Sulfolobus Spindle-Shaped Virus 1 Contains Glycosylated Capsid Proteins, a Cellular Chromatin Protein, and Host-Derived Lipids

Abstract

Geothermal and hypersaline environments are rich in virus-like particles, among which spindle-shaped morphotypes dominate. Currently, viruses with spindle- or lemon-shaped virions are exclusive to Archaea and belong to two distinct viral families. The larger of the two families, the Fuselloviridae, comprises tail-less, spindle-shaped viruses, which infect hosts from phylogenetically distant archaeal lineages. Sulfolobus spindle-shaped virus 1 (SSV1) is the best known member of the family and was one of the first hyperthermophilic archaeal viruses to be isolated. SSV1 is an attractive model for understanding virus-host interactions in Archaea; however, the constituents and architecture of SSV1 particles remain only partially characterized. Here, we have conducted an extensive biochemical characterization of highly purified SSV1 virions and identified four virus-encoded structural proteins, VP1 to VP4, as well as one DNA-binding protein of cellular origin. The virion proteins VP1, VP3, and VP4 undergo posttranslational modification by glycosylation, seemingly at multiple sites. VP1 is also proteolytically processed. In addition to the viral DNA-binding protein VP2, we show that viral particles contain the Sulfolobus solfataricus chromatin protein Sso7d. Finally, we provide evidence indicating that SSV1 virions contain glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, resolving a long-standing debate on the presence of lipids within SSV1 virions. A comparison of the contents of lipids isolated from the virus and its host cell suggests that GDGTs are acquired by the virus in a selective manner from the host cytoplasmic membrane, likely during progeny egress.

Importance: Although spindle-shaped viruses represent one of the most prominent viral groups in Archaea, structural data on their virion constituents and architecture still are scarce. The comprehensive biochemical characterization of the hyperthermophilic virus SSV1 presented here brings novel and significant insights into the organization and architecture of spindle-shaped virions. The obtained data permit the comparison between spindle-shaped viruses residing in widely different ecological niches, improving our understanding of the adaptation of viruses with unusual morphotypes to extreme environmental conditions.

FIG 1

Purification of SSV1. (A) Transmission electron micrograph of negatively stained SSV1 sample. Single particles as well as different aggregates are shown. (B) Depending on the concentration of NaCl in the SSV1 buffer, different stages of aggregation were observed: single particles (white columns), rosette-like structures containing between 2 and 5 particles (gray columns), and aggregates with more than 5 particles (black columns). The number of viruses in each category was determined from negatively stained electron micrographs obtained from three independent experiments. At least 1,000 particles were counted per condition, and error bars represent standard deviations. (C) Analysis of samples taken after each step of the 2× purification procedure. Absorbance at 260 nm, virus titer, recovery of infectivity, and specific infectivity are indicated.

FIG 2

Structural proteins of SSV1. (A) Protein profiles of 2× purified SSV1 virions compared to 2× purified His1 virions in a tricine-SDS-polyacrylamide gel stained with Coomassie blue. Molecular mass markers (M) are shown. The amounts of SSV1 and His1 samples loaded are comparable based on absorbance measurements at 260 nm. (B and C) 2× purified SSV1 and His1 virions analyzed in a tricine-SDS-PAGE gel stained with SYPRO Ruby protein stain (detecting all proteins) (B) and with Pro-Q Emerald 300 glycoprotein detecting reagent (detecting glycosylated proteins) (C). Candy-Cane glycoprotein molecular mass standard (labeled CC) contains a mixture of nonglycosylated and glycosylated proteins. Half of the amount loaded for panel A was added to the gel shown in panels B and C.

FIG 3

Identification of the structural proteins of SSV1. (A) Protein pattern of the 2× purified SSV1 virions in a tricine-SDS-PAGE stained with Coomassie blue. M indicates the molecular mass marker. Locations of protein bands processed for proteomic analyses are depicted with black boxes, and names of proteins identified are indicated on the right. (B) Proteins identified by N-terminal sequencing (NS) and mass spectrometry (MS). The NCBI accession number, theoretical molecular masses, putative functions, and peptide sequences determined during analysis are provided. (C) Sequence analysis of the SSV1 structural proteins VP1, VP3, and VP4. Sequences of the predicted transmembrane domains are highlighted in gray, whereas the theoretical glycosylation consensus motifs (N-X-S/T) are shown on the black background. The position of proteolytic cleavage in VP1 is indicated by a black arrowhead, and the N-terminal 65-amino-acid (aa) residues not shared with VP3 are boxed. Paralogous proteins VP1 and VP3 are aligned; identical and similar amino acid positions are indicated with asterisks and colons, respectively. At the bottom of the panel is the hydrophobicity profile of VP4. The broken line indicates the 0.9 probability threshold for the prediction of the transmembrane domains (TMD1-3). Predicted secondary structure elements are shown with green boxes (α-helixes) and blue arrows (β-strands).

FIG 4

Analysis of lipids of Sulfolobus solfataricus and SSV1. (A) Structures of lipids analyzed in this study, glycerol dibiphytanyl glycerol tetraethers (GDGTs). The numbers denote how many cyclopentane moieties are present within the isoprenoid chains. (B) Relative distribution of core lipid species identified by HPLC-APCI-MS in S. solfataricus cells and 2× purified SSV1 virions as described in Materials and Methods.