Cystoviridae

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Chapter Contents

Citation

A summary of this ICTV online (10th) report chapter has been published as an ICTV Virus Taxonomy Profile article in the Journal of General Virology, and should be cited when referencing this online chapter as follows:

Poranen, M.M., Mäntynen, S., and ICTV Report Consortium, 2017, ICTV Virus Taxonomy Profile: Cystoviridae, Journal of General Virology, (In Press).

Summary

The family Cystoviridae includes enveloped viruses with a tri-segmented dsRNA genome and a double-layered protein capsid. The innermost protein layer is a polymerase complex responsible for genome packaging, replication and transcription. Cystoviruses infect Gram-negative bacteria, primarily plant-pathogenic Pseudomonas syringae strains.

Table 1.Cystoviridae. Characteristics of the family Cystoviridae.

Currently, there is only one genus (Cystovirus) in the family. Therefore the description of the genus Cystovirus is the same as the family description.

Virion

Morphology

The enveloped virions are spherical, about 85 nm in diameter and covered by spikes (Figure 1.Cystoviridae). The envelope surrounds an isometric nucleocapsid, about 58 nm in diameter. The nucleocapsid surface shell follows T=13 isocahedral symmetry. Turret-like extrusions of the underlying polymerase complex span the nucleocapsid surface shell layer at the five-fold symmetry positions (Figure 1.Cystoviridae). The dodecahedral polymerase complex is about 50 nm in diameter. In the polymerase complex major capsid protein dimers are arranged on a T=1 icosahedral lattice [{Butcher et al., 1997:9250692RJOHTXButcher et al., 1997, Intermediates in the assembly pathway of the double-stranded RNA virus φ6, Embo j, 16, 14, 4477-87RJOMREFHuiskonen et al., 2006:16765897RJOHTXHuiskonen et al., 2006, Structure of the bacteriophage φ6 nucleocapsid suggests a mechanism for sequential RNA packaging, Structure, 14, 6, 1039-48RJOMREFSun et al., 2017:28287099RJOHTXSun et al., 2017, Double-stranded RNA virus outer shell assembly by bona fide domain-swapping, Nat Commun, 8, 14814}].

Figure 1.Cystoviridae. Schematic presentation of a cystovirus particle (Pseudomonas phage phi6) with the location of virion proteins (left). Three-dimensional reconstructions of the nucleocapsid (middle). Thin-section electron micrograph of Pseudomonas phage phi6 particles attached to the pilus receptor of the host (right). The bar represents 200 nm.

Physicochemical and physical properties

The molecular mass of Pseudomonas phage phi6 virion is 99 ×10⁶ and the nucleocapsid is 40 ×10⁶. Virion S_20,w is about 405S. The buoyant density of the virion is 1.27 g cm⁻³ in CsCl, 1.22 g cm⁻³ in Cs₂SO₄ and 1.24 g cm⁻³ in sucrose. Virions are sensitive to detergents, ether and chloroform but stable between pH 6.0 and 9.5.

Nucleic acid

Virions contain three segments of linear, double-stranded RNA: Segment L (6.4 kb) [{Mindich et al., 1988:3346944RJOHTXMindich et al., 1988, Nucleotide sequence of the large double-stranded RNA segment of bacteriophage φ6: genes specifying the viral replicase and transcriptase, J Virol, 62, 4, 1180-5}], Segment M (4.1 kb) [{Gottlieb et al., 1988:3347997RJOHTXGottlieb et al., 1988, Nucleotide sequence of the middle dsRNA segment of bacteriophage φ6: placement of the genes of membrane-associated proteins, Virology, 163, 1, 183-90}], and Segment S (2.9 kb) [{McGraw et al., 1986:3754015RJOHTXMcGraw et al., 1986, Nucleotide sequence of the small double-stranded RNA segment of bacteriophage φ6: novel mechanism of natural translational control, J Virol, 58, 1, 142-51}]. The complete genome is 13.4 kb and has a guanine+cytosine content of approximately 56%. All the genome segments are enclosed in a single particle and each virion contains a single copy of the genome. The genome constitutes approximately 10% of the virion weight.

