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. 2020 Aug 18;117(33):19643-19652.
doi: 10.1073/pnas.2011125117. Epub 2020 Aug 5.

Structures of filamentous viruses infecting hyperthermophilic archaea explain DNA stabilization in extreme environments

Fengbin Wang  1 , Diana P Baquero  2   3 , Leticia C Beltran  1 , Zhangli Su  1 , Tomasz Osinski  1 , Weili Zheng  1 , David Prangishvili  2   4 , Mart Krupovic  5 , Edward H Egelman  6
Affiliations

Structures of filamentous viruses infecting hyperthermophilic archaea explain DNA stabilization in extreme environments

Fengbin Wang et al. Proc Natl Acad Sci U S A. .

Abstract

Living organisms expend metabolic energy to repair and maintain their genomes, while viruses protect their genetic material by completely passive means. We have used cryo-electron microscopy (cryo-EM) to solve the atomic structures of two filamentous double-stranded DNA viruses that infect archaeal hosts living in nearly boiling acid: Saccharolobus solfataricus rod-shaped virus 1 (SSRV1), at 2.8-Å resolution, and Sulfolobus islandicus filamentous virus (SIFV), at 4.0-Å resolution. The SIFV nucleocapsid is formed by a heterodimer of two homologous proteins and is membrane enveloped, while SSRV1 has a nucleocapsid formed by a homodimer and is not enveloped. In both, the capsid proteins wrap around the DNA and maintain it in an A-form. We suggest that the A-form is due to both a nonspecific desolvation of the DNA by the protein, and a specific coordination of the DNA phosphate groups by positively charged residues. We extend these observations by comparisons with four other archaeal filamentous viruses whose structures we have previously determined, and show that all 10 capsid proteins (from four heterodimers and two homodimers) have obvious structural homology while sequence similarity can be nonexistent. This arises from most capsid residues not being under any strong selective pressure. The inability to detect homology at the sequence level arises from the sampling of viruses in this part of the biosphere being extremely sparse. Comparative structural and genomic analyses suggest that nonenveloped archaeal viruses have evolved from enveloped viruses by shedding the membrane, indicating that this trait may be relatively easily lost during virus evolution.

