''Bacillus virus Φ29'' (bacteriophage Φ29) is a double-stranded DNA (dsDNA)

bacteriophage A bacteriophage (), also known informally as a ''phage'' (), is a duplodnaviria virus that infects and replicates within bacteria and archaea. The term was derived from "bacteria" and the Greek φαγεῖν ('), meaning "to devour". Bacteri ...

with a

prolate A spheroid, also known as an ellipsoid of revolution or rotational ellipsoid, is a quadric surface obtained by rotating an ellipse about one of its principal axes; in other words, an ellipsoid with two equal semi-diameters. A spheroid has circu ...

icosahedral In geometry, an icosahedron ( or ) is a polyhedron with 20 faces. The name comes and . The plural can be either "icosahedra" () or "icosahedrons". There are infinitely many non- similar shapes of icosahedra, some of them being more symmetrica ...

head and a short tail that belongs to the genus ''

Salasvirus ''Salasvirus'' (synonyms ''Phi29-like phages'', ''Phi29-like viruses'', ''Phi29likevirus'') is a genus of viruses in the order ''Caudovirales'', in the family '' Salasmaviridae'', in the subfamily ''Picovirinae''. Bacteria serve as natural hosts. ...

'', order ''

Caudovirales ''Caudovirales'' is an order of viruses known as the tailed bacteriophages (''cauda'' is Latin for "tail"). Under the Baltimore classification scheme, the ''Caudovirales'' are group I viruses as they have double stranded DNA (dsDNA) genomes, ...

'', and family ''Salasmaviridae''. They are in the same order as phages PZA, Φ15, BS32, B103, M2Y (M2), Nf, and GA-1. First discovered in 1965, the Φ29 phage is the smallest ''

Bacillus ''Bacillus'' (Latin "stick") is a genus of Gram-positive, rod-shaped bacteria, a member of the phylum ''Bacillota'', with 266 named species. The term is also used to describe the shape (rod) of other so-shaped bacteria; and the plural ''Bacilli ...

'' phage isolated to date and is among the smallest known dsDNA phages. Φ29 has a unique DNA packaging motor structure that employs prohead packaging RNA (pRNA) to guide the translocation of the phage

genome In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...

during replication. This novel structure system has inspired ongoing research in

nanotechnology Nanotechnology, also shortened to nanotech, is the use of matter on an atomic, molecular, and supramolecular scale for industrial purposes. The earliest, widespread description of nanotechnology referred to the particular technological goal o ...

drug delivery Drug delivery refers to approaches, formulations, manufacturing techniques, storage systems, and technologies involved in transporting a pharmaceutical compound to its target site to achieve a desired therapeutic effect. Principles related to d ...

, and

therapeutics A therapy or medical treatment (often abbreviated tx, Tx, or Tx) is the attempted remediation of a health problem, usually following a medical diagnosis. As a rule, each therapy has indications and contraindications. There are many different ...

. In nature, the Φ29 phage infects ''

Bacillus subtilis ''Bacillus subtilis'', known also as the hay bacillus or grass bacillus, is a Gram-positive, catalase-positive bacterium, found in soil and the gastrointestinal tract of ruminants, humans and marine sponges. As a member of the genus ''Bacillu ...

'', a species of

gram-positive In bacteriology, gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their type of cell wall. Gram-positive bacte ...

, endospore-forming bacteria that is found in

soil Soil, also commonly referred to as earth or dirt, is a mixture of organic matter, minerals, gases, liquids, and organisms that together support life. Some scientific definitions distinguish ''dirt'' from ''soil'' by restricting the former te ...

, as well as the

gastrointestinal tracts The gastrointestinal tract (GI tract, digestive tract, alimentary canal) is the tract or passageway of the digestive system that leads from the mouth to the anus. The GI tract contains all the major organs of the digestive system, in humans and ...

of various

marine Marine is an adjective meaning of or pertaining to the sea or ocean. Marine or marines may refer to: Ocean * Maritime (disambiguation) * Marine art * Marine biology * Marine debris * Marine habitats * Marine life * Marine pollution Military * ...

and terrestrial organisms, including

human beings Humans (''Homo sapiens'') are the most abundant and widespread species of primate, characterized by bipedalism and exceptional cognitive skills due to a large and complex brain. This has enabled the development of advanced tools, culture, an ...

