Phi X 174


Phi X 174
Structure of phage phi X 174 capsid
Phi X 174
Virus classification
Group: Group II (ssDNA)
Family: Microviridae
Genus: Microvirus
Species: ΦX174 phage

The phi X 174 (or ΦX174) bacteriophage was the first DNA-based genome to be sequenced. This work was completed by Fred Sanger and his team in 1977.[1] In 1962, Walter Fiers had already demonstrated the physical, covalently closed circularity of phi X 174 DNA.[2]

In 2003, it was reported that the whole genome of ΦX174 had been assembled synthetically from scratch.[3] ΦX174 has also been successfully assembled in vitro[4]

Contents

Virology

This bacteriophage has a [+] circular single-stranded DNA genome of 5386 nucleotides encoding 11 proteins. Of these 11 genes, only 8 are essential to viral morphogenesis. The GC-content is 44% and 95% of nucleotides belong to coding genes.

Protein Function
A Stage II and stage III DNA replication
A* An unessential protein for viral propagation. It may play a role in the inhibition of host cell DNA replication and superinfection exclusion
B Internal scaffolding protein, required for capsid morphogenesis and the assembly of early morphogenetic intermediates. Sixty copies present in the procapsid
C Facilitates the switch from stage II to stage III DNA replication. Required for stage III DNA synthesis
D External scaffolding protein, required for procapsid morphogenesis. Two hundred and forty copies present in the procapsid.
E Host cell lysis
F Major coat protein. Sixty copies present in the virion and procapsid
G Major spike protein. Sixty copies present in the virion and procapsid
H DNA pilot protein need for DNA injection, also called the minor spike protein. Twelve copies in the procapsid and virion
J DNA binding protein, needed for DNA packaging. Sixty copies present in the virion
K An unessential protein for viral propagation. It may play a role optimizing burst sizes in various Hosts

Table from ΦX174 et al. the Microviridae by B.A. Fane et al.

Infection begins when H protein (or the DNA Pilot Protein) pilots the viral genome through the bacterial membrane of E.coli bacteria (Jazwinski et al. 1975) most likely via a predicted N-terminal transmembrane domain helix (Tusnady and Simon, 2001). However, it has become apparent that H protein is a multifunctional protein (Cherwa, Young and Fane, 2011). This is the only viral capsid protein of ΦX174 to lack a crystal structure for a couple of reasons. It has low aromatic content and high glycine content, making the protein structure very flexible and in addition, individual hydrogen atoms (the R group for glycines) are difficult to detect in protein crystallography. Additionally, H protein induces lysis of the bacterial host at high concentrations as the predicted N-terminal transmembrane helix easily pokes holes through the bacterial wall. By bioinformatics, this protein contains four predicted coiled-coil domains which has a significant homology to known transcription factors. Additionally, it was determined by Ruboyianes et al. (2009) that de novo H protein was required for optimal synthesis of other viral proteins. Interestingly, mutations in H protein that prevent viral incorporation, can be overcome when excess amounts of Protein B, the internal scaffolding protein, are supplied.

The DNA is ejected through a hydrophilic channel at the 5-fold vertex (McKenna et al. 1992). It is understood that H protein resides in this area but experimental evidence has not verified its exact location. Once inside the host bacterium, replication of the [+] ssDNA genome proceeds via a rolling circle mechanism. As D protein is the most abundant gene transcript, it is the most protein in the viral procaspid. Similarly, gene transcripts for F, J, and G are more abundant than for H as the stoichiometry for these structures proteins is 5:5:5:1.

Notes

Phi X is regularly used as a positive control in DNA sequencing due to its relatively small genome size in comparison to other organisms and the extensive work that has been done on it.

See also

References

  1. ^ Sanger F, Air GM, Barrell BG, Brown NL, Coulson AR, Fiddes CA, Hutchison CA, Slocombe PM, Smith M (1977-02-24). "Nucleotide sequence of bacteriophage phi X174 DNA". Nature 265 (5596): 687–695. doi:10.1038/265687a0. PMID 870828. 
  2. ^ Fiers W, Sinsheimer RL (1962). "The structure of the DNA of bacteriophage ΦX74. III. Ultracentrifuge evidence for a ring structure". J. Mol. Biol. 5 (4): 424–434. doi:10.1016/S0022-2836(62)80031-X. PMID 13945085. 
  3. ^ Smith, Hamilton O.; Clyde A. Hutchison, Cynthia Pfannkoch, J. Craig Venter (2003-12-23). "Generating a synthetic genome by whole genome assembly: {phi}X174 bacteriophage from synthetic oligonucleotides". Proceedings of the National Academy of Sciences 100 (26): 15440–15445. doi:10.1073/pnas.2237126100. PMID 14657399. http://www.pnas.org/cgi/content/abstract/100/26/15440. Retrieved 2007-10-08. 
  4. ^ Cherwa, James (2011). "In Vitro Assembly of the ΦX174 Procapsid from External Scaffolding Protein Oligomers and Early Pentameric Assembly Intermediates". Journal of Molecular Biology. 

1. B.A. Fane, et al. (2006). ØX174 et al., the "Microviridae" (The Bacteriophages, Oxford Press)

External links



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