Proteins

Proteins constitute about 70% of the virion weight. The viral genome (Figure 2.Cystoviridae) encodes structural (Figure 1.Cystoviridae) and non-structural proteins. The envelope contains four integral membrane proteins: P6, P9, P10 and P13 [{Gottlieb et al., 1988:3347997RJOHTXGottlieb et al., 1988, Nucleotide sequence of the middle dsRNA segment of bacteriophage φ6: placement of the genes of membrane-associated proteins, Virology, 163, 1, 183-90RJOMREFSinclair et al., 1975:1159897RJOHTXSinclair et al., 1975, Proteins of bacteriophage φ6, J Virol, 16, 3, 685-95}]. Receptor binding spike (P3) is anchored to the envelope via fusogenic protein P6 [{Bamford et al., 1987:3608985RJOHTXBamford et al., 1987, Membrane fusion in prokaryotes: bacteriophage φ6 membrane fuses with the Pseudomonas syringae outer membrane, Embo j, 6, 5, 1467-73}]. Protein P8 forms the nucleocapsid surface shell (Figure 1.Cystoviridae) [{Sun et al., 2017:28287099RJOHTXSun et al., 2017, Double-stranded RNA virus outer shell assembly by bona fide domain-swapping, Nat Commun, 8, 14814}] and protein P5 is a lytic enzyme [{Mindich and Lehman 1979:469991RJOHTXMindich and Lehman 1979, Cell wall lysin as a component of the bacteriophage φ6 virion, J Virol, 30, 2, 489-96RJOMREFDessau et al., 2012:23209446RJOHTXDessau et al., 2012, Selective pressure causes an RNA virus to trade reproductive fitness for increased structural and thermal stability of a viral enzyme, PLoS Genet, 8, 11, e1003102}] associated on the nucleocapsid surface. Major capsid protein P1 (120 copies per virion) is involved in single-stranded RNA binding [{Qiao et al., 2003:14563876RJOHTXQiao et al., 2003, Analysis of specific binding involved in genomic packaging of the double-stranded-RNA bacteriophage φ6, J Bacteriol, 185, 21, 6409-14}]. Protein P2 is the viral replicase and transcriptase and is located in the interior of the P1 shell [{Ilca et al., 2015:26534841RJOHTXIlca et al., 2015, Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes, Nat Commun, 6, 8843RJOMREFMakeyev and Bamford 2000:10619851RJOHTXMakeyev and Bamford 2000, Replicase activity of purified recombinant protein P2 of double-stranded RNA bacteriophage φ6, Embo j, 19, 1, 124-33RJOMREFButcher et al., 2001:11242087RJOHTXButcher et al., 2001, A mechanism for initiating RNA-dependent RNA polymerization, Nature, 410, 6825, 235-40}]. The turret-like extrusions of the polymerase complex are made by hexamers of P4 protein [{Sun et al., 2017:28287099RJOHTXSun et al., 2017, Double-stranded RNA virus outer shell assembly by bona fide domain-swapping, Nat Commun, 8, 14814}]. P4 is a nucleoside triphosphatase required for genome packaging and transcription [{Pirttimaa et al., 2002:12239286RJOHTXPirttimaa et al., 2002, Nonspecific nucleoside triphosphatase P4 of double-stranded RNA bacteriophage φ6 is required for single-stranded RNA packaging and transcription, J Virol, 76, 20, 10122-7RJOMREFSun et al., 2013:24089550RJOHTXSun et al., 2013, Rescue of maturation off-pathway products in the assembly of Pseudomonas phage φ6, J Virol, 87, 24, 13279-86}]. Minor capsid protein P7 is an assembly factor [{Poranen et al., 2001:11336707RJOHTXPoranen et al., 2001, Self-assembly of a viral molecular machine from purified protein and RNA constituents, Mol Cell, 7, 4, 845-54}]. Non-structural protein P12 is a morphogenetic protein that is probably involved in envelope assembly [{Johnson and Mindich 1994:8021194RJOHTXJohnson and Mindich 1994, Plasmid-directed assembly of the lipid-containing membrane of bacteriophage φ6, J Bacteriol, 176, 13, 4124-32}]. The function of the other non-structural protein P14 is unknown.

