In this article we will discuss about the replication of virus by lytic and lysogenic cycle.
Replication of Virus by Lytic Cycle:
This type of cycle is seen in T-even phages (T2, T4 etc.) which attack Escherichia coli.
The lytic cycle consists of five steps (Fig. 2.45):
(b) Infection (Fig. 2.44),
(c) Synthesis of phage components in host cell,
(d) Formation of new phage particle, and
(e) Liberation of phages from the host cell.
The interaction between the phage specific organelle — the tail and the receptor site of the host cell is called the adsorption. The adsorption is facilitated by the negatively charged carboxyl groups on the host surface and the positively charged amino-group of protein present at the tip of the phage tail.
In T-even phages, the tip of the tail fibre first attaches to the cell surface. The tail fibre then bends and allows the tail pins to attach on the host surface that makes an irreversible attachment (Fig. 2.44A, B).
After adsorption, the phage particle secretes an enzyme which hydrolyses the murin complex of the host cell wall and forms a pore. The sheath of the tail then contracts and pushes the central tubular part, i.e., core of the tail, into the host wall, like an injection needle (Fig. 2.44). The nucleic acid of the phage then passes through the core and enters the host bacterium.
The empty protein shell of the phage is called ghost, which may remain attached even after release of nucleic acid. Once the bacterial cell receives the nucleic acid of a phage, it becomes resistant to the other phages.
(c) Synthesis of Phage Components in Host Cell:
Once the phage nucleic acid takes the entry inside the bacterial cell, it suppresses the synthesis of bacterial protein and directs to synthesise the proteins of the phage particle (Fig. 2.45).
The DNA of phage replicates following the semi-conservative process. Majority of the DNA acts as a template for its own synthesis and the rest is used as template for the synthesis of viral specific m-RNA by utilizing the RNA-polymerase of the host.
The newly formed m-RNA directs the host cell to synthesise the proteins which are used to build up the protein coat of the phage particle (Fig. 2.45). Almost at the end of replication of phage nucleic acid, a protein, the phage lysozyme, is synthesised.
(d) Formation of New Phage Particle:
The new phage particles are formed by the assemblage of nucleic acid and protein. This process is called maturation, which is controlled by viral genome (Fig. 2.45). In this process, initially the condensation of nucleic acid molecule takes place.
The protein sub-units then aggregate around the nucleic acid molecule and form the head of the phage. By this time the tail formation starts. Initially the core tube is attached with the basal plate and then sheath becomes assembled around the core tube. In this stage, the tail becomes attached to the base of the head taking a collar in between. At last, the tail fibres are attached to the basal plate.
(e) Liberation of Phages from the Host Cell:
In a cycle of phage development, about 200 phages are formed which take about 30-90 minutes. In the host cell, the phage DNA secretes lysozyme (an enzyme) which causes the lysis of host cell wall. As a result of lysis the phage particles are liberated (Fig. 2.45).
During this process, initially the λ-phage gets attached to the bacterium with the help of tail fibre. The λ-phage then injects its DNA thread into the host bacterium (E. coli K12). After entry, the ds-DNA thread is converted into a circular DNA (described earlier, Fig. 2.47).
The circular DNA of phage then gets attached to the membrane at the specific site and starts replication. Replication is initiated near the origin and progress in both direction symmetrically and then terminates when the two replicating forks meet each other and form a typical theta (θ)-like structure.
The daughter DNA molecules synthesised and developed from the parental DNA undergo replication by ‘rolling-circular model’ and develop long thread-like concate-meters. These concate-meters contain several λ-genomes. The proper lengths with single stranded cohesive ends are cut off by enzymes and are packaged into the heads of λ-phages.
Transcription of the phage DNA develops coding messages that help to form the capsid and other proteins of phage. It is initiated by host polymerase which binds with the two promoters on λ-DNA that transcribes two different strands in opposite directions. More than 40 genes have been mapped in λ-DNA, having specific functions such as synthesis of viral DNA, head protein, tail protein, etc.
After the synthesis of sufficient number of virion components, they assemble and release by lysis of host bacterium. The released λ-phages then infect new bacterium and continue another lytic cycle or may enter into a lysogenic cycle.
Generally, the virus continues lytic cycle with a few numbers of infected cells, but major portion enters into lysogenic relationship and continues the lysogenic cycle.
Activity of the circular λ-DNA:
After circularisaion of λ-DNA inside the bacterial cell (E. coli K12), it functions in either of two alternative ways:
i) Lytic cycle:
In this way the DNA of λ- phage undergoes transcription, translation, assembly of progeny and the release by lysis of host bacterium.
ii) Lysogenic cycle:
In this way, the DNA of λ-phage is integrated with the bacterial DNA at a specific site to become a prophage and thus the infected host bacterium becomes lysogenic. Thus the phage DNA (prophage) replicates along with bacterial DNA. During this process the genes of phage DNA controlling lytic cycle becomes inhibited by a repressor — the λ-repressor.
