Q.19 Write short note on the following -
(a) replication in plant virus (2006)
(b) transduction (2006, 13)
(c) tobacco mosaic virus (2009, 06,15)
(d) episomes (2008, 2011)
(e) shape and symmetry of virus (2008)
(f) viroids (2009, 2011)
Ans. (a) Replication in Plant Virus: -
Replication: -
A single virus particle (virion) is in and of itself essentially inert. It lacks needed components that cells have to reproduce. Viruses are intracellular obligate parasites which means that they cannot reproduce or express their genes without the help of a living cell.
Once a virus has “infected” a cell, it will “marshal” the cell’s ribosomes, enzymes and much of the cellular machinery to reproduce. Unlike what we have seen in mitosis and meiosis, viral reproduction produces many, many progeny, that when complete, leave the host cell to infect other cells in the organism.
Bacteriophage binding to the cell wall of a bacterium.
Bacteriophage injecting its genetic material into the bacterium.
The exact nature of what happens after the host is infected varies depending on the nature of the virus. In most cases, the process depends on the form of the genome. The process for double-stranded DNA, single-stranded DNA, double-stranded RNA and single-stranded RNA will differ.
The bacteriophage genome replicates.
Self-Assembly: -
Interestingly enough, once the viral progeny components are produced by the cellular machinery, the assembly of the viral genome and the viral capsids is a non-enzymatic process. It is usually spontaneous.
The bacteriophage components and enzymes continue to be produced.
The components of the bacteriophage assemble.
Bacteriophage enzyme breaks down the bacterial cell wall causing the bacterium to split open.
Specificity: -
The “lock and key” mechanism is the most common explanation for this range. Certain proteins on the virus particle must fit certain receptor sites on the particular host’s cell surface. Once a virus has “infected” a cell, it will “marshal” the cell’s ribosomes, enzymes and much of the cellular machinery to reproduce. Unlike what we have seen in mitosis and meiosis, viral reproduction produces many, many progeny, that when complete, leave the host cell to infect other cells in the organism.
Bacteriophage binding to the cell wall of a bacterium.
Bacteriophage injecting its genetic material into the bacterium.
The exact nature of what happens after the host is infected varies depending on the nature of the virus. In most cases, the process depends on the form of the genome. The process for double-stranded DNA, single-stranded DNA, double-stranded RNA and single-stranded RNA will differ.
The bacteriophage genome replicates.
(b) Transduction: -
Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. This is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell’s genome.
When bacteriophages (viruses that infect bacteria) infect a bacterial cell, their normal mode of reproduction is to harness the replicational, transcriptional, and translation machinery of the host bacterial cell to make numerous virions, or complete viral particles, including the viral DNA or RNA and the protein coat.
Lytic and Lysogenic (Temperate) Cycles: -
Transduction happens through either the lytic cycle or the lysogenic cycle.
If the lysogenic cycle is adopted, the phage chromosome is integrated into the bacterial chromosome, where it can remain dormant for thousands of generations. If the lysogen is induced (by UV light for example), the phage genome is excised from the bacterial chromosome and initiates the lytic cycle, which culminates in lysis of the cell and the release of phage particles. The lytic cycle leads to the production of new phage particles which are released by lysis of the host.
Transduction as a Method of Transfer Genetic Material: -
The packaging of bacteriophage DNA has low fidelity and small pieces of bacterial DNA, together with the bacteriophage genome, may become packaged into the bacteriophage genome. At the same time, some phage genes are left behind in the bacterial chromosome.
There are generally two types of recombination events that can lead to this incorporation of bacterial DNA into the viral DNA, leading to two modes of recombination.
Generalized Transduction: -
Generalised transduction may occur in two main ways, recombination and headful packaging.
If bacteriophages undertake the lytic cycle of infection upon entering a bacterium, the virus will take control of the cell’s machinery for use in replicating its own viral DNA. If by chance bacterial chromosomal DNA is inserted into the viral capsid used to contain the viral DNA, while this lytic pathway is proceeding, the mistake will lead to generalized transduction.