History

In 1965,

American American(s) may refer to: * American, something of, from, or related to the United States of America, commonly known as the "United States" or "America" ** Americans, citizens and nationals of the United States of America ** American ancestry, pe ...

microbiologist A microbiologist (from Ancient Greek, Greek ) is a scientist who studies microscopic life forms and processes. This includes study of the growth, interactions and characteristics of Microorganism, microscopic organisms such as bacteria, algae, f ...

Dr. Bernard Reilly discovered the Φ29 phage in Dr. John Spizizen’s lab at the

University of Minnesota The University of Minnesota, formally the University of Minnesota, Twin Cities, (UMN Twin Cities, the U of M, or Minnesota) is a public university, public Land-grant university, land-grant research university in the Minneapolis–Saint Paul, Tw ...

. Due to its small size and complex morphology, it has become an ideal model for the study of many processes in

molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...

, such as

morphogenesis Morphogenesis (from the Greek ''morphê'' shape and ''genesis'' creation, literally "the generation of form") is the biological process that causes a cell, tissue or organism to develop its shape. It is one of three fundamental aspects of devel ...

, viral

DNA packaging A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...

, viral replication, and

transcription Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including: Genetics * Transcription (biology), the copying of DNA into RNA, the fir ...

Structure

The structure of Φ29 is composed of seven main

proteins Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...

: the terminal protein (p3), the head or

capsid protein A capsid is the protein shell of a virus, enclosing its genetic material. It consists of several oligomeric (repeating) structural subunits made of protein called protomers. The observable 3-dimensional morphological subunits, which may or may ...

(p8), the head or capsid fiber protein (p8.5), the distal tail knob (p9), the portal or connector protein (p10), the tail tube or lower collar proteins (p11), and the tail fibers or appendage proteins (p12*). The main difference between Φ29's structure and that of other phages is its use of pRNA in its DNA packaging motor.

DNA packaging motor

The Φ29 DNA packaging motor packages the phage genome into the

procapsid A capsid is the protein shell of a virus, enclosing its genetic material. It consists of several oligomeric (repeating) structural subunits made of protein called protomers. The observable 3-dimensional morphological subunits, which may or may ...

during viral replication. The Φ29 packaging motor is structurally composed of the procapsid and the connector proteins, which interact with the pRNA, the packaging

enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...

(gp16), and the packaging substrate (genomic DNA-gp3). Because the process of genome packaging is

energy In physics, energy (from Ancient Greek: ἐνέργεια, ''enérgeia'', “activity”) is the quantitative property that is transferred to a body or to a physical system, recognizable in the performance of work and in the form of heat a ...

-intensive, it must be facilitated by an

ATP ATP may refer to: Companies and organizations * Association of Tennis Professionals, men's professional tennis governing body * American Technical Publishers, employee-owned publishing company * ', a Danish pension * Armenia Tree Project, non ...

-powered motor that converts

chemical energy Chemical energy is the energy of chemical substances that is released when they undergo a chemical reaction and transform into other substances. Some examples of storage media of chemical energy include batteries, Schmidt-Rohr, K. (2018). "How ...

mechanical energy In Outline of physical science, physical sciences, mechanical energy is the sum of potential energy and kinetic energy. The principle of conservation of mechanical energy states that if an isolated system is subject only to conservative forces, t ...

through

ATP hydrolysis ATP hydrolysis is the catabolic reaction process by which chemical energy that has been stored in the high-energy phosphoanhydride bonds in adenosine triphosphate (ATP) is released after splitting these bonds, for example in muscles, by prod ...

. The Φ29 packaging motor is able to generate approximately 57 piconewtons (pN) of

force In physics, a force is an influence that can change the motion of an object. A force can cause an object with mass to change its velocity (e.g. moving from a state of rest), i.e., to accelerate. Force can also be described intuitively as a p ...

