Introduction
Taxonomic Structure of the Family
Virion Properties
Morphology
Virions in this family are rods or filaments which contain a single-stranded circular DNA genome within a cylindrical protein shell. There are no lipid components. Virion length is determined by both the exact number of nucleotides in the DNA, usually between 5 kb and 10 kb and the DNA conformation maintained by the specific protein shell. Virion length can change during evolution either through insertions or deletions in the genomes, as well as through mutations that alter the coat proteins. In the genus Inovirus, virions are all only about 7 nm in diameter, but lengths vary almost three-fold, from 700 nm (Pseudomonas phage Pf3 (Pf3)) to 2,000 nm (Pseudomonas phage Pf1 (Pf1)). Electron micrographs reveal different structures at the ends, one small and blunt, and the other larger and more variable. Engineered virions of Ff (the collective designation for Enterobacteria phage M13 (M13), Enterobacteria phage f1 (f1) and Enterobacteria phage fd (fd)), in which the shell protein (gp8) or the adsorption protein (gp3) are fusions with foreign peptides, are longer than wild type, due to the extra DNA, and can have other changes in shape, depending on the size and properties of the fusions. Wild type DNA conformations are diverse, different spectroscopic properties and dramatically different extensions, with axial projections of the distance between neighboring nucleotides (the rise-per-nucleotide) ranging from a low of 0.23 nm for Pf3, over 0.27 nm for Ff, to a high of 0.61 nm for Pf1. X-ray fiber diffraction shows some capsids to have 5-fold rotational and 2-fold screw symmetry (C5S2), and others to have 1-fold rotational and 5.4- to 5.5-fold screw symmetry (C1S5.4). Plectrovirus virions are nearly straight rods with one end rounded and the other more variable. Virions of Acholeplasma phages are 70 to 90 nm long and 14 to 16 nm in diameter, and Spiroplasma phages are 230 to 280 nm long and 10 to 15 nm in diameter. Very long rods are frequently observed. Negative stained images suggest a 4 ± 2 nm hollow core. Optical diffraction of images (Acholeplasma phage MV-L1 (L1)) suggests morphological units arranged in 2-fold rotational and 5.6-fold screw symmetry (C2S5.6). DNA conformations in this genus have apparent rise-per-nucleotide values in the range 0.02 to 0.03 nm (Fig. 1).
Physicochemical and Physical Properties
Within the family, virions are sensitive to chloroform and are generally resistant to heat and a wide range of pH. For the genus Inovirus, the buoyant densities in CsCl are 1.29 ± 0.01, and DNA contents range from 6% to 14%. The Mr range from 12-34 
106, almost a three-fold range, whereas the S20w fall in a narrow range, 41-44S; the closely similar sedimentation rates are largely determined by closely similar mass-per-length. Translational and rotational diffusion constants are consistent with essentially rigid rods, yet Inovirus virions can bend considerably without breaking. Two species (Enterobacteria phage C2 (C2) and Enterobacteria phage X (X)) appear to be more flexible than the others according to electron microscopy. Spectroscopic measurements reveal similar protein conformations, all highly helical, but various DNA conformations, some base-stacked and some not. For the genus Plectrovirus, buoyant densities of 1.39 g/cm3 in CsCl and 1.21 g/cm3 in metrizamide were reported for Spiroplasma phage 1 (SpV1).
Nucleic Acid
Virions contain one molecule of infectious, circular, positive sense ssDNA. Inovirus genomes range from 6 kb to 9 kb. Plectrovirus genomes are 4.5 kb for Acholeplasma phages and about 8 kb for Spiroplasma phages. DNA sequences of Inovirus species fd, M13, f1, Ike (Enterobacteria phage Ike), I2-2 (Enterobacteria phage I2-2), Pf1, Pf3, Cf1c (Xanthomonas phage Cf1c) (and Cf1t; Xanthomonas phage Cf1t), and Vibrio phage fs1 (fs1), as well as Plectrovirus species Spiroplasma phage 1-R8A2B (SpV1-R8A2B) and L1 are available in the GenBank or EMBL databases.
Proteins
In genus Inovirus, Ff (M13, f1, fd) virions, the long shells are composed of 2700 copies of gp8 (Mr 5.2 103) , the adsorption end has several copies (probably 5 each) of gp3 (Mr 43 103) and gp6 (Mr 12 103), and several copies (probably 5 each) of gp7 (Mr 3.5 103) and gp9 (Mr 3.3 103) form the assembly nucleation end. Six non-structural proteins have been identified: morphogenetic proteins gp1 (Mr 35 103), gp11 (Mr 8 103) and gp4 (Mr 50 103) , and DNA replication proteins gp2 (Mr 46 103), gp10 (Mr 12 103), and gp5 (Mr 9.8 103). In genus Plectrovirus L1 and L51 virions, the major capsid protein is probably of Mr 19 103, a protein with a strong tendency to aggregate, and there is at least one minor protein. The genome has only four ORFs. In genus Plectrovirus SpV1-R8A2B and Spiroplasma phage 1-T78 (SpV1-T78) the major capsid protein is of Mr 7.5 103.