If the virus replicates using ‘headful packaging’, it attempts to fill the nucleocapsid with genetic material. If the viral genome results in spare capacity, viral packaging mechanisms may incorporate bacterial genetic material into the new virion.
The new virus capsule now loaded with part bacterial DNA continues to infect another bacterial cell. This bacterial material may become recombined into another bacterium upon infection.
When the new DNA is inserted into this recipient cell it can fall to one of three fates
(1) The DNA will be absorbed by the cell and be recycled for spare parts.
(2) If the DNA was originally a plasmid, it will re-circularize inside the new cell and become a plasmid again.
(3) If the new DNA matches with a homologous region of the recipient cell’s chromosome, it will exchange DNA material similar to the actions in conjugation.
This type of recombination is random and the amount recombined depends on the size of the virus being used.
It is worth asking whether generalized transduction can occur by lysogenic phages. Two possible scenarios might be imagined to cause generalized transduction though literature references have not been found to confirm or dispute them:
(1) A lysogenic phage whose site of integration is randomly chosen, which occasionally brings along adjacent DNA because of an erroneous excision process.
(2) A lysogenic phage that goes into its lytic phase and randomly incorporates cell DNA.
Specialized Transduction: -
The second type of recombination event is called specialized transduction. If a virus removes itself from the chromosome incorrectly, some of the bacterial DNA can be packaged into the virion. Mistakes in this process of viral DNA going from the lysogenic to the lytic cycle lead to specialized transduction. There are three possible results from specialized transduction:
(1) DNA can be absorbed and recycled for spare parts.
(2) The bacterial DNA can match up with a homologous DNA in the recipient cell and exchange it. The recipient cell now has DNA from both itself and the other bacterial cell.
(3) DNA can insert itself into the genome of the recipient cell as if still acting like a virus resulting in a double copy of the bacterial genes.
Example of specialized transduction is ë phages in Escherichia coli.
RNA, DNA: -
Viruses with RNA genomes are not able to package DNA and so do not usually make this mistake.
Upon lysis of the host cell, the mispackaged virions containing bacterial DNA can attach to other bacterial cells and inject the DNA they have packaged, thus transferring bacterial DNA from one cell to another. This DNA can become part of the new bacterium’s genome and thus be stably inherited.
(c) Tobacco Mosaic Virus: -
Tobacco mosaic virus (TMV) is an RNA virus that infects plants, especially tobacco and other members of the family Solanaceae. The infection causes characteristic patterns (mottling and discoloration) on the leaves (thence the name). TMV was the first virus to be discovered. Although it was known from the late 19th century that an infectious disease was damaging tobacco crops, it was not until 1930 that the infectious agent was determined to be a virus.
Structure: -
Tobacco mosaic virus has a rod-like appearance. Its capsid is made from 2130 molecules of coat protein (see image above) and one molecule of genomic RNA 6400 bases long. The coat protein self-assembles into the rod like helical structure (16.3 proteins per helix turn) around the RNA which forms a hairpin loop structure. The protein monomer consists of 158 amino acids which are assembled into four main alpha-helices, which are joined by a prominent loop proximal to the axis of the virion. Virions are ~300 nm in length and ~18 nm in diameter. Negatively stained electron microphotographs show a distinct inner channel of ~4 nm. The RNA is located at a radius of ~6 nm and is protected from the action of cellular enzymes by the coat protein. There are three RNA nucleotides per protein monomer. TMV is a thermostable virus. On a dried leaf, it can withstand up to 120 degrees Fahrenheit (50 °C) for 30 minutes.
TMV has an index of refraction of about 1.57.
Replication: -
Following entry into its host via mechanical inoculation, the TMV RNA genome is not immediately translated. Instead the RNA is processed by a mechanism that is not yet understood. The resulting mRNAs encode several proteins, including the coat protein and an RNA-dependent RNA polymerase (RdRp). Thus TMV can replicate its own genome. After the coat protein and RNA genome of TMV have been synthesized, they spontaneously assemble into complete TMV virions in a highly organized process. The protomers come together to form disks composed of two layers of protomers arranged in a helical spiral. The helical capsid grows by the addition of protomers to the end of the rod. As the rod lengthens, the RNA passes through a channel in its center and forms a loop at the growing end. In this way the RNA can easily fit as a spiral into the interior of the helical capsid.