, making it one of the most powerful biomotors studied to date.

pRNA

The Φ29 pRNA is a highly versatile

molecule A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and bioch ...

that can

polymerize In polymer chemistry, polymerization (American English), or polymerisation (British English), is a process of reacting monomer molecules together in a chemical reaction to form polymer chains or three-dimensional networks. There are many for ...

into dimers, trimers,

tetramers A tetramer () (''tetra-'', "four" + '' -mer'', "parts") is an oligomer formed from four monomers or subunits. The associated property is called ''tetramery''. An example from inorganic chemistry is titanium methoxide with the empirical formula Ti ...

, pentamers, and

hexamers In chemistry and biochemistry, an oligomer () is a molecule that consists of a few repeating units which could be derived, actually or conceptually, from smaller molecules, monomers.Quote: ''Oligomer molecule: A molecule of intermediate relative ...

. Early studies such as Anderson (1990) and Trottier (1998) hypothesized that pRNA formed intermolecular hexamers, but these studies had a solely genetic basis rather than a

microscopy Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of micr ...

based approach. In the year 2000, a study by Simpson et al. employed

cryo-electron microscopy Cryogenic electron microscopy (cryo-EM) is a cryomicroscopy technique applied on samples cooled to cryogenic temperatures. For biological specimens, the structure is preserved by embedding in an environment of vitreous ice. An aqueous sample sol ...

to determine that, ''

in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...

'', only a pentamer or smaller polymer could spatially fit in the virus. Ultimately, single isomorphous replacement with

anomalous scattering Anomalous X-ray scattering (AXRS or XRAS) is a non-destructive determination technique within X-ray diffraction that makes use of the anomalous dispersion that occurs when a wavelength is selected that is in the vicinity of an absorption edge of one ...

(SIRAS)

crystallography Crystallography is the experimental science of determining the arrangement of atoms in crystalline solids. Crystallography is a fundamental subject in the fields of materials science and solid-state physics (condensed matter physics). The wor ...

was used to determine that the ''in vivo'' structure is a tetramer ring. This discovery aligned with what was known about the structural geometry and necessary

flexibility Stiffness is the extent to which an object resists deformation in response to an applied force. The complementary concept is flexibility or pliability: the more flexible an object is, the less stiff it is. Calculations The stiffness, k, of a bo ...

of the packaging motor’s three-way junction. When pRNA is in this tetramer ring form, it works as a part of the DNA packaging motor to transport DNA molecules to their destination location within the prohead capsule. Specifically, the functional domains of pRNA bind to the gp16 packaging enzyme and the structural connector molecule to aid in the translocation of DNA through the prohead channel. After DNA packaging is complete, the pRNA dissociates and is degraded.

Genome and replication

The Φ29 phage has a linear dsDNA genome consisting of 19,285 bases. Both 5’ ends of the genome are capped with a

covalently bonded A covalent bond is a chemical bond that involves the sharing of electrons to form electron pairs between atoms. These electron pairs are known as shared pairs or bonding pairs. The stable balance of attractive and repulsive forces between atoms ...

terminal protein (p3) that complexes with

DNA polymerase A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...

during replication. Φ29 is one of many phages with a DNA

polymerase A polymerase is an enzyme ( EC 2.7.7.6/7/19/48/49) that synthesizes long chains of polymers or nucleic acids. DNA polymerase and RNA polymerase are used to assemble DNA and RNA molecules, respectively, by copying a DNA template strand using base- ...

that has a different structure and function compared to standard DNA polymerases in other organisms. Φ29 forms a replication complex involving the p3 terminal protein, the dAMP nucleotide, and its own DNA polymerase to synthesize DNA in a 5’ to 3’ direction. This replication process also employs a sliding-back mechanism towards the 3’ end of the genome that uses a repeating TTT motif to move the replication complex backward without altering the template sequence. This allows the initiation of

DNA replication In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritanc ...

to be more accurate by having the polymerase complex check a specific sequence before beginning the elongation process.

