The below mentioned article provides a study note on viral genomes.
Viruses are a special class of infectious agents that are so small that they can be viewed only under electron microscope. A complete “viral particle” or “virion consists of a block of genetic material (DNA or RNA) surrounded by a protein coat and, sometimes by an additional membranous envelope.
The viruses contain neither cytoplasm nor exhibit any growth or metabolic activity. But when their genetic material enters into a suitable host cell, virus-specific protein synthesis replication of the viral chromosome occurs; these processes utilize both cellular (of host) and viral enzymes.
On the basis of the host organisms, viruses are divided into three main groups:
(1) Animal viruses,
(2) Bacterial viruses and
(3) Plant viruses.
Morphological Features of Viruses:
The viral chromosome is enclosed within a protein shell called capsid. The viral chromosome and its protein coat together are called nucleocapsid. Viruses vary considerably in their morphological features (Table 5.4).
1. Icosahedral virions:
Their capsid is icosahedral, i.e., the virion is a regular polyhedron with 20 triangular faces and 12 corners. Examples are, adenoviruses and bacteriophage φX174.
2. Helical virions:
The nucleic acid of such virions is enclosed in a cylindrical, rod shape capsid that forms a helical structure, e.g., TMV, bacteriophage M13.
3. In some cases, the nucleocapsid is icosahedral while in others, it is helical in some components. Such viruses are enveloped.
4. Coplexvrions:
These viruses do not have a clearly identifiable capsid. The viral nucleic acid is present in the centre of the shell which is made up of protein molecules. Some of the shells are complex while others are simple. In Herpes, an animal virus that contains DNA as genetic material, the capsid has a diameter of 1000A; it is further surrounded by an envelope making its diameter 1500A. (Fig. 5.19).
The capsid is mode up of protein subunits (capsomers) which form an icosahedron.
Bacteriophages have relatively complex structures: they contain a head, a tail, a base plate and several tail fibres (Fig. 5.20). The head is hexagonal (lateral side) and contains the viral DNA. The tail has a core tube surrounded by a sheath. At the tail end, there is a basal plate with 6 spikes from which 6 tail fibres emerge.
At the time of infection, the tail fibres bind to specific receptor sites on the host cell. The base plate is drawn to the cell surface and contraction of tube sheath occurs along with the removal of the base plate plug. The core of the tail penetrates the cell wall which is weakened by some hydrolytic enzymes present in the phage and the viral tail. DNA enters into the host cell through the core tube of the tail.
In the case of tobacco mosaic virus (TMV; multiplying in tobacco plant cells) and some small bacterial viruses (e.g., F2, R17, QB), the protein coat contains a single type of protein. These protein molecules are arranged in either a helical symmetry or a cubical symmetry.
The shell of TMV contains about 2150 protein molecules which are identical, each molecule having the molecular weight of-17,000. These molecules are helically arranged around the RNA genome which contains 6,000 nucleotides.
Lytic Viruses:
The viruses which lyse or disrupt the host cell following infection are called lytic viruses. During infection, the nucleic acid is injected into the host cell. The enzymes required for viral DNA replication are then synthesized so that replication of DNA occurs to produce numerous copies of the viral chromosome.
The protein components of the capsid are synthesized in the later stages leading to the formation of heads and tails; the viral DNA is then packed into the heads. In the end, the cell wall ruptures and the progeny phage particles are released (Fig. 5.21).
Lysogenic Viruses (Temperate Phages):
Lysogeny involves a symbiotic relationship between a temperate phage and its bacterial host. The viral chromosome becomes inserted into the bacterial chromosome, where it remains and replicates along with the latter. The viral DNA integrated into the bacterial genome is called a provirus or prophage (Fig. 5.22). The bacterium containing a prophage is immune to the infection by the same virus.
Viral Chromosomes:
Viruses contain either DNA or RNA as their genetic material. These nucleic acids may be either single or double-stranded (Table 5.5). Small viruses may contain 3 kb (kb =,kilo-bases = 1000 bases), while large viruses could have about 300 kb. in their genome. Thus the number of genes in viral genome may vary from only 3 to hundreds. The retroviruses arc diploid (have two copies of the genome per capsid), while the others are haploid.
Double-stranded DNA:
Several viruses possess double-stranded DNA as their genetic material. The base composition of different viruses is modified leading to change in the physical properties of DNA such melting temperature, buoyant density in caesium chloride (CsCl) etc.
