Microviridae

Microviridae
Virus classification
Group:	Group II (ssDNA)
Family:	Microviridae
Subfamily
Gokushovirinae Microvirinae

The Microviridae are a family of bacteriophages with a single stranded DNA genome. The name of this family is derived from the Greek word 'micro' meaning small. This refers to the size of their genomes which are among the smallest of the DNA viruses.

Virology

The virons are non-enveloped, round with an icosahedral symmetry (T = 1). They have a diameter between 25-27 nanometers and lack tails. Each viron has 60 copies each of the F, G, and J proteins and 12 copies of the H protein. They have 12 pentagonal trumpet-shaped pentomers (~7.1 nm wide x 3.8 nm high) each of which composed of 5 copies of G and one of the H protein.

Viruses in this family replicate their genomes via a rolling circle mechanism and encode dedicated RCR initiation proteins.^[1][2]

Although the majority of species in this family have lytic life cycles, a few may have temperate life cycles.

Genome

The genome sizes range from 4.5 - 6kb and is circular. It encodes 11 genes (in order: A, A*, B, C, K, D, E, J, F, G and H), nine of which are essential. The non essential genes are E and K.^[3][4] Several of the genes have overlapping reading frames.^[5][6] Protein A* is encoded within protein A. It lacks ~1/3 of the amino acids from the N terminal of the A protein and is encoded in the same frame as the A protein. It is translated from an internal start site within the messenger RNA. Gene E is encoded with gene D with a +1 frameshift. Gene K overlaps genes A, B and C. The origin of replication lies within a 30 base sequence.^[7] The entire 30 base sequence is required for replication.^[8]

Molecular biology

The major capsid protein (F) has 426 amino acids, the major spike protein (G) has 175 amino acids, the small DNA-binding protein (J) has 25 - 40 amino acids and the DNA pilot protein (H) has 328 amino acids.^[9] The major folding motif of protein F is the eight-stranded antiparallel beta barrel common to many viral capsid proteins.^[10] The G protein is a tight beta barrel with its strands running radially outwards. The G proteins occur in groups of five forming 12 spikes that enclose a hydrophilic channel. The highly basic J protein lacks any secondary structure and is situated in an interior cleft of the F protein. It has no acidic amino acid residues in the protein and the twelve basic residues are concentrated in two clusters in the N-terminus separated by a proline-rich region.

Assembly of the virion uses two scaffolding proteins, internal scaffolding protein B and external scaffolding protein D. The function of protein B seems to be to lower the amount of protein D needed by the virion for assembly.^[11] Protein H is a multifunctional structural protein required for piloting the viral DNA into the host cell interior during the entry process. Protein E is a 91 amino acid membrane protein which causes host cell lysis by inhibiting the host translocase MraY.^[12] This inhibitory activity is located within the N terminal 29 amino acids.^[13]Protein A is a single strand endonuclease^[14] and is responsible for the initation of viral DNA replication.^[15] It catalyses cleavage and ligation of a phosphodiester bond between a G and A nucleotide residue pair at the phi X origin.^[16] It may not be essential for phage viability but busrt sizs are reduced by 50% when it is mutated.^[7] Protein A* inhibits host DNA replication.^[17] Unlike protein A it is capable of cleaving the phi X viral DNA in the presence of single stranded binding protein of the host.^[18] Protein A*, like Protein A, may not be required for phage viability.^[19] Protein C increases the fidelity of the termination and reinitiation reactions and is required for the packagaing of the viral DNA in to the protein shell.^[20] Protein K has 56 amino acids and is found in the membrane of the host cell. It appears to be able to increase the burst size of the virus.^[21]

Taxonomy

This family is divided into two subfamilies - subfamilies Gokushovirinae and Microvirinae. These groups differ in their hosts, genome structure and viron composition. The name Gokushovirinae is derived from the Japanese for very small.

Subfamily Gokushovirinae:

• Genus Bdellomicrovirus; type species: Bdellovibrio phage MAC1
• Genus Chlamydiamicrovirus; type species: Chlamydia phage 1
• Genus Spiromicrovirus; type species: Spiroplasma phage 4

Subfamily Microvirinae:

• Genus Microvirus; type species: Enterobacteria phage φX174

A putative third subfamily has been proposed - Alpavirinae - which infect the order Bacteroidales.^[22] Additional work on these viruses seems indicated before subfamily status is granted.

