Mechanism of oncogenic viruses and Prevention.

How do virus causes cancer.

Most virus-induced cancers develop after a long period of persistant infection with an oncogenic virus. (for T cell leukaemia around 60 years).

Although many humans are persistently infected by the virus that are potentially oncogenic, only small percentages develop virus-linked cancers. So it is clear that virus alone do not cause cancer. Other factors are involved:

  • Complex interplay between the state of the host.
  • Environmental factors to which the host is exposed.
  • Host genetic factors.
  • Cellular changes induced by the virus.
  • Virus proteins synthesized during persistent infections play roles in the conversion of normal to cancer cells. 

 

Mechanisms Involved:

(a) ‘Delibrate’ interference with control of the cell cycle.

Many cells in the animal body grow and divide either slowly or not at all; the latter are arrested in the G1 phase of the cell cycle and are said to be in the G0 state. The control of the cell cycle is mediated by many proteins; two that play key role in humans are p53 and retinoblastoma protein (pRb).

Virus proteins that can interfere with the contol of the cell cycle include the HPV early proteins E6 and E7. Papillomavirus have small genomes about 8kbp and require the cell’s DNA synthesizing functions that are available in the S phase of the cell cycle. E6 and E7 make these functions available by localizing to the nucleus and binding to cell proteins.

E6 binds to p53, promoting its degradation, and E7 binds to pRb-E2F complexes causing their dissociation. E2F is an elongation factor which enhance DNA synthesis. As long as pRb is bound to E2F, no DNA syntheis will occur. The outcomes of this dissociation makes E2F free from pRb-E2F complexe and this transition will make the cell to move into S phase synthesizing viral DNA.

(b) ‘Accidental’ activation of cell genes.

Some virus proteins are able to bind to cell proteins that may not be the intended targets and may trigger events that are of no value to the virus, but may be harmful to host. 

A virus protein might inadvertently push a cell towards a cancerous state by activating a cell gene that is switched off, or by enhancing the rate of transcription of a gene that is being expressed at a low level. 

For example: Tax protein of HTLV-1, it activates the expression of its gene known as tax. this tax may interact with cell genes and some of proto-oncogenes (eg:c-myc) can be activated or their may be a boost in the expression resulting in cancer. 

 

(c) Retroviral Oncogenes 

Some retroviruses have the ability to cause cancer because of the presence in the viral genome of an oncogene. The retroviral genes were drived from cell proto-oncogenes and are closely related to them. 

When a proto-oncogene is mutated or deleted or aberrantly expressed it becomes an oncogene. The oncogene-carrying retroviruses have the ability to induce rapid formation of tumors, usually 1-6 weeks post infection.

Interaction between cell proteins and proteins produced by oncogenic viruses can lead to breakdown of immune defenses that may allow development of cancer,

1st mechanism: RNA by Reverse transcriptase is converted in to DNA which gets integrated into host genome. If Viral DNA gets integrated near proto-oncogene it gets activated and gives hyper expression and result into oncogene and then cancer.

2nd mechanism: It posses a viral oncogenes. Viral DNA which contain V-onc genes get integrated into host genome resulting in cancer. 

 

Prevention of Viral induced Cancer: 

  • prevent transmission of viruses.
  • strategies aimed at reducing the risk of transmission 
  • vaccination
  • Attempted elimination from the body of persistent infections with oncogenic viruses.

Hepatocellular carcinoma

Hepatocellular carcinoma (HCC, also called malignant hepatoma) is the most common type of liver cancer. Most cases of HCC are secondary to either a viral hepatitis infection or cirrhosis (alcoholism being the most common cause of hepatic cirrhosis).

In HCC number of factors are implicated as causative agents including consuption of mould toxins in food, and two viruses: Hepatitis B virus (HBV) and Hepatitis C virus (HCV).

HBV is the most significant agent. HBV DNA is integrated into genome, and in most cases the virus DNA has undergone rearrangements, including deletions.

When HBV genome is integrated into human chromosomes, possible out comes are as follows: activates oncognes; cayses chromosome instability and integration disrupts, which all leads to tumor cell.

 

Human T cell leukemia/lymphotropic virus type 1 (HTLV-1)

Adult T-cell leukemia/lymphoma (ATLL) is a rare and  aggressive T-cell lymphoma that is linked to infection by the human T-cell lymphotropic virus 1 (HTLV-1). Human
T-cell lymphotropic virus 1 is a retrovirus—meaning that  these viruses do not contain gentic material made of DNA, but instead carry RNA.

These viruses selectively infect only T-cells. Only about 2 percent to 5 percent of patients infected with the HTLV-1 virus will develop ATLL. Currently, physicians have no way of telling which infected patients will develop the lymphoma.

The HTLV-1 virus is in the same class of virus as the HIV/ AIDS virus and is endemic to certain parts of the world such as Japan, the Caribbean, South and Central America, West Africa and the southeastern United States. The HTLV-1 virus is believed to be transmitted through sexual contact,  exposure to contaminated blood from either a blood transfusion or used needles and can be passed from mother to child through breastfeeding.