In some of the viruses, such as. T-even coliphages, cytosine (C) is modified into 5-hydroxymethyl- cytosine (HMC). In certain cases, thymine is converted into 5-hydroxy-methyl uracil or 5-di-hydroxymethyluracil, e.g., in B. subtilisbacteriophges. Certain physical properties of DNA, such as, buoyant density in CsCl or melting temperature are changed due to these substitutions.
Some of the viruses contain linear DNA, while others contain circular (cyclic) DNA (Table 5.5). In the case of phage lambda (λ), DNA can exist in both linear and cyclic forms. When isolated from a viral particle, the λ DNA is linear, but when it enters into the host cell, becomes circular. However, it enters into the host cell in its linear form.
The A. chromosome is a double- stranded DNA molecule containing 47,000 nucleotides; it is 17 pm in length. There is single- stranded projection of 12 nucleotides at each 5′-end; these projections are complementary to each other and thus they are called cohesive ends.
These cohesive ends are responsible for the circularization of the chromosome. Circularization of the chromosome protects it from degradation by the host exonucleases. Further, the linear DNA cannot replicate vegetatively; the circularity therefore, provides an advantage in replication as well.
Single-stranded DNA:
Single-stranded DNA occurs in very small bacteriophages (Table 5.4). The single-stranded DNA found in the virion is called the positive (+) strand; as a rule only the plus (+) strand is found in the phage particles. However, in adeno-associated viruses, two complementary strands exist in different virions. The single-stranded DNA contains inverted repeating sequences that form hair pins. The hairpin structures have important role in circularization of the linear strands and in replication.
Double-stranded RNA:
Double-stranded RNAs are found in several icosahedral viruses of animals and plants. The genomes of such viruses are segmented (Table 5.5). The different segments may be connected short stretches of base pairs. Transcription of each segment occurs separately and the enzyme involved is “Double-stranded RNA transcriptase”. Each mRNA, on translation produces a separate polypeptide chain.
Single-stranded RNA:
Single-stranded RNA is the genetic material in a number of viruses (Table 5.5). Some viruses contain a single RNA molecule in their genome, while some other viruses contain several segments, e.g., influenza virus has 8 segments. The viruses contain either positive (+) or negative (-) strands of RNA in their capsids.
Positive (+) Strand:
The viral RNA strand that functions as mRNA in the host cell is called the plus (+) strand or positive strand. The RNA genomes of animal viruses have a cap at their 5′-end and a poly (A) sequence at the 3′-end. However, in Picornavirus RNA, there is a special sequence at the 5′-end to which a small protein is covalently attached.
The RNA genomes of plant viruses possess a cap at the 5′-end but they do not contain the poly (A) at their 3′-ends; their 3′-end is similar to tRNA. Each retrovirus particle contains two copies of the (+) RNA strand representing its genome; these copies are held together near the 5′-end.
Negative (-) strand:
These RNAs do not contain a cap but terminate into a nucleoside triphosphate at their 5′-ends. These strands do not function as mRNA directly. Instead, they are transcribed by the enzyme “single-stranded RNA transcriptase” present in the virion, to produce the mRNA.
Packaging of Nucleic Acids in the Viruses:
Viral genome (DNA/RNA) is tightly packed into the protein shell (capsid). The density of the nucleic acid in the protein shell is higher than 500 mg/ml, which is much greater than the density of DNA in other organisms. For example, density of DNA in bacterium is about 10 mg/ml, while in the eukaryotic nucleus, it is about 100 mg/ml. This shows that the nucleic acid is very tightly packaged in the viral particles.
The genetic material of TMV is single-stranded RNA containing 6400 nucleotides, making up a length of 2 pm. This RNA is packaged into the rod-shaped compartment of 0.3 x 0.008 pm. Adenoviruses contain 11 pm long double-stranded DNA consisting of 35,000 bp: this is packaged into an icosahedron type capsid of 0.07 pm diameter.
Phage T4 has a very long double-stranded DNA molecule (55 pm) having 170,000 bp. The capsid containing this rather long DNA is an icosahedron with the dimensions of 1.0 x 0.065 pm. Unlike eukaryotic nucleus and bacterial nucleoid, the volume of the capsid is fully packaged with the nucleic acid.
Packaging of nucleic acid to form a nucleocapsid occurs in two general ways. In one mechanism, the protein molecules assemble around the nucleic acid, e.g., in TMV. In the other mechanism, the protein coat is formed first and then the nucleic acid is inserted in it. In TMV, a duplex hairpin structure occurs in the RNA.