Another virus has been isolate from the turkey gut with features similar to other microviruses but quite distinct from the known species.^[23]

Notes

Members of the subfamily Microvirinae have four structural proteins: major capsid protein F, major spike protein G, a small DNA-binding protein J (25 - 40 amino acids in length) and DNA pilot protein H. Assembly of the viron uses two scaffolding proteins, internal scaffolding protein B and external scaffolding protein D. Protein H is a multifunctional structural protein required for piloting the viral DNA into the host cell interior during the entry process. The genomes are between 5.3 and 6.2 kilobases (kb) in length.

Members of this subfamily can be separated into three main clades according to genome sizes.^[24] Size variability within the groups mainly occurs as a result of insertions and deletions of the intergenic regions. Viruses are assigned according to their similarity to known lab based strains – the ΦX174-like clade, G4-like clade and the α3-like clade. The ΦX174-like clade of microviridae have the smallest and least variable genomes (5,386-5,387 bp); the G4-like clade varies in size from 5,486-5,487 bp; while the largest genome sized group is the α3-like clade with genomes ranging from 6,061-6259bp.

Members of the subfamily Gokushovirus have only two structural proteins: capsid proteins F (Virus Protein 1) and DNA pilot protein H (Virus Protein 2) and do not use scaffolding proteins. They also possess 'mushroom-like' protrusions positioned at the three-fold axes of symmetry of their icosahedral capsids. These are formed by large insertion loops within the protein F of gokushoviruses and are absent in the microviruses. They lack both the external scaffolding protein D and the major spike protein G of the species in the genus Microvirus. The genomes of this group tend to be smaller - about 4.5 kb in length. This subfamily includes the genera Bdellomicrovirus, Chlamydiamicrovirus and Spiromicrovirus.

Life cycle

There are a number of steps in the life cycle

1. Adsorbion to the host via specific receptor(s)

2. Movement of the viral DNA into the host cell

3. Conversion of the single strand form to a double stranded intermediate

This is known as the replicative form I.

4. Transcription of early genes

5. Replication of the viral genome

Viral protein A cleaves replicative form I DNA strand at the origin of replication (ori) and covalently attaches itself to the DNA, generating replicative form II molecule. Replication of the genome now begins via a rolling circle mechanism. The host's DNA polymerase converts the single stranded DNA into double stranded DNA.

6. Late genes are now transcribed by the host's RNA polymerase.

7. Synthesis of the new virons

Viral protein C binds to replication complex, inducing packaging of new viral positive stranded DNA into procapsids.^[25] The preinitiation complex consists of the host cell protein rep and viral A and C proteins.^[26] These associate with the procapsid forming a 50S complex.

7. Maturation of the virons in the host cytoplasm

8. Release from the host

This requires the viral lysozyme which attacks the peptidoglycan wall of the host.

Popular culture

The Star Trek episode The Vengeance Factor includes a reference to a microvirus that is genetically engineered to attack nerves of the parasympathetic system.

