| Abstract: | The virophage Sputnik is a satellite virus of the giant mimivirus and is the only satellite virus reported to date whose propagation adversely affects its host virus'' production. Genome sequence analysis showed that Sputnik has genes related to viruses infecting all three domains of life. Here, we report structural studies of Sputnik, which show that it is about 740 Å in diameter, has a T=27 icosahedral capsid, and has a lipid membrane inside the protein shell. Structural analyses suggest that the major capsid protein of Sputnik is likely to have a double jelly-roll fold, although sequence alignments do not show any detectable similarity with other viral double jelly-roll capsid proteins. Hence, the origin of Sputnik''s capsid might have been derived from other viruses prior to its association with mimivirus.Mimivirus is the largest virus known to date and has a 1.2-Mbp genome. It was discovered in a British water tower while searching for the cause of a hospital-acquired pneumonia outbreak (14). Although mimivirus'' natural host is amoeba, it could be a potential human pathogen (2, 7, 8, 15). Recently, a smaller virus named Sputnik was isolated from amoeba infected with mamavirus, a new strain of mimivirus (10). Sputnik utilizes the virus factory formed by mamavirus for replication and cannot reproduce on its own in amoeba. Furthermore, the coinfection of Sputnik with mamavirus reduces the yield of mamavirus by about 70% and causes the formation of many types of defective mamavirus virions.Sputnik has an 18-kbp, double-stranded, circular, highly AT-rich genome, which is predicted to encode 21 proteins ranging from 88 to 779 amino acids in size. Of these 21 proteins, 13 do not have detectable homologues in current sequence databases. The other eight genes have homologues in viruses whose hosts are from all three domains of life, the Eukarya, Archaea, and Bacteria. The chimeric characteristics of the Sputnik genome implies that it is involved in lateral gene transfer between viruses. It was proposed that Sputnik represents a new family of viruses termed virophage (10).Because of the mosaic nature of viral genomes and the lack of 16S rRNA for traditional phylogenetic tree analysis, the classification of viruses has been difficult. Recent structural studies of viral capsid proteins have led to the idea of structure-based viral lineages, which classify viruses based on the organization and structure of the viral capsids (3, 9). One of these lineages is the PRD1-adenovirus lineage, which is comprised of icosahedral double-stranded DNA (dsDNA) viruses including adenovirus, bacteriophage PRD1, Sulfolobus turreted icosahedral virus, the marine bacteriophage PM2, and the nucleocytoplasmic large DNA viruses (NCLDVs) such as mimivirus and Paramecium bursaria Chlorella virus 1 (PBCV-1). All these viruses have major capsid proteins (MCPs) whose polypeptides have a similar fold and in some cases, such as the NCLDVs, have significant sequence similarity. The MCP structures of the above-mentioned viruses, excluding mimivirus, have been determined to atomic resolution and were shown to have two consecutive “jelly-roll” domains (double jelly-roll fold) (1, 4, 13, 16, 17). A jelly-roll domain is an antiparallel β barrel consisting of eight β strands named B, C, …, I. The MCPs are organized into “capsomers” that are arranged into hexagonal arrays. Each viral capsomer contains three monomers that have a double jelly-roll fold, resulting in a pseudohexameric shape at the base, appropriate for packing into the hexagonal arrays. However, there are often large insertions in the loops between β strands D and E as well as between strands F and G of each jelly-roll fold (loops “DE” and “FG”). This gives the capsomers a triangular appearance on the surface. The thickness of capsomers is about 75 Å, and the diameter varies between 74 Å and 85 Å.Here, we report the cryo-electron microscopy (cryoEM) three-dimensional (3D) reconstruction of Sputnik to 10.7-Å resolution. We show that the MCP is organized into a hexagonal surface lattice characterized by a T=27 triangulation number. We also show that the capsomer structure in Sputnik is trimeric and that the MCP structure of PBCV-1 can be fitted into the cryoEM map of Sputnik. Thus, the MCP of Sputnik is probably a double jelly-roll fold as in viruses belonging to the PRD1-adenovirus lineage. However, there is no significant sequence similarity between the MCP of Sputnik and other members of the PRD1-adenovirus lineage, suggesting that Sputnik is a member of a separate branch from the NCLDVs (mimivirus and PBCV-1, etc.). |