The assembly of protein monomers begins at this nucleation centre and proceeds in both the directions, reaching the ends. A total of 17 protein units form a circular layer and two such layers together form a unit of capsid. This structure interacts with the RNA which is coiled to form a helix inside the shell.
In bacteriophage T4 and λ etc., the protein shell is formed first. The nucleic acid is inserted into the coat from one end and then the tail is joined to the head. In case of circular DNA, it must be first converted into a linear molecule for packaging.
The lambda (λ) genome is circular and contains two “cos” sites, cosL and cosR. The free end in λ DNA is produced by enzymatic cleavage at the cosL site. Insertion of DNA occurs from this end and continues till the cosR site enters the capsid; a cleavage then occurs at the cosR site to produce the other end of the λ genome.
Some of the viruses, e.g., phage T4 and λ. have terminal redundancy in their genomes. In these viruses, multiple genomes join end-to-end to produce “concatemeric structure.” In case of T4, insertion of the viral chromosome starts at a “random” point and continues until the required amount of DNA has been inserted into the head. The DNA inserted into the head has a terminal redundancy.
One likely origin of the “concatermeric” DNA is recombination. Recombination between two chromosomes combines two genomes end-to-end. Then recombination with a third genome produces a concatemer through successive recombination’s (Fig. 5.23).
Another mechanism suggested for concatemer formation is the rolling circle replication. Specific endonuclease cuts the concatemer at the points that produce the genome of the “required length.” The genomic DNA has homologous ends due to the terminal redundancy. Therefore, some chromosomes may be heterozygous for the terminal genes.
Mechanisms of Lysogenic and Lytic Pathways:
Bacteriophage λ is a temperate phage that maintains a lysogenic relationship with its bacterial host. However, it can undergo lytic cycle also. Infection, as a rule, occurs in the linear form, but the chromosome converts into a circular one once it enters the host cell. A generalized map of the X chromosome showing different functions is presented in Fig. 5.24.
Genes related to similar functions are clustered. On the linear chromosome, genes for head formation are located on left end, while those for lysis are located at the right end. The regulatory region lies between the region for recombination and the region for replication. The genes present in the regulatory region are responsible for determining whether the X will enter into a lysogenic relationship with its host or it will follow the lytic pathway.
Lytic pathway:
Regulatory genes are clustered and flanked by genes for recombination on their left side and those for replication on the right side (Fig. 5.25). Genes N (anti-terminator) and era (anti-repressor) are located within the regulatory region. These genes are called “immediate early genes”; they are transcribed by the host RNA polymerase.
In the presence of anti-termination factor (pN), transcription of both the genes (N and era) continues. These two genes are transcribed from different DNA strands in the opposite direction, the gene N being transcribed towards the left, while era is transcribed towards the right.
The transcription extends to other region of the genome for different functions (Fig. 5.25). In the absence of cl repressor protein, the host RNA polymerase binds to PL/OL sites so that the transcription of the “late genes” is initiated; as a result, phage particles are produced and the cell is lysed.
Lysogenic pathway:
The regulatory region contains the cl gene which is responsible for the lysogenic pathway. A mutation in this region causes the phage to undergo lytic cycle.
The cl gene is transcribed to produce mRNA; the enzyme involved in transcription is RNA polymerase that binds to the promoter for repressor maintenance (PRM). The transcription occurs from right to left. This cl mRNA is translated to produce the repressor monomer (Fig. 5.25).
Repressor dimers are formed that bind to the PL/OR and PL/OL sites, thus preventing the RNA polymerase from binding to these promoters. This leads to the inhibition of transcription of N and cro genes. Later, the X chromosome is integrated into the bacterial chromosome; its delayed early genes are not expressed and the phage remains as a “provirus”. Delayed early genes are the genes for recombination, replication and Q (anti-terminator). Late genes are tail, head and lysis genes.
When the cl repressor is bound to the 0L and 0R sites, RNA polymerase initiates transcription of the cl gene, and synthesis of repressor protein is continued. But in absence of the repressor, RNA polymerase binds to PL/OL and Pr/Or sites and transcription of N and cro genes begins.
Thus the presence of cl repressor itself is necessary for its synthesis. Continuous production of cl repressor is necessary for lysogeny to be maintained. During this period, the OL and OR sites are always bound by repressor.
When the lysogenized cell is infected by another phage X, the cl repressor protein produced by the “prophage” immediately binds to the OL and 0R sites of the infecting X genome. The function of the infecting X genes is thus inhibited and the cell remains immune to X infection.