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Virology 404 (2010) 1–4

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Virology

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Rapid Communication The Sulfolobus rod-shaped virus 2 encodes a prominent structural component of the unique virion release system in Archaea

Tessa E.F. Quax a, Mart Krupovič a,b, Soizick Lucas a, Patrick Forterre a, David Prangishvili a,⁎

a Institut Pasteur, Molecular Biology of the Gene in Extremophiles Unit, rue du Dr. Roux 25, 75724 Paris Cedex 15, France b Department of Biosciences and Institute of Biotechnology, Viikki Biocenter 2, PO Box 56 (Viikinkaari 5), University of Helsinki, FIN-00014 Helsinki, Finland

article info abstract

Article history: Recently a unique mechanism of virion release was discovered in Archaea, different from lysis and egress Received 23 February 2010 systems of bacterial and eukaryotic viruses. It involves formation of pyramidal structures on the host cell Returned to author for revision surface that rupture the S-layer and by opening outwards, create apertures through which mature virions 8 March 2010 escape the cell. Here we present results of a protein analysis of Sulfolobus islandicus cells infected with the Accepted 20 April 2010 rudivirus SIRV2, which enable us to postulate SIRV2-encoded protein P98 as the major constituent of these Available online 20 May 2010 exceptional cellular ultrastructures. Keywords: © 2010 Elsevier Inc. All rights reserved. Virus–host interaction Archaea SIRV2 Virus release Lysis Sulfolobus Hyperthermophile

Viruses of the Archaea, one of the three domains of life, are apertures. The lysed cells persist in the form of empty spheres which, morphologically distinct from viruses of the Bacteria and Eukarya apart from the apertures, appear to be intact, with both the S-layer (Prangishvili et al., 2006a). In addition, they carry genomes where more and the membrane being visible on electron micrographs (Bize et al., than 90% of the genes are without predictable functions and detectable 2009; Brumfield et al., 2009, Fig. 1C). The apertures are delimited by homologues in other viruses or cellular life forms (Prangishvili et al., polygonal shaped ring structures, which sometimes detach from the 2006b). The lack of knowledge about gene functions can be partly cell envelope (Bize et al., 2009). The observations indicated that the attributed to very limited understanding of life cycles of archaeal viruses VAPs represent a distinct proteinatious structure. The large number of and special features of their interactions with the host cells. Recent the VAPs per cell, a dozen or more (Bize et al., 2009), suggested a high discovery of the novel mechanism of virion release in Archaea provided abundance of their protein constituents within the membrane the first example of such exceptional features of virus–host interactions. fraction of infected cells. We aimed at their identification by analyzing This mechanism is employed by two hyperthermophilic archaeal the protein content of SIRV2-infected cells of Sulfolobus islandicus,asa viruses, Sulfolobus rod-shaped virus 2, SIRV2 (Bize et al., 2009), and function of cellular fraction and time. Sulfolobus turreted icosahedral virus, STIV (Brumfieldetal.,2009). Growth and infection of S. islandicus with SIRV2 were performed as Infection with the viruses SIRV2 and STIV leads to the formation described in Bize et al. (2009), and the previous characterization of of pyramid-shaped ultrastructures, virus-associated pyramids (VAPs), the viral life cycle guided the selection of time points for protein on the Sulfolobus host cell. They are localized on the cell envelope, and analysis and electron microscopy. Samples were collected at following point outwards perforating the S-layer (Bize et al., 2009; Brumfield time points post infection (p.i.): 0 h (start of infection), 3 h, 7 h (prior et al., 2009, Fig. 1B). Such projections have not been documented for to virion release), 10 h (middle of virion release period) and 26 h any bacterial or eukaryal virus–host system. Moreover, the pyramids (after virion release). At each time point the uninfected cell culture appear to have seven-fold rotational symmetry, representing a was used as a control. After mechanical disruption of cells by French peculiar case in the living world. press, three fractions were collected for protein analysis: the total cell At later stages of infection, the VAPs open and the virions that lysate, the membrane fraction and the cytosol fraction. The latter two have been preassembled in the cytoplasm are released through the fractions were separated from each other by high-speed centrifuga- tion at 100,000×g, as described by Albers et al. (1999). Proteins in each fraction were solubilized by incubation for 2 h at 37 °C in the ⁎ Corresponding author. Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, β France. Fax: +33 145 68 8834. presence of 1% n-Dodecyl -D-maltoside. Following heat denaturation E-mail address: [email protected] (D. Prangishvili). (90 °C, 10 min, 0.5% SDS, and 1.25% β-mercaptoethanol), proteins