Infection: -
When TMV infects a tobacco plant, the virus enters mechanically (For example through a ruptured plant cell wall) and replicates. After its multiplication, it enters the neighboring cells through plasmodesmata. For its smooth entry, TMV produces a 30 kDa movement protein called P30 which tends to enlarge the plasmodesmata. TMV most likely moves from cell-to-cell as a complex of the RNA, P30, and replicase proteins. The first symptom of this virus disease is a light green coloration between the veins of young leaves. This is followed quickly by the development of a “mosaic” or mottled pattern of light and dark green areas in the leaves. These symptoms develop quickly and are more pronounced on younger leaves. Mosaic does not result in plant death, but if infection occurs early in the season, plants are stunted. Lower leaves are subjected to “mosaic burn” especially during periods of hot and dry weather. In these cases, large dead areas develop in the leaves. This constitutes one of the most destructive phases of tobacco mosaic virus infection. Infected leaves may be crinkled, puckered, or enlongated.
Transference to Humans: -
Consumption of tobacco products infected with the Tobacco Mosaic Virus has been found to have no effect on humans.
(d) Episomes: -
An episome is a portion of genetic material that can exist independent of the main body of genetic material (called the chromosome) at some times, while at other times is able to integrate into the chromosome.
Examples of episomes include insertion sequences and transposons. Viruses are another example of an episome. Viruses that integrate their genetic material into the host chromosome enable the viral nucleic acid to be produced along with the host genetic material in a nondestructive manner. As an autonomous unit (i.e., existing outside of the chromosome) however, the viral episome destroys the host cell as it commandeers the host’s replication apparatuses to make new copies of itself.
Another example of an episome is called the F factor. The F factor determines whether genetic material in the chromosome of one organism is transferred into another organism. The F factor can exist in three states that are designated as FPLUS, Hfr, and F prime.
FPLUS refers to the F factor that exists independently of the chromosome. Hfr stands for high frequency of recombination, and refers to a factor that has integrated into the host chromosome. The F prime factor exists outside the chromosome, but has a portion of chromosomal DNA attached to it.
An episome is distinguished from other pieces of DNA that are independent of the chromosome (i.e.,plasmids) by their large size.
Plasmids are different from episomes, as plasmid DNA cannot link up with chromosomal DNA. The plasmid carries all the information necessary for its independent replication. While not necessary for bacterial survival, plasmids can be advantageous to a bacterium. For example, plasmids can carry genes that confer resistance to antibiotics or toxic metals, genes that allow the bacterium to degrade compounds that it otherwise could not use as food, and even genes that allow the bacterium to infect an animal or plant cell. Such traits can be passed on to another bacterium.
Transposons and insertion sequences are episomes. These are also known as mobile genetic elements. They are capable of existing outside of the chromosome. They are also designed to integrate into the chromosome following their movement from one cell to another. Like plasmids, transposons can carry other genetic material with them, and so pass on resistance to the cells they enter. Class 1 transposons, for example, contain drug resistance genes. Insertion sequences do not carry extra genetic material. They code for only the functions involved in their insertion into chromosomal DNA.
Transposons and insertion sequences are useful tools to generate changes in the DNA sequence of host cells. These genetic changes that result from the integration and the exit of the mobile elements from DNA, are generically referred to as mutations. Analysis of the mobile element can determine what host DNA is present, and the analysis of the mutated host cell can determine whether the extra or missing DNA is important for the functioning of the cell.
(l) Shape and Symmetry of Virus: -
The amount and arrangement of the proteins and nucleic acid of viruses determine their size and shape. The nucleic acid and proteins of each class of viruses assemble themselves into a structure called a nucleoprotein, or nucleocapsid. Some viruses have more than one layer of protein surrounding the nucleic acid; still others have a lipoprotein membrane (called an envelope), derived from the membrane of the host cell, that surrounds the nucleocapsid core. Penetrating the membrane are additional proteins that determine the specificity of the virus to host cells. The protein and nucleic acid constituents have properties unique for each class of virus; when assembled, they determine the size and shape of the virus for that specific class.
