Phi X 174

{{Short description|Single-stranded DNA virus that infects bacteria}} {{virusbox | name = Bacteriophage phiX174 | image = Bacteriophage_Phi_X_174_Electron_micrograph.gif | image_caption = Electron micrograph of phage ΦX174 | parent = Sinsheimervirus | species = Sinsheimervirus phiX174 }}

thumb|225 px| Structure of phage ΦX174 capsid thumb|225 px|Schematic drawing of a ''Sinsheimervirus'' (aka ''Phix174microvirus'') virion The '''phi X 174''' (or '''ΦX174''') bacteriophage is a single-stranded DNA (ssDNA) virus that infects ''Escherichia coli''. This virus was isolated in 1935 by Nicolas Bulgakov<ref>{{Cite journal |last1=Lacković |first1=Zdravko |last2=Toljan |first2=Karlo |date=2020-12-20 |title=Vladimir Sertić: forgotten pioneer of virology and bacteriophage therapy |journal=Notes and Records: The Royal Society Journal of the History of Science |language=en |volume=74 |issue=4 |pages=567–578 |doi=10.1098/rsnr.2019.0010 |issn=0035-9149 |pmc=7653334 |pmid=33177747}}</ref> in Félix d'Hérelle's laboratory at the Pasteur Institute, from samples collected in Paris sewers. Its characterization and the study of its replication mechanism were carried out from the 1950s onwards. It was the first DNA-based genome to be sequenced. This work was completed by Fred Sanger and his team in 1977.<ref name=":0">{{cite journal | vauthors = Sanger F, Air GM, Barrell BG, Brown NL, Coulson AR, Fiddes CA, Hutchison CA, Slocombe PM, Smith M | display-authors = 6 | title = Nucleotide sequence of bacteriophage phi X174 DNA | journal = Nature | volume = 265 | issue = 5596 | pages = 687–95 | date = February 1977 | pmid = 870828 | doi = 10.1038/265687a0 | s2cid = 4206886 | bibcode = 1977Natur.265..687S }}</ref> In 1962, Walter Fiers and Robert L. Sinsheimer had already demonstrated the physical, covalently closed circularity of ΦX174 DNA.<ref name="pmid13945085">{{cite journal | vauthors = Fiers W, Sinsheimer RL | title = The structure of the DNA of bacteriophage phi-X174. III. Ultracentrifugal evidence for a ring structure | journal = Journal of Molecular Biology | volume = 5 | issue = 4 | pages = 424–34 | date = October 1962 | pmid = 13945085 | doi = 10.1016/S0022-2836(62)80031-X }}</ref> 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.<ref>National Library of Medicine Profiles in Science. The Arthur Kornberg Papers. "Creating Life in the Test Tube," 1959-1970. [https://web.archive.org/web/20110810163854/http://profiles.nlm.nih.gov/ps/retrieve/Narrative/WH/p-nid/209/p-docs/true link]{{primary source inline|date=February 2013}}</ref><ref>{{cite journal | vauthors = Goulian M, Kornberg A, Sinsheimer RL | title = Enzymatic synthesis of DNA, XXIV. Synthesis of infectious phage phi-X174 DNA | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 58 | issue = 6 | pages = 2321–8 | date = December 1967 | pmid = 4873588 | pmc = 223838 | doi = 10.1073/pnas.58.6.2321 | bibcode = 1967PNAS...58.2321G | jstor = 58720 | doi-access = free }}</ref> In 1972–1974, Jerard Hurwitz, Sue Wickner, and Reed Wickner with collaborators identified the genes required to produce the enzymes to catalyze conversion of the single stranded form of the virus to the double stranded replicative form.<ref name="pmid4610569">{{cite journal | vauthors = Wickner S, Hurwitz J | title = Conversion of phiX174 viral DNA to double-stranded form by purified Escherichia coli proteins | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 71 | issue = 10 | pages = 4120–4 | date = October 1974 | pmid = 4610569 | pmc = 434340 | doi = 10.1073/pnas.71.10.4120 | doi-access = free | bibcode = 1974PNAS...71.4120W }}</ref> 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.<ref>{{cite journal | vauthors = Smith HO, Hutchison CA, Pfannkoch C, Venter JC | title = Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 100 | issue = 26 | pages = 15440–5 | date = December 2003 | pmid = 14657399 | pmc = 307586 | doi = 10.1073/pnas.2237126100 | bibcode = 2003PNAS..10015440S | jstor = 3149024 | doi-access = free }}</ref> The ΦX174 virus particle has also been successfully assembled ''in vitro''.<ref>{{cite journal | vauthors = Cherwa JE, Organtini LJ, Ashley RE, Hafenstein SL, Fane BA | title = In VITRO ASSEMBLY of the øX174 procapsid from external scaffolding protein oligomers and early pentameric assembly intermediates | journal = Journal of Molecular Biology | volume = 412 | issue = 3 | pages = 387–96 | date = September 2011 | pmid = 21840317 | doi = 10.1016/j.jmb.2011.07.070 }}</ref> In 2012, it was shown how its highly overlapping genome can be fully decompressed and still remain functional.<ref name=":1">{{cite journal | vauthors = Jaschke PR, Lieberman EK, Rodriguez J, Sierra A, Endy D | title = A fully decompressed synthetic bacteriophage øX174 genome assembled and archived in yeast | journal = Virology | volume = 434 | issue = 2 | pages = 278–84 | date = December 2012 | pmid = 23079106 | doi = 10.1016/j.virol.2012.09.020 | doi-access = free }}</ref> {{Clear|left}}

