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Structure of the Archaeal Head-Tailed Virus HSTV-1 Completes the HK97 Fold Story

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Structure of the Archaeal Head-Tailed Virus HSTV-1 Completes the HK97 Fold Story Structure of the archaeal head-tailed virus HSTV-1 completes the HK97 fold story Maija K. Pietiläa,b, Pasi Laurinmäkib, Daniel A. Russellc, Ching-Chung Koc, Deborah Jacobs-Serac, Roger W. Hendrixc, Dennis H. Bamforda,b, and Sarah J. Butcherb,1 aDepartment of Biosciences and bInstitute of Biotechnology, University of Helsinki, FI-00014, Helsinki, Finland; and cDepartment of Biological Sciences, Pittsburgh Bacteriophage Institute, University of Pittsburgh, Pittsburgh, PA 15260 Edited by Michael G. Rossmann, Purdue University, West Lafayette, IN, and approved May 13, 2013 (received for review March 16, 2013) It has been proposed that viruses can be divided into a small number particles are most likely nanocompartments for enzyme storage, of structure-based viral lineages. One of these lineages is exemplified not viruses because they contain no nucleic acids (24, 25). by bacterial virus Hong Kong 97 (HK97), which represents the head- To date, only approximately 30 high-resolution protein struc- tailed dsDNA bacteriophages. Seemingly similar viruses also infect tures from archaeal viruses have been determined, and only 5 are archaea. Here we demonstrate using genomic analysis, electron MCPs. However, none come from head-tailed viruses (26). All cryomicroscopy, and image reconstruction that the major coat protein known archaeal head-tailed viruses infect either extreme halophiles fold of newly isolated archaeal Haloarcula sinaiiensis tailed virus 1 or anaerobic methanogens belonging to the phylum Euryarchaeota has the canonical coat protein fold of HK97. Although it has been (20). Here we set out to screen a previously isolated group of anticipated previously, this is physical evidence that bacterial and haloarchaeal head-tailed viruses (19) to find a suitable candidate archaeal head-tailed viruses share a common architectural principle. for the determination of the MCP fold by electron cryomicro- The HK97-like fold has previously been recognized also in herpesvi- scopy (cryo-EM). A podovirus, Haloarcula sinaiiensis tailed virus 1 ruses, and this study expands the HK97-like lineage to viruses from all (HSTV-1) (19), was chosen because it was also stable at low sa- three domains of life. This is only the second established lineage to linity and could be readily purified. The genome of HSTV-1 was include archaeal, bacterial, and eukaryotic viruses. Thus, our findings sequenced, and the major structural proteins were identified. The support the hypothesis that the last common universal ancestor of 3D reconstruction of the HSTV-1 capsid at subnanometer reso- cellular organisms was infected by a number of different viruses. lution revealed that the virus adopts the HK97-like fold. Thus, the BIOPHYSICS AND HK97-like lineage can be extended to archaeal head-tailed viru- COMPUTATIONAL BIOLOGY archaeal virus | virus evolution | major capsid protein fold ses, suggesting a common ancestor for prokaryotic head-tailed viruses and eukaryotic herpesviruses. very organism is constantly under the attack of viral infections Ebecause viruses outnumber cellular organisms by at least an Results order of magnitude. It has been estimated that viruses kill ap- Virus Production and Morphology. HSTV-1 virus sloppy agar stocks 11 proximately one fifth of the marine biomass on a daily basis (1, 2). had high titers, on average 3 × 10 pfu/mL. Turbidity measure- Furthermore, viruses are the leading cause of infectious diseases ments of the infected cultures demonstrated that the virus is lytic (3). Thus, viruses have a crucial role in controlling the evolution of (Fig. S1). Virus titers in liquid cultures, however, reached only ap- 9 cellular organisms owing to their high abundance and genetic di- proximately 6 × 10 pfu/mL, and for this reason the virus was pu- versity (1). However, cryoelectron microscopic and crystallographic rified directly from the stocks. The specific infectivity of the purified 12 studies have revealed that this genetic diversity may be reduced to HSTV-1 virions was ∼9 × 10 pfu/mg of protein, and the density in a limited number of structure-based viral lineages (2, 4–6). CsCl was ∼1.45 g/mL, which is typical of head-tailed viruses (27). The discovery that a bacterial and an animal virus have the Cryo-EM micrographs of HSTV-1 virions at high and low salinity same major capsid protein (MCP) fold and virion architecture (5, showed that the overall morphotype remained unaltered despite 7) led to the hypothesis that viruses infecting cells that belong to changes in salinity (Fig. 1A). HSTV-1 was inactivated at low salinity, different domains of life (in this case Bacteria and Eukarya) may but the infectivity was regained when the high salinity conditions have a common origin and be related, although there is no de- were restored (Fig. 1B).HSTV-1hadanisometric,genome-filled