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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.
Pseudomonas phage phi6 (Segment S, M12921; Segment M, M17462; Segment L, M17461), species Pseudomonas virus phi6, genus Cystovirus
Enveloped virions (~85 nm) with two concentric, icosahedrally symmetric protein layers: the nucleocapsid surface shell (T=13) and the polymerase complex core (T=1). Spikes protrude from the virion surface.
Three segments of linear, double-stranded RNA, totaling 13.4 kb, encoding 13 genes
Single-stranded genomic precursor molecules are packaged into the viral polymerase complex. The packaged RNA molecules are replicated and transcribed within the particle.
Viral proteins are translated from polycistronic messenger RNAs.
Gram-negative bacteria, mostly Pseudomonas species
One genus (Cystovirus) and one species (Pseudomonas virus phi6)
Currently, there is only one genus (Cystovirus) in the family. Therefore the description of the genus Cystovirus is the same as the family description.
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, Huiskonen et al., 2006, Sun et al., 2017).
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.
The molecular mass of Pseudomonas phage phi6 virion is 99 ×106 and the nucleocapsid is 40 ×106. Virion S20,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.
Virions contain three segments of linear, double-stranded RNA: Segment L (6.4 kb) (Mindich et al., 1988), Segment M (4.1 kb) (Gottlieb et al., 1988), and Segment S (2.9 kb) (McGraw et al., 1986). 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 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, Sinclair et al., 1975). Receptor binding spike (P3) is anchored to the envelope via fusogenic protein P6 (Bamford et al., 1987). Protein P8 forms the nucleocapsid surface shell (Figure 1.Cystoviridae) (Sun et al., 2017) and protein P5 is a lytic enzyme (Mindich and Lehman 1979, Dessau et al., 2012) associated on the nucleocapsid surface. Major capsid protein P1 (120 copies per virion) is involved in single-stranded RNA binding (Qiao et al., 2003). Protein P2 is the viral replicase and transcriptase and is located in the interior of the P1 shell (Ilca et al., 2015, Makeyev and Bamford 2000, Butcher et al., 2001). The turret-like extrusions of the polymerase complex are made by hexamers of P4 protein (Sun et al., 2017). P4 is a nucleoside triphosphatase required for genome packaging and transcription (Pirttimaa et al., 2002, Sun et al., 2013). Minor capsid protein P7 is an assembly factor (Poranen et al., 2001). Non-structural protein P12 is a morphogenetic protein that is probably involved in envelope assembly (Johnson and Mindich 1994). 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).
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). The lipid compositions of viral envelope and host plasma membrane are similar.
Distinct noncoding regions at the termini of the three genome segments contain signals for genome packaging and replication (Mindich et al., 1988, Gottlieb et al., 1988, McGraw et al., 1986). Genes are clustered into functional groups (Figure 2.Cystoviridae).
Virions adsorb to pili of the host bacterium (Bamford et al., 1976, Roine et al., 1998) (Figure 3.Cystoviridae). Viral envelope fuses with the host outer membrane (Bamford et al., 1987) and the nucleocapsid associated lytic enzyme locally digests the peptidoglycan layer (Mindich and Lehman 1979). Viral polymerase complex delivery into the host cytoplasm involves an endocytic-like process at the host plasma membrane (Poranen et al., 1999). 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, Emori et al., 1983). 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).
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). These package the three positive-strand transcripts (Frilander and Bamford 1995). 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). 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, Olkkonen et al., 1991). 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).
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.
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.
Cysto: from Greek kystis, “bladder”, “sack”.
In terms of genome replication strategy, cystoviruses resemble eukaryotic double-stranded RNA viruses (Mertens 2004, Poranen and Bamford 2012). 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.
Pseudomonas phage phi7
Pseudomonas phage phi8
[L:AF226851; M:AF226852; S:AF226853]
Pseudomonas phage phi9
Pseudomonas phage phi10
Pseudomonas phage phi11
Pseudomonas phage phi12
[L:AF408636; M:AY039807; S:AY034425]
Pseudomonas phage phi13
[L:AF261668; M:AF261667; S:AF261666]
Pseudomonas phage phi14
Pseudomonas phage phi2954
[L:FJ608823; M:FJ608824; S:FJ608825]
Pseudomonas phage phiNN
[L:KJ957164; M:KJ957165; S:KJ957166]
Pseudomonas phage phiYY
[L:KX07420; M:KX074202; S:KX074203]
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