Figure 2.Cystoviridae. Genome organization of Pseudomonas phage phi6. The gene and protein numbers are the same. Colourings indicate genes encoding constituents of the polymerase complex (green), nucleocapsid (blue), envelope associated proteins (beige), and non-structural proteins (red).

Lipids

Virions are composed of 20% lipids by weight. There is enough lipid to cover about one-half of the envelope surface area (the rest being protein). Viral lipids are derived from host plasma membrane [{Laurinavičius et al., 2007:17658459RJOHTXLaurinavičius et al., 2007, Transbilayer distribution of phospholipids in bacteriophage membranes, Biochim Biophys Acta, 1768, 10, 2568-77}]. The lipid compositions of viral envelope and host plasma membrane are similar.

Genome organization and replication

Distinct noncoding regions at the termini of the three genome segments contain signals for genome packaging and replication [{Mindich et al., 1988:3346944RJOHTXMindich et al., 1988, Nucleotide sequence of the large double-stranded RNA segment of bacteriophage φ6: genes specifying the viral replicase and transcriptase, J Virol, 62, 4, 1180-5RJOMREFGottlieb et al., 1988:3347997RJOHTXGottlieb et al., 1988, Nucleotide sequence of the middle dsRNA segment of bacteriophage φ6: placement of the genes of membrane-associated proteins, Virology, 163, 1, 183-90RJOMREFMcGraw et al., 1986:3754015RJOHTXMcGraw et al., 1986, Nucleotide sequence of the small double-stranded RNA segment of bacteriophage φ6: novel mechanism of natural translational control, J Virol, 58, 1, 142-51}]. Genes are clustered into functional groups (Figure 2.Cystoviridae).

Virions adsorb to pili of the host bacterium [{Bamford et al., 1976:798023RJOHTXBamford et al., 1976, Ultrastructure and life cycle of the lipid-containing bacteriophage φ6, J Gen Virol, 32, 2, 249-59RJOMREFRoine et al., 1998:9805392RJOHTXRoine et al., 1998, Characterization of type IV pilus genes in Pseudomonas syringae pv. tomato DC3000, Molecular plant-microbe interactions : MPMI, 11, 11, 1048-56}] (Figure 3.Cystoviridae). Viral envelope fuses with the host outer membrane [{Bamford et al., 1987:3608985RJOHTXBamford et al., 1987, Membrane fusion in prokaryotes: bacteriophage φ6 membrane fuses with the Pseudomonas syringae outer membrane, Embo j, 6, 5, 1467-73}] and the nucleocapsid associated lytic enzyme locally digests the peptidoglycan layer [{Mindich and Lehman 1979:469991RJOHTXMindich and Lehman 1979, Cell wall lysin as a component of the bacteriophage φ6 virion, J Virol, 30, 2, 489-96}]. Viral polymerase complex delivery into the host cytoplasm involves an endocytic-like process at the host plasma membrane [{Poranen et al., 1999:10545509RJOHTXPoranen et al., 1999, A novel virus-host cell membrane interaction. Membrane voltage-dependent endocytic-like entry of bacteriophage φ6 nucleocapsid, J Cell Biol, 147, 3, 671-82}]. The viral genome is transcribed by virion-associated RNA-dependent RNA polymerase within the polymerase complex. Early in the infection approximately equal amounts of messenger RNA molecules are produced from L, M and S [{Coplin et al., 1975:1055383RJOHTXCoplin et al., 1975, Intermediates in the biosynthesis of double-stranded ribonucleic acids of bacteriophage φ6, Proceedings of the National Academy of Sciences, USA, 72, 3, 849-53RJOMREFEmori et al., 1983:6827650RJOHTXEmori et al., 1983, Transcriptional regulation of three double-stranded RNA segments of bacteriophage φ6 in vitro, J Virol, 46, 1, 196-203}]. Later in the infection cycle transcripts of M and S typically predominate. Transcription is semi-conservative and produces full-length copies of the genome segments [{Usala et al., 1980:7379123RJOHTXUsala et al., 1980, Displacement of parental RNA strands during in vitro transcription by bacteriophage φ6 nucleocapsids, Cell, 19, 4, 855-62}].

