Phi X 174
|Phi X 174|
|Group:||Group II (ssDNA)|
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. In 1962, Walter Fiers and Robert Sinsheimer had already demonstrated the physical, covalently closed circularity of ΦX174 DNA. Nobel prize winner Arthur Kornberg used ΦX174 as a model to first prove that DNA synthesized in a test tube by purified enzymes could produce all the features of a natural virus, ushering in the age of synthetic biology. In 2003, it was reported by Craig Venter's group that the genome of ΦX174 was the first to be completely assembled in vitro from synthesized oligonucleotides. The ΦX174 virus particle has also been successfully assembled in vitro. Recently, it was shown how its highly overlapping genome can be fully decompressed and still remain functional.
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.
|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 G protein binds to lipopolysaccharides on the bacterial host cell surface. 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 negative sense DNA intermediate. This is done as the phage genome supercoils and the secondary structure formed by such supercoiling attracts a primosome protein complex. This translocates once around the genome and synthesises a [-]ssDNA from the positive original genome. [+]ssDNA genomes to package into viruses are created from this by a rolling circle mechanism. This is the mechanism by which the double stranded supercoiled genome is nicked on the negative strand by a virus-encoded A protein, also attracting a bacterial DNA Polymerase to the site of cleavage. DNAP will use the negative strand as a template to make positive sense DNA. As it translocates around the genome it displaces the outer strand of already-synthesised DNA, which is immediately coated by ssBP proteins. The A protein will cleave the complete genome every time it recognises the origin sequence.
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 structural proteins is 5:5:5:1.The primosome are protein complexes which attach/bind the enzyme helicase on the template. primosomes gives RNA primers for DNA synthesis to strands.
- Sanger, F.; Air, G. M.; Barrell, B. G.; Brown, N. L.; Coulson, A. R.; Fiddes, J. C.; Hutchison, C. A.; Slocombe, P. M.; Smith, M. (1977). "Nucleotide sequence of bacteriophage ΦX174 DNA". Nature 265 (5596): 687–95. Bibcode:1977Natur.265..687S. doi:10.1038/265687a0. PMID 870828.
- Fiers, Walter; Sinsheimer, Robert L. (1962). "The structure of the DNA of bacteriophage ΦX174". Journal of Molecular Biology 5 (4): 424. doi:10.1016/S0022-2836(62)80031-X.
- National Library of Medicine Profiles in Science. The Arthur Kornberg Papers. "Creating Life in the Test Tube," 1959-1970. link[non-primary source needed]
- Goulian, Mehran; Kornberg, Arthur; Sinsheimer, Robert L. (1967). "Enzymatic Synthesis of DNA, XXIV. Synthesis of Infectious Phage ΦX174 DNA". Proceedings of the National Academy of Sciences 58 (6): 2321–2328. Bibcode:1967PNAS...58.2321G. doi:10.1073/pnas.58.6.2321. JSTOR 58720. PMC 223838. PMID 4873588.
- Smith, Hamilton O.; Hutchison, Clyde A.; Pfannkoch, Cynthia; Venter, J. Craig (2003). "Generating a Synthetic Genome by Whole Genome Assembly: ΦX174 Bacteriophage from Synthetic Oligonucleotides". Proceedings of the National Academy of Sciences 100 (26): 15440–5. Bibcode:2003PNAS..10015440S. doi:10.1073/pnas.2237126100. JSTOR 3149024. PMC 307586. PMID 14657399.
- Cherwa, James E.; Organtini, Lindsey J.; Ashley, Robert E.; Hafenstein, Susan L.; Fane, Bentley A. (2011). "In Vitro Assembly of the ΦX174 Procapsid from External Scaffolding Protein Oligomers and Early Pentameric Assembly Intermediates". Journal of Molecular Biology 412 (3): 387–96. doi:10.1016/j.jmb.2011.07.070. PMID 21840317.
- Jaschke, Paul R.; Lieberman, Erica K.; Rodriguez, Jon; Sierra, Adrian; Endy, Drew (2012). "A fully decompressed synthetic bacteriophage ΦX174 genome assembled and archived in yeast". Virology 434 (2): 278–84. doi:10.1016/j.virol.2012.09.020. PMID 23079106.