Keywords: cryo-EM; extremophiles; filamentous viruses; hyperthermophilic archaea.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Cryo-EM of the S. solfataricus rod-shaped virus (SSRV1). (A) Representative cryo-electron micrograph of SSRV1 virions. A Saccharolobus solfataricus Type IV pilus, indicated by black arrowhead, is the cellular receptor for SSRV1. (Scale bar, 50 nm.) (B) Averaged power spectrum of the segments used in the IHRSR reconstruction. The layer lines that were used to initially estimate the helical symmetry are indicated, along with the Bessel orders for the correct symmetry found. (C) Surface of the SSRV1 cryo-EM reconstruction at 2.8-Å resolution. The right-handed ∼43-Å pitch 1-start helix passing through every asymmetric unit is shown. (D) Helical net of SSRV1, using the convention that the surface is unrolled and we are viewing it from the outside. Since there are 14.67 subunits per turn of the right-handed 1-start helix, subunits S14 and S15 will be above the reference subunit S0 indicated. (E) Top and side views of the SSRV1 atomic model fit into the EM map. The side view is from a central slice of the map/model, indicated by the dashed line. A-form DNA is colored magenta, and MCP dimers are cyan and yellow. (F) Ribbon models for the SSRV1 protein dimer and 36 bp of A-DNA, fit into the EM map. The clear separation of DNA base pairs is seen in the map, despite averaging over the entire genomic sequence with the imposition of helical symmetry.
Fig. 2.
Fig. 2.
Cryo-EM of the S. islandicus filamentous virus (SIFV). (A) Representative cryo-electron micrograph of SIFV. Most of the virions are enveloped by a membrane, indicated by a black arrowhead. Some virions have lost their membrane, and one is indicated by a white arrowhead. This results in a narrower diameter, and these virions appear to have an increased flexibility. (Scale bar, 50 nm.) (B) Averaged power spectrum of the segments used in the IHRSR reconstruction. The layer lines that were used to initially estimate the helical symmetry are indicated, along with the Bessel orders for the correct symmetry found. (C) Top and side views of the SIFV atomic model fit into the EM map. The side view is from a central slice of the map/model, indicated by the dashed line. A-form DNA is colored magenta, and MCP dimers are in blue and yellow. The membrane has been filtered to 7-Å resolution and cylindrically averaged in this figure. The radial density profile of the cylindrically averaged and filtered membrane is shown. (D) Helical net of SIFV, using the convention that the surface is unrolled and we are viewing from the outside. Since there are 9.35 subunits per turn of the right-handed 1-start helix, subunits S9 and S10 will be above the reference subunit S0 indicated. (E) The surface of the 3D reconstruction of SIFV at 4.0-Å resolution, with the membrane removed. (F) Ribbon models of the SIFV MCP dimer (cyan and yellow) and 36 bp of A-DNA (magenta), fit into the EM map. (G) An 11-aa loop (residues 122 to 132) of SIFV-1 associated with the membrane. An unambiguous backbone trace in this region was not possible due to the low resolution resulting from structural flexibility. The membrane shown here is not cylindrically averaged.
Fig. 3.
Fig. 3.
Extensive MCP–DNA interactions in SSRV1 and SIFV. (A) N-terminal residues of SSRV1 MCP, SIFV MCP1 (SIFV-1), and SIFV MCP2 (SIFV-2) inserting into a groove of the A-DNA. The corresponding EM density in this region is shown. For SIFV, these residues are poorly ordered and therefore only a backbone trace is shown. Protein is red, and DNA is yellow. (B) Schematic indicating all of the polar protein–DNA contacts in SSRV1. The MCPs form a symmetrical dimer, so residues colored blue are related by the dihedral symmetry to those colored orange. (C) Schematic indicating all of the polar protein–DNA contacts in SIFV. Residues from SIFV-1 are colored blue, and those from SIFV-2 are colored orange. (D) Water molecules around A-DNA. The 12-bp A-DNA (wrapped by the SSRV1 homodimer) is shown in a yellow ribbon representation, and the water molecules are shown as red spheres. Several close-up views of water and nearby amino acids are shown with the EM density map.
Fig. 4.
Fig. 4.
The flexibility of archaeal filamentous virions. (A) Estimates of the persistence length of the six archaeal filamentous virions. The measurements for each virus were from 100 filaments randomly selected from cryo-EMs. The measurements are shown in box-and-whisker plots that display five summary statistics (the median, two hinges, and two whiskers), and all “outlying” points individually. (B) The filamentous protein–DNA models of SSRV1 and SIFV, with DNA in magenta and protein in gray. (C) The parameters of all six archaeal filamentous virus structures, including helical rise and twist, 1-start pitch, A-DNA diameter, pitch angle θ, and the presence/absence of membrane. The DNA diameter is taken as the distance from the axis of the DNA on one side to the other, as this is more precisely defined than something like the outer diameter. (D) The electrostatic potential surface of all six archaeal filamentous virus structures, calculated by APBS (63).
Fig. 5.
Fig. 5.
Structural conservation and diversity of the filamentous virus MCPs. (A) Structure-based sequence alignments of 10 MCP sequences from six archaeal filamentous virus structures. The α-helices are indicated by blue rounded rectangles, and β-sheets are indicated by orange rectangles. (B) Representative domain architecture of the MCP in known filamentous virus. An SSRV1 dimer with 36-bp A-DNA is shown: One MCP is shown in rainbow coloring and the other in white; DNA is colored magenta. N-terminal α-helices wrap DNA on the luminal side and C-terminal α-helix bundles wrap DNA facing the outer solvent or the membrane. (C) MCP comparison of SSRV1, SIRV2, SFV1, PFV2, AFV1, and SIFV. SSRV1 and SIRV2 are homodimers, while the other four MCP dimers are heterodimers. (D) All-against-all comparison of the six MCP dimers. The matrix is based on the pairwise TM score calculated from the MM-align server.
Fig. 6.
Fig. 6.
Inferred phylogenomic tree of archaeal filamentous viruses. The tree is based on whole-genome VICTOR analysis at the amino acid level. The tree is rooted with tristromaviruses, and the branch length is scaled in terms of the Genome BLAST Distance Phylogeny (GBDP) distance formula D6. The numbers above branches are GBDP pseudobootstrap support values from 100 replications. For each genome, the abbreviated virus name and GenBank accession number are indicated. The tree is divided into colored blocks according to the taxonomy of the compared viruses.
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