==Genome== [[File:Genome map of the bacteriophage ΦX174 showing overlapping genes.svg|thumb|right|250px|Genome of the bacteriophage ΦX174 showing its 11 genes <ref name="NCBI NC_001422">Enterobacteria phage phiX174 ''sensu lato'', complete genome. [https://www.ncbi.nlm.nih.gov/nuccore/NC_001422 "Complete genome: accession NC_001422"], ''National Center for Biotechnology Information''. Retrieved on 30 January 2016.</ref>]] This bacteriophage has a [+] sense circular single-stranded DNA genome of 5,386 nucleotides.<ref name="NCBI NC_001422" /> The genome GC-content is 44% and 95% of nucleotides belong to coding genes. Because of the balance base pattern of the genome, it is used as the control DNA for Illumina sequencers.{{citation needed|date=November 2022}}

===Genes=== ΦX174 encodes 11 genes, named as consecutive letters of the alphabet in the order they were discovered, with the exception of A* which is an alternative start codon within the large A genes. Only genes A* and K are thought to be non-essential, although there is some doubt about A* because its start codon could be changed to ATT but not any other sequence.<ref>{{cite journal | vauthors = Baas PD, Liewerink H, van Teeffelen HA, van Mansfeld AD, van Boom JH, Jansz HS | title = Alteration of the ATG start codon of the A protein of bacteriophage phi X174 into an ATT codon yields a viable phage indicating that A protein is not essential for phi X174 reproduction | journal = FEBS Letters | volume = 218 | issue = 1 | pages = 119–25 | date = June 1987 | pmid = 2954853 | doi = 10.1016/0014-5793(87)81030-x | s2cid = 24174007 | doi-access = free }}</ref> It is now known that the ATT is still likely capable of producing protein within ''E. coli''<ref>{{cite journal | vauthors = Hecht A, Glasgow J, Jaschke PR, Bawazer LA, Munson MS, Cochran JR, Endy D, Salit M | display-authors = 6 | title = Measurements of translation initiation from all 64 codons in E. coli | journal = Nucleic Acids Research | volume = 45 | issue = 7 | pages = 3615–3626 | date = April 2017 | pmid = 28334756 | pmc = 5397182 | doi = 10.1093/nar/gkx070 }}</ref> and therefore this gene may in fact be essential.