Replication of Virus by Lysogenic Cycle:
A. Lwoff (1953) discovered this type of cycle in Lambda (W phages that attack E. coli. The phage involved in this cycle is called temperate phage, the bacterium is the lysogenic strain and the entire process is called lysogeny (Fig. 2.46).
At first the phage is adsorbed on the wall of the host bacterium and its DNA becomes injected into the host cell. Here the phage DNA, like the lytic cycle, does not take over the protein synthesis machinery of the host cell, instead, it becomes integrated with the nucleoid of the host genome.
This integrated phage DNA is called a prophage (Fig. 2.45). Thus the new composite genome replicates as one unit. The composite genome then multiplies for indefinite number of times and produces daughter lysogenic bacteria.
After a number of generations the viral genome gets detached from the composite genome and releases in the cytoplasm. This dissociation is called induction (Fig. 2.45). The viral genome then enters the lytic cycle and forms temperate phages that are released by lysis of wall of the host bacterium.
Detailed molecular mechanism of lysogeny and induction by lambda (λ) phage:
Being non-cellular the viral particles have no capacity of independent metabolism and reproduction like other organisms. Thus, they need others’ help for their multiplication almost like the activity of a terrorist of present day. Multiplication of viruses commonly takes place by infection, multiplication and lysis of the host bacterium, called lytic cycle.
But in many others, the mechanism is different, those show different modes of parasitism including infection, integration with genome, multiplication along with host genome; and, later, it gets separated from the host genome, causes multiplication and comes out through lysis of host cell, called as lysogenic cycle.
The phage involved in this cycle is called temperate phage, the bacterium as lysogenic strain, the injected phage DNA integrated with the host genome as prophage and the entire process is called lysogeny.
The temperate phage, after infection, may undergo any one option of multiplication i.e., either lytic cycle like a virulent phage, or lyso- genic cycle. Artificial disruption of lysogenic host cell does not show the presence of infectious phages. In other way, it indicates that phages must be present in non-infectious state.
Before going through the cycle one has to know the structure and circularisation of genome of λ-phage.
Structure of λ-phage and circularisation of its genome:
A λ-phage is a double stranded DNA virus with an icosahedral head of about 55 nm in diameter and a long tail (180 nm) without any contractile sheath (Fig. 2.47). The tail has a thin tail fibre at its distal end. The double stranded DNA of virus is a linear thread-like structure with 12 nucleotides long, single stranded cohesive ends at the 5′-(p) ends. The cohesive ends are complementary to each other.
The single stranded complementary regions of both ends come to each other and form a circular double stranded DNA. The 5′-(p) and 3′-(OH) ends of both the thread then rejoin in vivo by DNA ligase. The circularisaion of DNA takes place after its entry inside the host bacterium (Fig. 2.47).
After attachment of λ-phage with the host bacterium (E. coli K12), the phage pushes its DNA inside the cytoplasm of host cell. The phage DNA is then circularised in the usual pattern. The cl gene of λ-phage then produces the λ-repressor, an acidic protein (made up of 20 amino acid residues of molecular weight 26,000 dalton) which inhibits the action of gene controlling phage multiplication and lysis.
The λ-repressor binds with the two different operators of its own genome, the OL and OR, those are involved in the initiation of transcription of phage controlling the phage multiplication. So the two essential proteins required for the initiation of phage multiplication are not synthesised. Thus the operation lytic cycle stops and ensures clearance to establish the lysogenic state.
The circular λ-phage then becomes integrated with the bacterial DNA. The int gene of λ-phage produces an enzyme integrase which helps in integration.
During this process the circular DNA of λ-phage is inserted as a linear DNA into the bacterial DNA at a specific site between galactose and biotin operons (Fig. 2.48). The inserted DNA of λ-phage is called prophage. The λ-DNA remains with bacterial DNA for long time and replicates along with bacterial DNA.
In course of time, the lysogenic bacteria are able to produce phage particles through the process of induction either spontaneously at a very low frequency (one out of 102 or more) or due to action of different agents like X-ray, y- ray, UV ray etc. (Fig. 2.46).
Due to induction, the prophage becomes released from bacterial chromosome and again becomes circular. The excision of prophage is catalysed by an enzyme excisionase produced by the xix gene of λ-phage. The rec A gene of the host bacterium produces a proteolytic enzyme which degrades the λ-repressor.
At the same time, the cro gene becomes activated and produces croprotein, which inhibits the synthesis of λ-repressor. The circular λ-DNA then proceeds through lytic cycle and develops new crop of phage particle.
Significance of Lysogeny:
The lysogeny has an important role in the transfer of genetic material from one bacterium to the other. The temperate phage acts as an agent for gene transfer through the process known as specialised transduction. In this process, when a temperate phage comes out from host bacterium as prophage, it may include a part of bacterial DNA along with its DNA by mistake.
After lysis, the newly developed phage can infect a new host bacterium and thus transfers the portion of previous bacterial DNA to the newly infected bacterium, and, thereby, recombination takes place.