these contacts are well conserved (29). SIRV2, Sulfolobus islandicus rod-shaped virus 2; ARV1, Acidianus rod-shaped virus 1; SRV, Stygiolobus rod-shaped virus; SMRV1, Sulfolobales Mexican rudivirus 1. The yellow boxes indicate the residues that are interacting with the DNA backbones.

Small acid-soluble proteins (SASPs) are responsible for protecting DNA in Gram-positive bacterial spores (20) and are largely unstructured in solution (2124), but they become α helical upon binding dsDNA (25, 26). Almost half of the SIRV2 capsid protein is unstructured in solution (17), and this portion becomes α helical when bound to DNA in the virion. The binding of SASPs to DNA is saturable, with saturation occurring at an SASP:DNA weight ratio of ~3:1 (27). In the SIRV2 virion, we have now shown that the weight ratio of capsid protein to DNA is 3.5:1. The binding of the SASPs to DNA induces a dimerization of the SASPs (25), and the asymmetric unit in the virion contains a symmetrical dimer of the coat protein. The binding of SASPs to DNA induces a transition from B-DNA to A-DNA (27), and an en masse transition of DNA from B-form to A-form can be induced in bacterial cells, suggesting that the A-form plays an unrecognized role in stabilizing DNA under adverse conditions such as dessication (28). Sequence analysis and structural comparison (with PDB ID 2Z3X) do not show obvious homology, suggesting that these similarities could have arisen as a result of convergent evolution.

Supplementary Materials

www.sciencemag.org/content/348/6237/914/suppl/DC1

Materials and Methods

Figs. S1 to S5

References (3037)

References and Notes

  1. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
  2. Acknowledgments: This work was supported by NIH grant GM035269 (to E.H.E.). The map and model have been deposited in the Electron Microscopy Data Bank (accession number EMD-6310) and in PDB (ID 3J9X), respectively. We thank K. Dryden for assistance with the microscopy.
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