The first half of the ΦX174 genome features high levels of gene overlap<ref name=":2">{{Cite journal|last1=Wright|first1=Bradley W.|last2=Molloy|first2=Mark P.|last3=Jaschke|first3=Paul R.|date=2021-10-05|title=Overlapping genes in natural and engineered genomes|journal=Nature Reviews Genetics|volume=23 |issue=3 |language=en|pages=154–168|doi=10.1038/s41576-021-00417-w|issn=1471-0064|pmc=8490965|pmid=34611352}}</ref> with eight out of 11 genes overlapping by at least one nucleotide.<ref name=":0" /> These overlaps have been shown to be non-essential<ref name=":1" /> although the refactored phage with all gene overlaps removed had decreased fitness from wild-type.<ref name=":3" />

Phage ΦX174 has been used to try to establish the absence of undiscovered genetic information through a "proof by synthesis" approach.<ref>{{cite journal | vauthors = Jaschke PR, Dotson GA, Hung KS, Liu D, Endy D | title = Definitive demonstration by synthesis of genome annotation completeness | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 116 | issue = 48 | pages = 24206–24213 | date = November 2019 | pmid = 31719208 | pmc = 6883844 | doi = 10.1073/pnas.1905990116 | doi-access = free | bibcode = 2019PNAS..11624206J }}</ref>

===Transcriptome=== In 2020, the transcriptome of ΦX174 was generated.<ref>{{cite journal | vauthors = Logel DY, Jaschke PR | title = A high-resolution map of bacteriophage ϕX174 transcription | journal = Virology | volume = 547 | pages = 47–56 | date = August 2020 | pmid = 32560904 | doi = 10.1016/j.virol.2020.05.008 | s2cid = 219459208 | doi-access = free }}</ref> Notable features of the ΦX174 transcriptome is a series of up to four relatively weak promoters in series with up to four Rho-independent (intrinsic) terminators and one Rho-dependent terminator.{{citation needed|date=November 2022}}

===Proteins=== ΦX174 encodes 11 proteins.

{| class="wikitable" |- ! Protein !! Copies !! Function<ref>{{cite book |last1=Fane |first1=Bentley A. |last2=Brentlinger |first2=Karie L. |last3=Burch |first3=April D. |last4=Chen |first4=Min |last5=Hafenstein |first5=Susan |last6=Moore |first6=Erica |last7=Novak |first7=Christopher R. |last8=Uchiyama |first8=Asako |editor1-last=Calender |editor1-first=Richard | name-list-style = vanc |title=The Bacteriophages |date=2006 |publisher=Oxford Univ. Press |location=New York |isbn=978-0195148503 |page=130 |edition=2nd |chapter=ɸX174 et al., the ''Microviridae'' }}</ref> |- | A || — || Nicks RF DNA to initiate rolling circle replication; ligates ends of linear phage DNA to form single-stranded circular DNA |- | A* || — || Inhibits host cell DNA replication; blocks superinfecting phage; not essential |- | B || 60 in procapsid || Internal scaffolding protein involved in procapsid assembly |- | C || — || DNA packaging |- | D || 240 in procapsid || External scaffolding protein involved in procapsid assembly |- | E || — || Host cell lysis |- | F || 60 in virion || Major capsid protein |- | G || 60 in virion || Major spike protein |- | H || 12 in virion || DNA pilot protein (or minor spike protein) |- | J || 60 in virion || Binds to new single-stranded phage DNA; accompanies phage DNA into procapsid |- | K || — || Optimizes burst size; not essential |}

=== Proteome === Identification of all ΦX174 proteins using mass spectrometry has recently been reported.<ref name=":3">{{cite journal | vauthors = Wright BW, Ruan J, Molloy MP, Jaschke PR | title = Genome Modularization Reveals Overlapped Gene Topology Is Necessary for Efficient Viral Reproduction | journal = ACS Synthetic Biology | volume = 9 | issue = 11 | pages = 3079–3090 | date = November 2020 | pmid = 33044064 | doi = 10.1021/acssynbio.0c00323 | s2cid = 222300240 }}</ref>