0042-6822/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2010.04.020 2 Rapid Communication

Fig. 1. Coomassie-stained SDS-PAGE gels of different protein fractions of S. islandicus cells infected with the virus SIRV2. Proteins from the three fractions, (“membrane”, “total”, and “cytotosol”) from uninfected control cells (“c”) and infected cells (“i”) were prepared as described in the text, and analyzed at time points post infection indicated on the top of each gel. Positions of proteins with known molecular masses (in kDa) are indicated with bars. Asterisks highlight protein bands in membrane and cytosol fractions which appear as a result of infection. Electron micrographs of thin sections of infected cells (A, B, and C) are displayed at corresponding time points. Bars, 100 nm. were separated by electrophoresis on 4–12% polyacrylamide gradient protocol. The SDS-PAGE gels are shown in Fig. 1,alongwith gel (Invitrogen™), with 2-(N-morpholino)ethanesulfonicacid SDS transmission electron micrographs of representative cells. running buffer (Invitrogen™) and visualized with Coomassie-based At time point 0, no difference was observed in protein patterns of Instant Blue™ staining (Expedeon) according to the manufacturer's infected and uninfected control cells (Fig. 1). However, at later time points, Rapid Communication 3 additional protein bands appeared in all fractions of infected cells. The most dramatic change was observed in the membrane fraction, where from 3 h p.i. onwards, an abundant protein with an apparent molecular mass of 10 kDa was detected in infected cells (Fig. 1). The relative intensity of this band, in comparison with the total protein content of the membrane fraction, increased from 0 at the time of infection to about 0.1 at 3 h p.i. and gradually reached about 2.0 at 26 h p.i., when all cells were perforated. This protein was identified as a product of SIRV2-ORF98 (NCBI RefSeq ID: NP_666583) by trypsin digestion and Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spec- trometry (MS) and MS/MS analyses. MS was performed as described by Stingl et al. (2008), and protein identifications were obtained using a combination of MS and MS/MS data from a 4800 Proteomics Analyzer (Applied Biosystem, USA) and the NCBI (20100119) protein database. The protein P98 was not observed in the cytosol fraction. However, on an SDS-PAGE gel of this fraction, four protein bands appeared from 10 h p.i. onwards, in SIRV2 infected cells. The four proteins had estimated molecular masses of ∼124, 35, 16, and 15 kDa (Fig. 1) and were analyzed by MALDI-TOF-MS and MS/MS. The 35 and 16 kDa proteins were identified as host proteins: The thermosome and the S-adenosyl-L- methionine-dependent methyltransfease of S. islandicus (NCBI RefSeq ID: YP_002829404 and YP_002829621, respectively). The 15 and 124 kDa proteins were identified as SIRV2 proteins: The product of ORF131b, without putative function, but conserved in all rudiviruses, and the product of ORF1070, a minor structural protein of the virion (NCBI RefSeq ID: NP_666551 and NP_666572, respectively). Edman (1950) degradation was used to determine the N-terminal sequence of the protein P98 at 10 h p.i. This confirmed the identity of the protein, and also revealed that P98 starts with AITLLE and therefore lacks the initiator methionine, as is often the case for archaeal proteins Fig. 2. Coomassie-stained SDS-PAGE gels of proteins from protease treated cells of “ ” “ ” (Falb et al., 2006). We also found that P98 at 10 h p.i. is not glycosylated, S. islandicus. (A) Proteins from uninfected cells ( c ) and SIRV2-infected cells ( i ) were separated by SDS-PAGE after incubation of intact cells with α-chymotrypsin (▼) or, as a as determined using the Pro-Q® Emerald 300 Glycoprotein Gel Stain Kit control, after incubation with the assay mixture without the enzyme (●). (B) Proteins (Invitrogen) (Supplementary material). By contrast, virion proteins of from SIRV2-infected cells were incubated with α-chymotrypsin after mechanical the Rudiviridae family, to which SIRV2 belongs, are known to be disruption of cells. Conditions of protease treatment in (A) and (B) were identical. extensively glycosylated (Vestergaard et al., 2008). Positions of proteins with known molecular masses (in kDa) are indicated with bars. An asterisk highlights the position of P98. In order to establish whether P98 is exposed on the surface of SIRV2- infected cells, its accessibility to externally added proteases was tested. Infected and uninfected cells were harvested at 10 h p.i. and washed different in their properties. They radically differ in morphology and twice with 100 mM Tris–HCl, pH 7.5, resuspended in the same buffer and share just three genes, from which only the pair SIRV2-P98/STIV-C92 incubated with α-chymotrypsin