Structural conservation in a membrane-enveloped filamentous virus infecting a hyperthermophilic acidophile

Abstract

Different forms of viruses that infect archaea inhabiting extreme environments continue to be discovered at a surprising rate, suggesting that the current sampling of these viruses is sparse. We describe here Sulfolobus filamentous virus 1 (SFV1), a membrane-enveloped virus infecting Sulfolobus shibatae. The virus encodes two major coat proteins which display no apparent sequence similarity with each other or with any other proteins in databases. We have used cryo-electron microscopy at 3.7 Å resolution to show that these two proteins form a nearly symmetrical heterodimer, which wraps around A-form DNA, similar to what has been shown for SIRV2 and AFV1, two other archaeal filamentous viruses. The thin (∼ 20 Å) membrane of SFV1 is mainly archaeol, a lipid species that accounts for only 1% of the host lipids. Our results show how relatively conserved structural features can be maintained across evolution by both proteins and lipids that have diverged considerably.

Fig. 1

Electron micrographs of SFV1 virions. a Negatively stained purified virions. b Negatively stained virions with an elongated “neck;” mop-like terminal structures are shown in the inset. c Cryo-EM of SFV1. Scale bars, 200 nm (50 nm in the inset, b)

Fig. 2

Genes, proteins, and lipids of SFV1. a Genome map of SFV1. The ORFs are represented with arrows, indicating the direction of transcription. Genes encoding structural proteins are in blue, and ORFs containing predicted transmembrane domains are indicated by asterisks. b SDS-PAGE of SFV1 virion proteins stained with Coomassie brilliant blue. The VPs which were identified in proteins bands are indicated. The protein bands marked by asterisks contained a mixture of different VPs in comparable proportion. c Distribution of lipid species identified in S. shibatae BEU9 cells (Host) and SFV1 virions. d Chemical diagrams for the lipid species identified in SFV1 virions and S. shibatae BEU9 cells. M molecular mass standards

Fig. 3

Three-dimensional reconstruction of SFV1. A top view (a) shows the membrane enveloping the nucleoprotein core. The membrane has been filtered to 7 Å resolution and cylindrically averaged. The nucleoprotein core has been filtered to 3.7 Å resolution. In the side view (b) the membrane has been removed. c A view from within the lumen of the virion shows the atomic model built into the density. The asymmetric unit contains VP5 (red), VP4 (yellow) and 12 basepairs of DNA. One strand of DNA is shown in magenta, the other in cyan. VP5 contains a small C-terminal domain that makes an extensive contact with a subunit in the helical turn above. d The radial density profile of the virion is shown, after cylindrically averaging the structure. Two slightly different estimates of the solvent density come from the inside and the outside of the structure. The higher density on the outside could arise from bound ions or glycosylation, particularly since it has been determined that the membrane protein VP3 is glycosylated (Supplementary Fig. 2)

Fig. 4

The resolution of the map allows for an unambiguous modeling of both the protein and the DNA. a A helix from VP5 shows how almost all side chains can be visualized and built into the model. b A section of the DNA is shown, with one chain in magenta and the other in cyan. Even though the reconstruction is averaging over the whole genome, most of the bases are clearly resolved (black arrows) and the discrete densities from the phosphate groups are seen (red arrows). A positively-charged side chain from VP4 (Lys20) is adjacent to the negatively-charged phosphate groups. c The wrapping of the DNA (cyan) by VP5 (red). Two positively-charged sidechains (Lys55 and Arg36) are in close proximity to the phosphate backbone, while two hydrophobic residues (Phe44 and Phe40) are inserted into the groove in the DNA and are in proximity to the bases

Fig. 5

N-terminal extensions penetrate the DNA grooves. a Six or seven residues at the N-terminus of VP5 (red) project deeply into the DNA. Sidechain density for these residues is absent, suggesting that the conformation of these sidechains may depend on the DNA sequence. b Two or three residues from VP4 (yellow) project into the DNA groove. No density for the first three VP4 residues is seen in the reconstruction. One strand of DNA is in magenta, the other in cyan

Fig. 6

Structural conservation and divergence among capsid proteins. Heterodimers (a, b, c, e) and homodimers (d) form the capsids in SFV1 (a, b, c), SIRV2 (d) and AFV1 (e). a, b VP5 (red) and VP4 (yellow) from SFV1 have been aligned to each other. The long α-helix that wraps around the DNA is structurally quite conserved between the two. The main differences between the two coat proteins is that VP5 has an insert (from ∼141 to 169) that contains the two β-strands seen in (b), and VP5 has a C-terminal extension that contains two short helices. This C-terminal extension crosses the helical groove and makes a large contact with a subunit in the next turn. The SFV1 heterodimer (c) is more similar in how it wraps DNA to the SIRV2 homodimer (d) than it is to the AFV1 heterodimer (e)

Fig. 7

Separation of fragments of Triton X-treated SFV1 virions by CsCl gradient cenrifugation. a The scheme shows collected fractions and their density, along with electron micrographs of the observed particles from two fractions negatively stained with 2% uranyl acetate. Scale bars: 500 nm in the upper panel, 100 nm in the lower panel. b SDS-PAGE of collected fractions after staining with Coomassie brilliant blue; M molecular mass standards, SFV1 SDS-PAGE of intact virions, with indication of proteins VP3, VP4, and VP5