The produced messenger RNA molecules are polycistronic. Translation of L transcripts produces the early proteins, which assemble to form empty polymerase complexes (Figure 3.Cystoviridae) [{Gottlieb et al., 1988:3275432RJOHTXGottlieb et al., 1988, Production of a polyhedral particle in Escherichia coli from a cDNA copy of the large genomic segment of bacteriophage φ6, J Virol, 62, 1, 181-7}]. These package the three positive-strand transcripts [{Frilander and Bamford 1995:7877165RJOHTXFrilander and Bamford 1995, In vitro packaging of the single-stranded RNA genomic precursors of the segmented double-stranded RNA bacteriophage φ6: the three segments modulate each other's packaging efficiency, J Mol Biol, 246, 3, 418-28}]. Negative-strand synthesis then takes place inside the polymerase complex. RNA packaging and replication induce structural changes in the polymerase complex (expansion) [{Nemecek et al., 2011:22019738RJOHTXNemecek et al., 2011, Stepwise expansion of the bacteriophage φ6 procapsid: possible packaging intermediates, J Mol Biol, 414, 2, 260-71}]. Transcription by these polymerase complexes produces messages for late protein synthesis. The nucleocapsid surface shell assembles on the polymerase complex (Figure 3.Cystoviridae) [{Poranen et al., 2001:11336707RJOHTXPoranen et al., 2001, Self-assembly of a viral molecular machine from purified protein and RNA constituents, Mol Cell, 7, 4, 845-54RJOMREFOlkkonen et al., 1991:2016747RJOHTXOlkkonen et al., 1991, Generation of infectious nucleocapsids by in vitro assembly of the shell protein on to the polymerase complex of the dsRNA bacteriophage φ6, J Mol Biol, 218, 3, 569-81}]. Nucleocapsid acquires protein P5 and the envelope. Spikes are assembled on the envelope surface. Mature virions are released upon virus-induced host cell lysis [{Bamford et al., 1976:798023RJOHTXBamford et al., 1976, Ultrastructure and life cycle of the lipid-containing bacteriophage φ6, J Gen Virol, 32, 2, 249-59}].

Figure 3.Cystoviridae. Schematic presentation of the Pseudomonas phage phi6 life cycle. (A) Adsorption. (B) Envelope fusion. (C) Peptidoglycan digestion. (D–E) Endocytotic uptake of the nucleocapsid. (F) Early transcription. (G) Polymerase complex assembly. (H) ssRNA packaging and replication. (I) Late transcription. (J) Nucleocapsid shell assembly. (K) Translocation of the viral envelope and assembly of spikes. (L) Host cell lysis and release of mature virions.

Biology

Cystoviruses are lytic bacteriophages that induce host cell lysis at the end of the viral reproduction cycle. Natural hosts are Gram-negative plant pathogenic bacteria.

Derivation of names

Cysto: from Greek kystis, “bladder”, “sack”.

Similarity with other taxa

In terms of genome replication strategy, cystoviruses resemble eukaryotic double-stranded RNA viruses [{Mertens 2004:15010213RJOHTXMertens 2004, The dsRNA viruses, Virus Res, 101, 1, 3-13RJOMREFPoranen and Bamford 2012:22297523RJOHTXPoranen and Bamford 2012, Assembly of large icosahedral double-stranded RNA viruses, Adv Exp Med Biol, 726, 379-402}]. The structure, organization and functions of the polymerase complex containing the genome are the major similarities among members of families Cystoviridae, Reoviridae, Totiviridae, Partitiviridae and Picobirnaviridae. The T=13 architecture of the surrounding capsid layer is also shared by cystoviruses and reoviruses.

Related, Unclassified Viruses

Virus names, the choice of exemplar isolates, and virus abbreviations, are not official ICTV designations.
Download GenBank/EMBL query for sequences listed in the table here.