from bovine pancreas (Sigma-Aldrich®; is exclusive to STIV, SIRV2, and close relatives of the latter from the 50 ng per 1.5×1010 cells)for3hat37°C.Afterincubation,theprotease family Rudiviridae (SIRV1 (Prangishvili et al., 1999), and Stygiolobus was inactivated by heating for 10 min at 80 °C. The cells were washed rod-shaped virus, SRV (Vestergaard et al., 2008)). Fig. 3 depicts a twice with 20 mM bis-Tris propane, pH 6, and mechanically disrupted by sequence alignment of SIRV2-P98 with homologous proteins. The French press. The cellular proteins were analyzed by SDS-PAGE (Fig. 2A). higher similarity of the SIRV2 protein to its homologue in STIV than to As a result of the protease treatment, P98 was no longer detectable that of the related rudivirus SRV (Fig. 3), suggests that the gene could among proteins of infected cells (Fig. 2A). In a control experiment, where have been transferred horizontally to STIV prior to the divergence SIRV2-infected cells were mechanically broken prior to protease of Sulfolobus and Stygiolobus rudiviruses. Sequence analysis of P98 treatment, all proteins were completely digested by α-chymotrypsin revealed the presence of an N-terminal transmembrane domain (Fig. 2B). These results demonstrate that intracellular content of the (TMD; position 10–30; predicted with TMHMM (Krogh et al., 2001) infected cells was not accessible to α-chymotrypsininthecourseofthe with probability cut-off of 0.7). P98 is predicted to be a Type II “protein shaving” experiment, and that P98 was degraded apparently membrane protein, with the major part facing the extracellular side of due to its exposure on the cell surface. the cytoplasmic membrane (probability of 0.92), by the Signal-Pred Based on the data reported above, it appears highly likely that P98 (Bagos et al., 2009), a hidden Markov model-based tool trained of SIRV2 is the major constituent of the virus-associated pyramids. specifically on archaeal protein sequences. These predictions are in Firstly, it is the only protein to appear specifically in the membrane accordance with the results obtained during the protease treatment fraction of infected cells, where its accumulation throughout the viral of the surface-exposed proteins of SIRV2-infected cells (Fig. 2A). cycle correlates well with the emergence of the VAPs, as can be judged The virion release mechanism exploited by SIRV2 and STIV from the TEM analysis (Fig. 1). Secondly, the protein is exposed on apparently is not universal for hyperthermophilic archaeal viruses. the cell surface and is not covered by the S-layer, in accordance with Except for STIV and the Rudiviridae (SIRV1/2, SRV), no other archaeal the data on rupturing of the S-layer by the VAPs (Fig. 1B). virus carries a homologue of the SIRV2-ORF98. Moreover, even in the An additional argument for the involvement of SIRV2-P98 in VAP family Rudiviridae, one out of the four known species, the Acidianus rod- formation is provided by the presence of its homologue, STIV-C92, shaped virus 1, ARV (Vestergaard et al., 2005), lacks a homologue of the on the genome of the virus STIV. As mentioned above, VAPs highly gene. A homologous gene is also absent from the genome of STIV2, a similar in size and shape to those produced by SIRV2, were also close relative of STIV (Happonen et al., 2010). It appears that in archaeal observed in STIV infected cells (Brumfield et al., 2009). Except sharing viruses morphogenetic and egress systems could be evolving indepen- the virion release mechanism, the two viruses are dramatically dently. This situation resembles the one observed for bacterial viruses, 4 Rapid Communication

Fig. 3. Sequence analysis of the P98-like proteins. The multiple sequence alignment was generated using PROMALS3D (Pei et al., 2008), manually edited and visualized using JalView (Waterhouse et al., 2009). The alignment is coloured according to the standard ClustalX colouring scheme. The sequence conservation at each position is indicated at the bottom of the figure (the height of each bar is proportional to the conservation of physico-chemical properties for each column of the alignment). The transmembrane domain (TMD) is indicated above the alignment, while the pairwise identity values of the aligned sequences to the SIRV2-P98 protein are indicated on the right of the alignment. Virus name abbreviations and protein accession numbers: SIRV2, Sulfolobus islandicus rod-shaped virus 2 (NP_666583); SIRV_XX, Sulfolobus islandicus rudivirus 1 variant XX (CAG38861); SIRV1_II, Sulfolobus islandicus rod-shaped virus 1 variant II (CAG28292); SIRV1, Sulfolobus islandicus rod-shaped virus 1 (NP_666630); STIV, Sulfolobus turreted icosahedral virus (YP_024995); SRV, Stygiolobus rod-shaped virus (CAQ58475).

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