Viruses vary in diameter from 20 nanometres (nm; 0.0000008 inch) to 250–400 nm. Only the largest and most complex viruses can be seen under the light microscope at the highest resolution. Any determination of the size of a virus also must take into account its shape, since different classes of viruses have distinctive shapes.
Shapes of viruses are predominantly of two kinds: rods, or filaments, so called because of the linear array of the nucleic acid and the protein subunits; and spheres, which are actually 20-sided (icosahedral) polygons. Most plant viruses are small and are either filaments or polygons, as are many bacterial viruses. The larger and more-complex bacteriophages, however, contain as their genetic information double-stranded DNA and combine both filamentous and polygonal shapes. The classic T4 bacteriophage is composed of a polygonal head, which contains the DNA genome and a special-function rod-shaped tail of long fibres. Structures such as these are unique to the bacteriophages.
The amount and arrangement of the proteins and nucleic acid of viruses determine their size and shape. The nucleic acid and proteins of each class of viruses assemble themselves into a structure called a nucleoprotein, or nucleocapsid. Some viruses have more than one layer of protein surrounding the nucleic acid; still others have a lipoprotein membrane (called an envelope), derived from the membrane of the host cell, that surrounds the nucleocapsid core. Penetrating the membrane are additional proteins that determine the specificity of the virus to host cells. The protein and nucleic acid constituents have properties unique for each class of virus; when assembled, they determine the size and shape of the virus for that specific class.
Viruses vary in diameter from 20 nanometres (nm; 0.0000008 inch) to 250–400 nm. Only the largest and most complex viruses can be seen under the light microscope at the highest resolution. Any determination of the size of a virus also must take into account its shape, since different classes of viruses have distinctive shapes.
(o) Viroids: -
Viroids are plant pathogens that consist of a short stretch (a few hundred nucleobases) of highly complementary, circular, single-stranded RNA without the protein coat that is typical for viruses. The smallest discovered is a 220 nucleobase scRNA (small cytoplasmic RNA) associated with the rice yellow mottle sobemovirus (RYMV).In comparison, the genome of the smallest known viruses capable of causing an infection by themselves are around 2 kilobases in size. The human pathogen hepatitis D is similar to viroids.
Viroids were discovered and given this name by Theodor Otto Diener, a plant pathologist at the Agricultural Research Service in Maryland, in 1971.
Viroid RNA does not code for any protein. The replication mechanism involves RNA polymerase II, an enzyme normally associated with synthesis of messenger RNA from DNA, which instead catalyzes “rolling circle” synthesis of new RNA using the viroid’s RNA as template. Some viroids are ribozymes, having catalytic properties which allow self-cleavage and ligation of unit-size genomes from larger replication intermediates
The first viroid to be identified was the potato spindle tuber viroid (PSTVd). Some 33 species have been identified.
Viroids and RNA Silencing: -
There has long been confusion over how viroids are able to induce symptoms in plants without encoding any protein products within their sequences. Evidence now suggests that RNA silencing is involved in the process. First, changes to the viroid genome can dramatically alter its virulence. This reflects the fact that any siRNAs produced would have less complementary base pairing with target messenger RNA. Secondly, siRNAs corresponding to sequences from viroid genomes have been isolated from infected plants. Finally, transgenic expression of the noninfectious hpRNA of potato spindle tuber viroid develops all the corresponding viroid like symptoms.
This evidence indicates that when viroids replicate via a double stranded intermediate RNA, they are targeted by a dicer enzyme and cleaved into siRNAs that are then loaded onto the RNA-induced silencing complex. The viroid sRNAs actually contain sequences capable of complementary base pairing with the plant’s own messenger RNAs and induction of degradation or inhibition of translation is what causes the classic viroid symptoms.