==Infection cycle== 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<ref>{{cite journal | vauthors = Jazwinski SM, Lindberg AA, Kornberg A | title = The lipopolysaccharide receptor for bacteriophage phiX174 and S13 | journal = Virology | volume = 66 | issue = 1 | pages = 268–82 | date = July 1975 | pmid = 1094681 | doi = 10.1016/0042-6822(75)90197-x }}</ref> most likely via a predicted N-terminal transmembrane domain helix.<ref>{{cite journal | vauthors = Tusnády GE, Simon I | title = The HMMTOP transmembrane topology prediction server | journal = Bioinformatics | volume = 17 | issue = 9 | pages = 849–50 | date = September 2001 | pmid = 11590105 | doi = 10.1093/bioinformatics/17.9.849 | doi-access = free }}</ref> However, it has become apparent that H protein is a multifunctional protein.<ref>{{cite journal | vauthors = Cherwa JE, Young LN, Fane BA | title = Uncoupling the functions of a multifunctional protein: the isolation of a DNA pilot protein mutant that affects particle morphogenesis | journal = Virology | volume = 411 | issue = 1 | pages = 9–14 | date = March 2011 | pmid = 21227478 | doi = 10.1016/j.virol.2010.12.026 | doi-access = free }}</ref> 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 that ''de novo'' H protein was required for optimal synthesis of other viral proteins.<ref>{{cite journal | vauthors = Ruboyianes MV, Chen M, Dubrava MS, Cherwa JE, Fane BA | title = The expression of N-terminal deletion DNA pilot proteins inhibits the early stages of phiX174 replication | journal = Journal of Virology | volume = 83 | issue = 19 | pages = 9952–6 | date = October 2009 | pmid = 19640994 | pmc = 2748053 | doi = 10.1128/JVI.01077-09 }}</ref> Mutations in H protein that prevent viral incorporation, can be overcome when excess amounts of protein B, the internal scaffolding protein, are supplied.{{citation needed|date=November 2022}}

The DNA is ejected through a hydrophilic channel at the 5-fold vertex.<ref>{{cite journal | vauthors = McKenna R, Xia D, Willingmann P, Ilag LL, Krishnaswamy S, Rossmann MG, Olson NH, Baker TS, Incardona NL | display-authors = 6 | title = Atomic structure of single-stranded DNA bacteriophage phi X174 and its functional implications | journal = Nature | volume = 355 | issue = 6356 | pages = 137–43 | date = January 1992 | pmid = 1370343 | pmc = 4167681 | doi = 10.1038/355137a0 | bibcode = 1992Natur.355..137M }}</ref> 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 synthesizes 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 positive strand by a virus-encoded A protein, also attracting a bacterial DNA polymerase (DNAP) to the site of cleavage. DNAP uses 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 cleaves the complete genome every time it recognises the origin sequence.{{citation needed|date=November 2022}}

As D protein is the most abundant gene transcript, it is the most abundant protein in the viral procapsid. 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 primosomes are protein complexes which attach/bind the enzyme helicase on the template. Primosomes gives RNA primers for DNA synthesis to strands.{{citation needed|date=November 2022}}

===Mutation rate===

The mutation rate of phiX174 is estimated to be 1.0 × 10<sup>−6</sup> substitutions per base per round of copying, a value that is consistent with Drake's rule (0.003 mutations per genome per round of copying in DNA-based microorganisms).<ref>{{cite journal |vauthors=Cuevas JM, Duffy S, Sanjuán R |title=Point mutation rate of bacteriophage PhiX174 |journal=Genetics |volume=183 |issue=2 |pages=747–9 |date=October 2009 |pmid=19652180 |pmc=2766332 |doi=10.1534/genetics.109.106005 |url=}}</ref>

===Recombination===

PhiX174 is able to undergo genetic recombination. Based on recombination frequencies obtained in genetic crosses, a genetic map was constructed.<ref name = Benbow1971>{{cite journal |vauthors=Benbow RM, Hutchison CA, Fabricant JD, Sinsheimer RL |title=Genetic Map of Bacteriophage phiX174 |journal=J Virol |volume=7 |issue=5 |pages=549–58 |date=May 1971 |pmid=16789129 |pmc=356162 |doi=10.1128/JVI.7.5.549-558.1971 |url=}}</ref> Recombination in phi X174 is associated with high negative interference, i.e., a positive correlation (negative interference) of recombinational events (see wikipedia crossover interference).<ref name = Benbow1971/>