Applications

Targeting_of_TNBC_molecules_by_bacteriophage_phi29_pRNA

Nanoparticle assembly

Versatility in

RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...

structure and function provides the ability to assemble

nanoparticles A nanoparticle or ultrafine particle is usually defined as a particle of matter that is between 1 and 100 nanometres (nm) in diameter. The term is sometimes used for larger particles, up to 500 nm, or fibers and tubes that are less than 1 ...

for nanomedicinal

. The pRNA in bacteriophage Φ29 can use its three-way junction in order to self-assemble into nanoparticles. One major challenge of using pRNA-derived nanoparticles is large-scale production, as most industries are currently unequipped to handle industrial pRNA synthesis. This is primarily because RNA nanotechnology is still an emerging field that lacks industrial application and manufacturing optimization of small RNAs.

Drug delivery

Φ29’s DNA packaging system, using pRNA, incorporates a motor for the delivery of therapeutic molecules like

ribozymes Ribozymes (ribonucleic acid enzymes) are RNA molecules that have the ability to catalyze specific biochemical reactions, including RNA splicing in gene expression, similar to the action of protein enzymes. The 1982 discovery of ribozymes demonst ...

and aptamers. The small size of pRNA-derived nanoparticles also helps to deliver

drugs A drug is any chemical substance that causes a change in an organism's physiology or psychology when consumed. Drugs are typically distinguished from food and substances that provide nutritional support. Consumption of drugs can be via inhalat ...

in tight spaces like

blood vessels The blood vessels are the components of the circulatory system that transport blood throughout the human body. These vessels transport blood cells, nutrients, and oxygen to the tissues of the body. They also take waste and carbon dioxide away f ...

. The main difficulty in using aptamer-based drug delivery is sourcing unique aptamers and other multimers for specific treatments for

diseases A disease is a particular abnormal condition that negatively affects the structure or function of all or part of an organism, and that is not immediately due to any external injury. Diseases are often known to be medical conditions that ar ...

that potentially degrade therapeutic multimers and nanoparticles ''in vivo.'' Nanoparticles need to be stabilized as delivery mechanisms in order to adapt to microenvironments that may result in loss of therapeutic cargo.

Triple-negative breast cancer treatment

Triple-negative breast cancer Triple-negative breast cancer (TNBC) is any breast cancer that lacks or show low levels of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) overexpression and/or gene amplification (i.e. the tum ...

(TNBC) is an aggressive form of

breast cancer Breast cancer is cancer that develops from breast tissue. Signs of breast cancer may include a lump in the breast, a change in breast shape, dimpling of the skin, milk rejection, fluid coming from the nipple, a newly inverted nipple, or a re ...

that accounts for ten to fifteen percent of all breast cancer cases.

Chemotherapy Chemotherapy (often abbreviated to chemo and sometimes CTX or CTx) is a type of cancer treatment that uses one or more anti-cancer drugs (chemotherapeutic agents or alkylating agents) as part of a standardized chemotherapy regimen. Chemotherap ...

is the only viable current treatment for TNBC because the loss of target receptors inherent to the disease causes

cancer cells Cancer cells are cells that divide continually, forming solid tumors or flooding the blood with abnormal cells. Cell division is a normal process used by the body for growth and repair. A parent cell divides to form two daughter cells, and these d ...

to resist therapeutic

pharmaceuticals A medication (also called medicament, medicine, pharmaceutical drug, medicinal drug or simply drug) is a drug used to diagnose, cure, treat, or prevent disease. Drug therapy (pharmacotherapy) is an important part of the medical field and rel ...

. The three-way junction in the Φ29 DNA packaging motor can help sensitize TNBC cells to chemotherapy using a

siRNA Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20-24 (normally 21) base pairs in length, similar to miRNA, and operating wi ...

drug delivery mechanism to inhibit TNBC growth and volume. This treatment can also be combined with anti-cancer drugs like

Doxorubicin Doxorubicin, sold under the brand name Adriamycin among others, is a chemotherapy medication used to treat cancer. This includes breast cancer, bladder cancer, Kaposi's sarcoma, lymphoma, and acute lymphocytic leukemia. It is often used togeth ...

to enhance therapeutic effects.

References

{{DEFAULTSORT:Phi 29 Phage Model organisms Podoviridae Bacillus phages