CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Virology 351 (2006) 280–290 www.elsevier.com/locate/yviro TTSV1, a new virus-like particle isolated from the hyperthermophilic crenarchaeote Thermoproteus tenax ⁎ Dae-Gyun Ahn a, Se-Il Kim a, Jin-Kyu Rhee a, Kwang Pyo Kim b, Jae-Gu Pan c, Jong-Won Oh a, a Department of Biotechnology, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-749, Korea b Department of Molecular Biotechnology, Konkuk University, Seoul 143-701, Korea c Korea Research Institute of Bioscience and Biotechnology, Taejon 305-600, Korea Received 29 October 2005; returned to author for revision 30 December 2005; accepted 28 March 2006 Available online 6 May 2006 Abstract A new virus-like particle TTSV1 was isolated from the hyperthermophilic crenarchaeote Thermoproteus tenax sampled at a hot spring region in Indonesia. TTSV1 had a spherical shape with a diameter of approximately 70 nm and was morphologically similar to the PSV isolated from a strain of Pyrobaculum. The 21.6 kb linear double-stranded DNA genome of TTSV1 had 38 open reading frames (ORFs), of which 15 ORFs were most similar to those of PSV. The remaining 23 ORFs showed little similarity to proteins in the public databases. Southern blot analysis demonstrated that the viral genome is not integrated into the host chromosome. TTSV1 consisted of three putative structural proteins of 10, 20, and 35 kDa in size, and the 10-kDa major protein was identified by mass spectrometry as a TTSV1 gene product. TTSV1 could be assigned as a new member of the newly emerged Globuloviridae family that includes so far only one recently characterized virus PSV. © 2006 Elsevier Inc. All rights reserved. Keywords: Archaea; Hyperthermophilic virus; Linear genome; Thermoproteus; Globuloviridae Introduction SIRV2, TTV4, and ARV1; stiff helical rod-shape), Lipothrix- viridae (SIFV, TTV1-3, and AFV1 and AFV2; flexible rod- Viruses infecting microorganisms are diverse and represent shape), and Guttaviridae (SNDV; droplet shape). In Fusellovir- the largest number of biological entities in the biosphere idae, a lysogenic virus SSV1 isolated from Sulfolobus shibatae (Forterre, 2001; Rohwer, 2003; Wommack and Colwell, has been studied most extensively (Palm et al., 1991; Schleper 2000). Currently, more than two dozens of thermophilic viruses et al., 1992). This virus has a 15.5-kb circular double-stranded or virus-like particles (VLPs) have been isolated from extreme DNA (Schleper et al., 1992). PAV1, lemon-shaped VLP with a thermal environments. These are mainly from Sulfolobus, short tail terminated by fibers was recently isolated from a Thermoproteus,andAcidianus, belonging to the Crenarchaeota hyperthermophilic euryarchaeote. The genome of PAV1 is phylum in the Archaea domain (Bettstetter et al., 2003; circular, double-stranded DNA of 18 kb in size, and is present in Prangishvili and Garrett, 2005; Prangishvili et al., 2001; Rachel a high copy number in a free form in host cells (Geslin et al., et al., 2002; Rice et al., 2001). From the Euryarchaeota, a second 2003). The second family, Rudiviridae,includesSIRV1 major phylum of the Archaea domain, so far only the VLP PAV1 (Sulfolobus islandicus rod-shaped virus) and SIRV2 isolated has been isolated from Pyrococcus abyssi (Geslin et al., 2003). from Iceland. These viruses are enveloped stiff rods containing The viruses and VLPs discovered from hyperthermophilic a linear double-stranded DNA genome (Blum et al., 2001; Peng archaea over the past two decades have been grouped into four et al., 2001; Prangishvili et al., 1999; Zillig et al., 1994). families on the basis of their unique morphologies and genome Similarly, TTV4 from Thermoproteus tenax and ARV1 from structures. These families include Fuselloviridae (SSV1, SSV2, Acidianus are stiff rods containing a linear double-stranded SSVK1, and SSVRH; spindle shape), Rudiviridae (SIRV1 and DNA genome (Vestergaard et al., 2005; Zillig et al., 1998). The third family, Lipothrixvirdae, includes SIFV from Sulfolobus ⁎ Corresponding author. Fax: +82 822 362 7265. (Arnold et al., 2000b), TTV1, TTV2, and TTV3 from T. tenax E-mail address: [email protected] (J.-W. Oh). (Janekovic et al., 1983), and AFV1 and AFV2 from Acidianus 0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2006.03.039 D.