Filamentous bacteriophage

Characteristics

Filamentous bacteriophages are among the simplest viruses known, with far fewer genes than the classical tailed bacteriophages studied by the phage group in the mid-20th century. The family contains 29 defined species, divided among 23 genera. However, mining of genomic and metagenomic datasets using a machine learning approach led to the discovery of 10,295 inovirus-like sequences in nearly all bacterial phyla across virtually every ecosystem, indicating that this group of viruses is much more diverse and widespread than originally appreciated.

Three filamentous bacteriophages, fd, f1, and M13, were isolated and characterized by three different research groups in the early 1960s, but they are so similar that they are sometimes grouped under the common name "Ff", which are members of the genus Inovirus, as acknowledged by the International Committee on Taxonomy of Viruses (ICTV). The molecular structure of Ff phages was determined using a number of physical techniques, especially X-ray fiber diffraction, solid-state NMR and cryo-electron microscopy. The structures of the phage capsid and of some other phage proteins are available from the Protein Data Bank. The single-stranded Ff phage DNA runs down the central core of the phage, and is protected by a cylindrical protein coat built from thousands of identical α-helical major coat protein subunits coded by phage gene 8. The gene 8 protein is inserted into the plasma membrane as an early step in phage assembly. Some strains of phage have a "leader sequence" on the gene 8 protein to promote membrane insertion, but others do not seem to need the leader sequence. The two ends of the phage are capped by a few copies of proteins that are important for infection of the host bacteria, and also for assembly of nascent phage particles. These proteins are the products of phage genes 3 and 6 at one end of the phage, and phage genes 7 and 9 at the other end. The fiber diffraction studies identified two structural classes of phage, differing in the details of the arrangement of the gene 8 protein. Class I has a rotation axis relating the gene 8 coat proteins, whereas for Class II this rotation axis is replaced by a helix axis. This technical difference has little noticeable effect on the overall phage structure, but the extent of independent diffraction data is greater for symmetry Class II than for Class I. This assisted the determination of the Class II phage Pf1 structure, and by extension the Class I structure.

Structural Class I includes strains fd, f1, M13 of genus Inovirus as well as If1 (of ICTV's species Infulavirus If1, genus Infulavirus) and IKe (of ICTV's species Lineavirus IKe, genus Lineavirus), whereas Class II includes strains Pf1 (of ICTV's species Primolicivirus Pf1 of genus Primolicivirus), and perhaps also Pf3 (of ICTV's species Tertilicivirus Pf3 of genus Tertilicivirus), Pf4 and PH75 (of NCBI's proposed species Thermus phage PH75, incertae sedis within Inoviridae).

The DNA isolated from fd phage (of genus Inovirus) is single-stranded, and topologically a circle. That is, the DNA single strand extends from one end of the phage particle to the other and then back again to close the circle, although the two strands are not base-paired. This topology was assumed to extend to all other filamentous phages, but it is not the case for phage Pf4, for which the DNA in the phage is single-stranded but topologically linear, not circular. During fd phage assembly, the phage DNA is first packaged into a linear intracellular nucleoprotein complex with many copies of the phage gene 5 replication/assembly protein. The gene 5 protein is then displaced by the gene 8 coat protein as the nascent phage is extruded across the bacterial plasma membrane without killing the bacterial host. This protein also binds with high affinity to G-quadruplex structures (although they are not present in the phage DNA) and to similar hairpin structures in phage DNA.

The p1 protein of Ff phage (i. e. genus Inovirus), which is required for phage assembly at the membrane, has a membrane-spanning hydrophobic domain with the N-terminal portion in the cytoplasm and the C-terminal portion in the periplasm (the reverse of the orientation of the gene 8 coat protein). Adjacent to the cytoplasmic side of the membrane-spanning domain is a 13- residue sequence of p1 having a pattern of basic residues closely matching the pattern of basic residues near the C terminus of p8, but inverted with respect to the sequence. This assembly mechanism makes this phage a valuable system with which to study transmembrane proteins. Gene 1, coding for an ATPase, is a conserved marker gene that (along with three additional genetic features) was used to automatically detect inovirus sequences.

Life cycle

Viral replication is cytoplasmic. Entry into the host cell is achieved by pilus-mediated adsorption into the host cell. Replication follows the ssDNA rolling circle model. DNA-templated transcription is the method of transcription. The virus exits the host cell by viral extrusion. Viral assembly occurs at the inner membrane (in case of Gram-negative bacteria), mediated by a membrane-embedded motor protein complex. This multimeric assembly complex, including p1 encoded by gene 1 (referred to as ZOT, zonula occludens toxin by researchers on Vibrio cholerae phage CTXΦ) is an ATPase containing functional and essential Walker motifs that are thought to mediate the hydrolysis of ATP providing the energy for the assembly of the phage filament. Filamentous phage Cf1t from Xanthomonas campestris (of NCBI's proposed species Xanthomonas phage Cf1t, incertae sedis within Inoviridae, likely misspelled as Cflt), was shown in 1987 to integrate into the host bacterial genome, and further such temperate filamentous phages have since been reported, many of which have been implicated in pathogenesis.