== Phylogenetics and diversity == ΦX174 is closely related to other microviruses, especially the NC phage (e.g. NC1, NC7, NC11, NC16, NC37, NC5, NC41, NC56, NC51, etc.) and more distantly related to the G4-like phages and even more distantly related to the α3-like phage. Rokyta et al. 2006 presented a phylogenetic tree of their relationships.<ref>{{cite journal | vauthors = Rokyta DR, Burch CL, Caudle SB, Wichman HA | title = Horizontal gene transfer and the evolution of microvirid coliphage genomes | journal = Journal of Bacteriology | volume = 188 | issue = 3 | pages = 1134–42 | date = February 2006 | pmid = 16428417 | pmc = 1347346 | doi = 10.1128/JB.188.3.1134-1142.2006 }}</ref>

==Uses==

===Experimental evolution=== ΦX174 has been used as a model organism in many evolution experiments.<ref>{{cite journal | vauthors = Wichman HA, Brown CJ | title = Experimental evolution of viruses: Microviridae as a model system | journal = Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences | volume = 365 | issue = 1552 | pages = 2495–501 | date = August 2010 | pmid = 20643739 | pmc = 2935103 | doi = 10.1098/rstb.2010.0053 }}</ref>

===Biotechnology=== ΦX174 is regularly used as a positive control in DNA sequencing due to its relatively small genome size in comparison to other organisms, its relatively balanced nucleotide content — about 23% G, 22% C, 24% A, and 31% T, i.e., 45% G+C and 55% A+T, see the accession NC_001422.1<ref name="NCBI NC_001422"/> for its 5,386 nucleotide long sequence. Illumina's sequencing instruments use ΦX174 as a positive control,<ref name="Illumina tech note">{{cite web |title=Using a PhiX Control for HiSeq® Sequencing Runs |url=https://support.illumina.com/sequencing/sequencing_kits/phix_control_v3.html |publisher=Illumina |access-date=8 January 2019 |archive-url=https://web.archive.org/web/20190109062332/https://support.illumina.com/sequencing/sequencing_kits/phix_control_v3.html |archive-date=9 January 2019 |url-status=dead }}</ref> and a single Illumina sequencing run can cover the ΦX174 genome several million times over, making this very likely the most heavily sequenced genome in history.{{Citation needed|date=March 2020}}

ΦX174 is also used to test the resistance of personal protective equipment to bloodborne viruses.<ref>{{Cite web|url=https://wwwn.cdc.gov/PPEInfo/Standards/Info/ASTMF181907(2013)|title=PPE-Info – Standard Details|website=wwwn.cdc.gov|access-date=2019-02-08|archive-date=3 October 2020|archive-url=https://web.archive.org/web/20201003024053/https://wwwn.cdc.gov/PPEInfo/Standards/Info/ASTMF181907(2013)|url-status=dead}}</ref>

ΦX174 has also been modified to enable peptide display (phage display) from the viral capsid G protein.<ref>{{cite journal | vauthors = Christakos KJ, Chapman JA, Fane BA, Campos SK | title = PhiXing-it, displaying foreign peptides on bacteriophage ΦX174 | journal = Virology | volume = 488 | pages = 242–8 | date = January 2016 | pmid = 26655242 | pmc = 6191337 | doi = 10.1016/j.virol.2015.11.021 }}</ref>

=== Synthetic biology === The ΦX174 genome was the first phage to be cloned in yeast,<ref name=":1"/> which provides a convenient drydock for genome modifications.<ref>{{cite journal | vauthors = Ando H, Lemire S, Pires DP, Lu TK | title = Engineering Modular Viral Scaffolds for Targeted Bacterial Population Editing | journal = Cell Systems | volume = 1 | issue = 3 | pages = 187–196 | date = September 2015 | pmid = 26973885 | pmc = 4785837 | doi = 10.1016/j.cels.2015.08.013 }}</ref> ΦX174 was also the first genome to be fully decompressed, having all gene overlaps removed.<ref name=":2" /> The effect of these changes resulted in significantly reduced host attachment, protein expression dysregulation, and heat sensitivity.<ref name=":3"/>

== See also == {{Portal|Viruses}} * Bacteriophage MS2

== References == {{Reflist|2}}

== External links ==  * {{cite web |url=http://www.rcsb.org/pdb/101/motm.do?momID=2 |title=Bacteriophage phiX174 |date=February 2000 |work=Molecule of the Month | vauthors = Goodsell D |publisher=RCSB-PDB}}

Category:Microviridae Category:Bacteriophages