-G. Ahn et al. / Virology 351 (2006) 280–290 281 (Bettstetter et al., 2003; Häring et al., 2005a). SIFV has a linear established with thermal environmental samples collected from virion containing a core of linear double-stranded DNA and various hot spring areas in Indonesia, for the presence of viral DNA-binding proteins. This core is wrapped in a membrane genomes in polyethyleneglycol (PEG) precipitates of culture consisting of host lipids and, possibly, hydrophobic viral supernatants. Viral genomes extracted from the PEG precipi- proteins. SIFV has a unique tail structure that may be involved tates were visualized by agarose gel electrophoresis followed by in adsorption of virus particles onto receptors of host cells ethidium bromide staining. Of a total of 58 cultures tested, we (Arnold et al., 2000b). T. tenax TTV1 is rod-shaped enveloped obtained one positive sample, which showed a prominent virus and has a 15.9-kb linear, double-stranded DNA genome putative viral genome of approximately 20 kb, from a Sileri hot (Janekovic et al., 1983; Neumann et al., 1989; Neumann and spring region. Subsequent analysis of the PEG precipitates of Zillig, 1990). The fourth family, Guttaviridae, includes an this sample by transmission electron microscope showed SNDV (Sulfolobus neozealandicus droplet-shaped virus) with a morphologically homogeneous VLPs (Fig. 1A). The VLPs bearded drop-like morphology (Arnold et al., 2000a). In were also detected in the precipitates directly obtained by addition to the above described four families, which were ultracentrifugation of culture supernatant (Fig. 1B). The sphere- approved by the International Committee for Taxonomy of shaped VLPs purified by sucrose density gradient centrifuga- Viruses, the fifth family, Globuloviridae, has been proposed for tion had a diameter of approximately 70 nm (Fig. 1C). Some of the sphere-shaped virus PSV that was isolated from anaerobic virus particles formed a short chain with 4–5 virus particles or hyperthermophilic archaeal genera Pyrobaculum (Häring et al., an aggregate (Fig. 1, panels A–C). The density of the purified 2004). The genome of PSV is linear, double-stranded DNA of VLP was estimated to be approximately 1.29 g/ml, similar to 28.3 kb in size, and the two DNA strands of the genome are that of enveloped viruses PAV1 (1.28 g/ml) (Geslin et al., 2003), probably linked at their termini. His1 (1.3 g/ml) (Bath and Dyall-Smith, 1998), AFV1 (1.3 g/ml) Considering the existence of diverse microorganisms in (Bettstetter et al., 2003), and SSV1 (1.24 g/ml) (Murphy et al., extremely hot environments (Breitbart et al., 2004; Noll and 1995). Vargas, 1997; Rhee et al., 2005), viruses infecting these hosts are also likely to be diverse in thermal environments. Indeed, TTSV1 host various morphologically unique VLPs have been discovered from thermal environments in Yellowstone National Park As observed by a phase contrast microscope, the host of the (YNP), USA, by two independent groups (Prangishvili et al., VLP, which was isolated by serial dilution of enrichment culture 2001; Rachel et al., 2002; Rice et al., 2001), indicating a followed by colony isolation in a semi-solid Gelrite-containing potential for a high viral diversity even in harsh thermal media, exhibited a rod-shaped morphology with a granule-like environments. Here, we describe the novel sphere-shaped VLP, particle at the tip or middle part of the cells (Fig. 2A, panel a). It named TTSV1, which was isolated from a strain of T. tenax had a mean length of 1–2 μm and a width of approximately collected from a Sileri hot spring region of Indonesia. Genome 0.2 μm, and its ends were angular with slightly rounded edges sequence analysis indicates that it is most similar to PSV in both when examined under a transmission electron microscope (Fig. morphology and protein sequence, suggesting that it could be 2A, panels b and c). During the exponential growth phase, assigned as a new member of the Globuloviridae family. spherical bodies protruding terminally from the rod were visible (panel c). These characteristics are similar to the morphological Results and discussion features of T. tenax (Janekovic et al., 1983; Rieger et al., 1997). By phylogenetic analysis using 16S rDNA sequence, the Isolation and analysis of virus-like particles purified host was identified as a T. tenax-related archaea in the Crenarchaeota phylum, named T. tenax YS44 (97% identity to In an attempt to isolate viruses from hyperthermophiles, we the T. tenax 16S rDNA) (Fig. 2B). The new VLP, named TTSV1 screened hyperthermophile enrichment cultures, which were for T. tenax spherical virus 1, was morphologically different Fig. 1. Transmission electron microscopy of TTSV1. Virus particles obtained by PEG precipitation of a culture supernatant (A), by ultracentrifugation of a culture supernatant (B), and by ultracentrifugation in a sucrose density gradient were stained with 2% uranyl acetate and observed by a transmission electron microscope. Scale bars indicate 100 nm. 282 D.-G. Ahn et al. / Virology 351 (2006) 280–290 Fig. 2. Morphology and phylogenetic tree of TTSV1 host. (A) Phase contrast (a) and thin-section transmission electron (b and c) microscopy images of T. tenax YS44. The scale bars represent 200 nm. (B) The phylogenetic tree was constructed by the neighbor-joining method with the Molecular Evolutionary Genetics Analysis 2.1 software (MEGA, version 2.1). The numbers associated with the branches refer to bootstrap values resulting from 1000 replicate resamplings. The scale bar indicates 2 substitutions per 100 nucleotides. The 16s rDNA sequence accession numbers of the aligned sequences are shown in parentheses.