References

1. ^ Keegstra W, Baas PD, Jansz HS (1979) Bacteriophage phi X174 RF DNA replication in vivo. A study by electron microscopy. J Mol Biol 135(1):69-89
2. ^ Fluit AC, Baas PD, Jansz HS (1986) Termination and reinitiation signals of bacteriophage phi X174 rolling circle DNA replication. Virology 154(2):357-368
3. ^ Tessman ES, Tessman I, Pollock TJ (1980) Gene K of bacteriophage phi X 174 codes for a nonessential protein. J Virol 33(1):557-560
4. ^ Bläsi U, Young R, Lubitz W (1988) Evaluation of the interaction of phi X174 gene products E and K in E-mediated lysis of Escherichia coli. J Virol 62(11):4362-4364
5. ^ Sander C, Schulz GE (1979) Degeneracy of the information contained in amino acid sequences: evidence from overlaid genes. J Mol Evol 13(3):245-252
6. ^ Fiddes JC, Godson GN (1979) Evolution of the three overlapping gene systems in G4 and phi X174. J Mol Biol 133(1):19-43
7. ^ ^{a b} Baas PD (1987) Mutational analysis of the bacteriophage phi X174 replication origin. J Mol Biol 198(1):51-61
8. ^ Fluit AC, Baas PD, Jansz HS (1985) The complete 30-base-pair origin region of bacteriophage phi X174 in a plasmid is both required and sufficient for in vivo rolling-circle DNA replication and packaging. Eur J Biochem 149(3):579-584
9. ^ McKenna R, Ilag LL, Rossmann MG (1994) Analysis of the single-stranded DNA bacteriophage phi X174, refined at a resolution of 3.0 A. J Mol Biol 237(5):517-543
10. ^ McKenna R, Xia D, Willingmann P, Ilag LL, Krishnaswamy S, Rossmann MG, Olson NH, Baker TS, Incardona NL (1992) Atomic structure of single-stranded DNA bacteriophage phi X174 and its functional implications. Nature 355(6356):137-143
11. ^ Chen M, Uchiyama A, Fane BA (2007) Eliminating the requirement of an essential gene product in an already very small virus: scaffolding protein B-free øX174, B-free. J Mol Biol 373(2):308-314
12. ^ Zheng Y, Struck DK, Young R (2009) Purification and functional characterization of phiX174 lysis protein E. Biochemistry 48(22):4999-5006
13. ^ Buckley KJ, Hayashi M (1986) Lytic activity localized to membrane-spanning region of phi X174 E protein. Mol Gen Genet 204(1):120-125
14. ^ Eisenberg S (1980) Cleavage of phi X174 single-stranded DNA by gene A protein and formation of a tight protein-DNA complex. J Virol 35(2):409-413
15. ^ van Mansfeld AD, Langeveld SA, Baas PD, Jansz HS, van der Marel GA, Veeneman GH, van Boom JH (1980) Recognition sequence of bacteriophage phi X174 gene A protein--an initiator of DNA replication. Nature 288(5791):561-566
16. ^ van Mansfeld AD, van Teeffelen HA, Baas PD, Jansz HS (1987) Two juxtaposed tyrosyl-OH groups participate in phi X174 gene A protein catalysed cleavage and ligation of DNA. Nucleic Acids Res 14(10):4229-4238
17. ^ Eisenberg S, Ascarelli R (1981) The A* protein of phi X174 is an inhibitor of DNA replication. Nucleic Acids Res 9(8):1991-2002
18. ^ van Mansfeld AD, van Teeffelen HA, Fluit AC, Baas PD, Jansz HS (1986) Effect of SSB protein on cleavage of single-stranded DNA by phi X gene A protein and A* protein. Nucleic Acids Res 14(4):1845-1861
19. ^ Colasanti J, Denhardt DT (1987) Mechanism of replication of bacteriophage phi X174. XXII. Site-specific mutagenesis of the A* gene reveals that A* protein is not essential for phi X174 DNA replication. J Mol Biol 197(1):47-54
20. ^ Goetz GS, Englard S, Schmidt-Glenewinkel T, Aoyama A, Hayashi M, Hurwitz J (1988) Effect of phi X C protein on leading strand DNA synthesis in the phi X174 replication pathway. J Biol Chem 263(31):16452-16460
21. ^ Gillam S, Atkinson T, Markham A, Smith M (1985) Gene K of bacteriophage phi X174 codes for a protein which affects the burst size of phage production. J Virol 53(2):708-709
22. ^ Krupovic M, Forterre P (2011) Microviridae goes temperate: Microvirus-related proviruses reside in the genomes of Bacteroidetes. PLoS ONE 6(5): e19893. doi:10.1371/journal.pone.0019893
23. ^ Zsak L, Day JM, Oakley BB, Seal BS (2011) The complete genome sequence and genetic analysis of ΦCA82 a novel uncultured microphage from the turkey gastrointestinal system. Virol J 8:331.
24. ^ Kodaira K, Nakano K, Okada S, Taketo A (1992) Nucleotide sequence of the genome of the bacteriophage alpha 3: interrelationship of the genome structure and the gene products with those of the phages, phi X174, G4 and phi K. Biochim Biophys Acta 1130(3):277-288
25. ^ Aoyama A, Hayashi M (1986) Synthesis of bacteriophage phi X174 in vitro: mechanism of switch from DNA replication to DNA packaging. Cell 47(1):99-106
26. ^ Hafenstein S and Fane BA (2002) X174 Genome-capsid interactions influence the biophysical properties of the virion: Evidence for a scaffolding-like function for the genome during the final stages of morphogenesis. J Virol 76(11): 5350–5356 doi: 10.1128/JVI.76.11.5350-5356.2002

Additional reading

Roykta, D.R. et al., 2006. Horizontal Gene Transfer and the Evolution of Microvirid Coliphage Genomes. Journal of Bacteriology, 118(3) p1134-1142

External links

• ViralZone: Microviridae
• List of sequenced genomes [1]