tectable sequence similarity. Further examination of viral capsid capsid and a short, podovirus-like tail with at least five long fibers protein structures has revealed that this double-β-barrel MCP of attached to the central tail structure (Fig. 1, Inset). tailless icosahedral viruses is also present in viruses infecting ar- chaea (5, 8, 9). These observations have led to the hypothesis of Genome Sequence and Structural Proteins. The genome of HSTV-1 viral structure-based lineages unifying viruses previously consid- contained 53 putative open reading frames (ORFs) (Table S1). As ered unrelated and leading to a paradigm shift in how we see the has been seen in the few head-tailed archaeal viruses sequenced viral universe (2, 4, 5). previously (21, 28, 29), the organization of the genome is remi- A second lineage has been proposed containing head-tailed niscent of the genomes of tailed bacteriophages: the predicted bacterial viruses and herpesviruses sharing the so-called canonical genes occupy 90% of the genome and are closely spaced and in Hong Kong 97 (HK97)-like MCP fold (4, 10). This fold was first determined for bacteriophage HK97 (10) and has since been dis- covered in seven additional phages and in herpes simplex virus type Author contributions: M.K.P., R.W.H., D.H.B., and S.J.B. designed research; M.K.P., P.L., D.A.R., and C.-C.K. performed research; M.K.P., P.L., D.J.-S., R.W.H., D.H.B., and S.J.B. 1 (11-18). Head-tailed viruses morphologically similar to phages analyzed data; and M.K.P., R.W.H., D.H.B., and S.J.B. wrote the paper. are also known to infect archaeal organisms (19, 20). Comparative The authors declare no conflict of interest. genomics indicate that these archaeal viruses share common genes This article is a PNAS Direct Submission. with bacteriophages for virion assembly and maturation, as well as Data deposition: The sequence reported in this paper has been deposited in the GenBank genome packaging (21, 22). The fundamental issue is whether ar- database (accession no. KC117378). The 3D reconstruction has been deposited with the chaeal head-tailed viruses also share the canonical HK97-like Electron Microscopy Data Bank (accession no. EMD-2279). MCP fold, as suggested by bioinformatics (21, 23). Interestingly, 1To whom correspondence should be addressed. E-mail: sarah.butcher@helsinki.fi. the HK97-like fold has been recognized in icosahedral particles This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. produced by hyperthermophilic archaea (24, 25). However, these 1073/pnas.1303047110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1303047110 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Fig. 1. Podovirus HSTV-1 tolerates salinity changes. (A) Cryo-EMs of HSTV-1 at 4-μm underfocus. (I) Virions incubated in 9% (wt/vol) SW containing 1.2 M NaCl, (II) virions in 20 mM Tris·HCl (pH 7.2) buffer, and (III) virions first incubated in 20 mM Tris·HCl (pH 7.2) and then restored in 9% (wt/vol) SW. (Inset) (6-μm underfocus) A particle viewed along the tail and showing possible tail fibers. (Scale bars, 50 nm.) (B) Specific infectivity of HSTV-1 virions at a logarithmic scale. (I, II, and III) High and low salinity conditions as in A. some cases slightly overlapping (Fig. 2A). The majority of the pre- The HSTV-1 genome is 32,189 bp long. The sequence assembles dicted proteins of HSTV-1 make no sequence matches to the se- as a circle, and there is no indication in the sequence data for quence databases in a BlastP search, and of the ones that do match, discrete ends, which argues that the genome is circularly permuted more than half match “hypothetical proteins” with unknown func- across the population of virions. As base pair 1 we have chosen the tion. The predicted proteins that do match proteins of known first base pair of the large terminase subunit gene. We identified function are also typical of the tailed bacteriophages, with functions four genes encoding structural proteins and two more as genes such as DNA replication, nucleotide metabolism, and structural encoding enzymes (terminase and protease) required for capsid proteins of the virion. Some of the structural proteins can be assembly and maturation: the terminase large subunit gene (gene 1) identified by a simple BlastP search, and some are found with more and the portal gene (gene 3) are identified directly by the se- extensive searching (PSI-Blast, HHpred). We confirmed a few of quence similarity of their protein products to the corresponding these identifications by N-terminal sequencing of virion compo- proteins of tailed bacteriophages. The portal similarity extends nents (Fig. 2 B and C). Strikingly, the order of the genes for struc- only through the first two-thirds of the sequence; the last one-third tural proteins identified in this way is the same as the order almost is homologous to a minor head protein found in some bacter- always observed for the corresponding genes in the genomes of iophages (gpF in phage Mu; gp7 in phage SPP1), which is encoded tailed bacteriophages.
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