Lipids
None reported.
Carbohydrates
None reported.
Genome Organization and Replication
Genomes replicate either independently, by the rolling circle mechanism like free plasmids, or with the chromosome if the viral genome becomes integrated. In the normal productive infectious cycle there are five steps: phage adsorption and uptake of the infecting ssDNA circle, conversion of the ssDNA circle to a parental replicative form (RF) by host cell enzymes, semiconservative RF replication initiated by a viral endonuclease, synthesis of progeny ssDNA sequestered by ssDNA binding protein, and the membrane based assembly process that extrudes progeny virions into the medium. Genome organization reflects this sequence in that viral genes for DNA replication, for virion structure, and for virion morphogenesis are grouped in succession around the circle (Fig. 2).
The most detailed studies have been done on Inovirus Ff (M13, f1 and fd). In each of the other systems examined, closely similar or parallel phenomena have been observed. Genes are closely spaced; several genes are translated from overlapping reading frames or from alternate starts in the same frame. Intergenic regions contain the complementary- and viral-strand replication origins and DNA packaging signals. Phage adsorption involves specific interaction between the F pilus and a domain of gp3 on the infecting phage, and ssDNA translocation into the cytoplasm involves specific interactions between another domain of gp3 with other host membrane proteins. The ssDNA is converted to a supercoiled dsDNA replicative form (RF) by cellular enzymes. Phage DNA replication begins when the viral endonuclease gp2 expressed from parental RF nicks this RF at a specific, high symmetry site. Progeny RF produced via ssDNA intermediates in rolling circle replication become templates for further RF replication and further mRNA synthesis. Gp10 and gp5 can down-regulate the nicking activity of gp2. When sufficient gp5 is made, complementary strand synthesis is blocked and complexes of gp5 and progeny viral ssDNA accumulate. Assembly is initiated at the membrane by concerted interactions of gp7, gp9, gp1, and gp11 and a specific packaging signal in a hairpin on the ssDNA in the gp5 complex. Assembly proceeds at the inner membrane where about 1500 subunits of gp5 are displaced by 2700 subunits of gp8. Both gp1 and gp11 appear to be involved in this transfer of the DNA from gp5-ssDNA complexes into the assembling virion. Gp1 may also function in the formation of adhesion zones between the inner and outer membranes by interacting with outer membrane pores formed by subunits of viral protein gp4. Assembly of virions is completed by addition of gp6 and gp3. There are notable exceptions to this overall pathway. Lysogenic strains encode integrases, and viral sequences, partial or complete, are found integrated at several chromosomal sites. In Cf1t and Cf16 (Xanthomonas phage Cf16) variants of Cf1c, the first Inovirus lysogens characterized, the gene for site specific integration shows no homology with any Ff genes. Another species with a lysogenic phase is the Vibrio phage CTX (CTX), the genome of which encodes the two subunits of cholera toxin. Upon conversion of the lysogen to the nonlytic productive cycle the toxin genes become highly expressed and the toxin is released. The Plectrovirus species Acholeplasma phage MV-L51 (L51) has been shown to be similar to this scheme with respect to DNA replication pathways, and virions are assembled at the membranes as they are released into the medium without lysis. This is presumably true for all species in the genus replicating as independent plasmids and producing virus by extrusion. The genomes of two viruses (L1 and SpV1) have both been found integrated into the host chromosome at one or more sites.
Biological Properties
Members of the family infect their natural hosts without causing lysis, and the infected cells continue to divide and produce virus indefinitely. The hosts are plant and animal pathogens. In several systems the phage enter into lysogenic phases. Cell growth rates are slowed marginally by infection. On plates the slower growth usually allows the formation of turbid plaques. Sometimes there is phage multiplication but no plaque formation. Inovirus hosts are all Gram-negative bacteria (i.e., Escherichia coli, Salmonella, Pseudomonas, Vibrio, Xanthomonas, etc.). Host ranges are determined primarily by host cell receptors which are usually conjugative pili. Some pili are encoded chromosomally and some are encoded on plasmids of different incompatibility groups, i.e., phage Ff (M13, f1, fd) adsorbs to IncF pili, Pf3 to IncP pili, tf-1 to IncT pili, X to IncX pili, etc. Transmission of the plasmids to new bacterial species usually transfers phage sensitivity. Additional host range determinants include restriction - modification systems, host periplasmic proteins involved in viral ssDNA translocation into the cytoplasm, and host protein(s) involved in membrane assembly. Transfections of non-natural hosts with naked ssDNA or dsDNA are sometimes possible. When Vibrio cholera phage lysogens colonize the human intestine, states of elevated cholera toxin expression and release, and of progeny filamentous choleraphage extrusion are induced. Thus Inovirus lysogeny is a critical virulence factor in cholera pathogenesis. Plectrovirus species infect wall-less Acholeplasma and Spiroplasma and their receptors may contain both polysaccharide and protein components, but they are not well characterized. The indications of lysogeny in the L1 and SpV1-R8A2B systems suggest that the two potential modes of carrier states, as free plasmids with virus extrusion or as lysogens, might be generally true for all members of the family Inoviridae.
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