Kaposi’s sarcoma-associated herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the eighth human herpesvirus. This virus causesKaposi’s sarcoma, a cancer commonly occurring in AIDS patients, as well as primary effusion lymphoma and some types of multicentric Castleman’s disease. It is one of seven currently known human cancer viruses, or oncoviruses.

 

Structure and Genome

KSHV is a herpesvirus, and is a large double-stranded DNA virus with a protein covering that packages its nucleic acids, called the capsid, which is then surrounded by an amorphous protein layer called the tegument, and finally enclosed in a lipid envelope derived in part from the cell membrane. KSHV has a genome which is approximately 165,000 nucleic acid bases in length. It is a rhadinovirus, and is remarkable since it has stolen numerous genes from host cells including genes that encode for complement-binding protein, IL-6, BCL-2, cyclin-D, a G protein-coupled receptor, interferon regulatory factor and Flice inhibitory protein (FLIP), as well as DNA synthesis proteins including dihydrofolate reductase, thymidine kinase, thymidylate synthetase, DNA polymerase and many others.

History

Kaposi’s sarcoma was first described in the 19th century as a rare skin cancer that effected elderly men in the Maditteranean region. In 1994 it was discovered that the tumor cells contain the DNA of a new herpesvirus. The virus was subsequently isolated and was named Kaposi’s sarcoma-associated herpesvirus (KSHV).

 

Epidemiology

It is transmitted through sexsual contact and via organ transplant. Healthy individuals can be infected with the virus and show no signs or symptoms, due to the immune system’s ability to keep the infection in check. Kaposi’s sarcoma occurs when someone who has been infected with KSHV becomes immunocompromised due to AIDS, medical treatment or very rarely aging.

Its tumor cells are derived from endothelial cells. Two other human cancers linking with KSHV is : primary effusion lymphomas and Multicentric Castleman’s disease. Both are tumor derived from B cells,

Epstein – Barr Virus

The Epstein–Barr virus (EBV), also called human herpesvirus 4 (HHV-4), is a virus of the herpes family, and is one of the most common viruses in humans.

Structure and genome

The virus is approximately 122 nm to 180 nm in diameter and is composed of a double helix of DNA wrapped in a protein capsid. The capsid is surrounded by a tegument made of protein, which in turn is surrounded by a envelope made from lipids.The EBV DNA is about 192,000 base pairs long and contains about 85 genes.The viral envelope contains glycoproteins, which are essential to infection of the host cell.

 

Replication

It infects primarily lymphocytes and epithelial cells. In lymphocytes, the infection is usually non-productive, while virus is shed (produce infection) from infected epithelial cells.

EBV is casually associated with:

  • Burkitt’s Lymphoma in the tropics.
  • Nasopharyngeal cancer, particularly in china and south Asia, where certain diets acts as co-carcinogens.
  • B cell lymphomas in Immune suppressed individuals (as in organ transplantation or HIV).
  • Hodgkin’s lymphoma (in 40% of affected patients).
  • X-linked lymphoproliferative Disease (Duncan’s syndrome).
  • Non-Hodgkin’s lymphoma in AIDS.

Burkitt’s lymphoma is a B cell tumor that occurs with a high frequency in children in central Africa. The Epstein–Barr virus is named after Michael Anthony Epstein, a professor emeritus at the University of Bristol, and Yvonne Barr (born 1932 in London), a 1966 Ph.D graduate from theUniversity of London, who together discovered and documented the virus, So was named Esptein – Barr Virus.

A constant abnormality in BL tumor cells is a chromosomal rearrangement that results in the c-myc gene being placed next to an enhancer of an immunoglobulin gene. This results in the expression of c-myc at abnormally high levels. 

Phage Display

Phage Display Cycle

One of the laboratory techniques employed in studying different protein interactions is Phage Display. With this in vitro screening method, protein ligands and macromolecules can be easily identified and interactions between protein and protein, peptide and protein, & DNA and protein can be studied further.

History of Phage Display

The first described instance of Phage Display occurred in 1985, when George P. Smith fused a peptide with a gene III from a filamentous phage. He filed a patent detailing the process of generating phage display libraries in the same year. Eventually, further development of Phage Display technology led by different groups from the MRC Laboratory of Molecular Biology, as well as from The Scripps Research Institute, led to the possibility of displaying proteins for the purpose of therapeutic protein engineering. The technique has been continuously improved to screen and amplify huge collections of proteins showing the connection of phenotype and genotype better.

Structure

A filamentous phage has a diameter of around 6.5 nanometers, with a length that depends on the size of its genome. It comes from a huge family of bacterial viruses that also infect other forms of bacteria. It contains a small genome with an intergenic region containing the necessary sequences for the replication and encapsidation of DNA.

A phage particle consists of five coat proteins. The particle has a hollow tube that houses so many copies of the primary coat protein. There are also binding interactions between the adjacent subunits’ hydrophobic midsections. One end of the particle is blunt, and the other is sharp. The blunt end contains plenty of copies of the two tiniest ribosomally translated proteins, while the sharp end contains around only 5 copies of the pIII and pVI genes, which are necessary for the detachment of the phage from the cell membrane.

How it works

Phage Display is a method wherein a library of phage particles that express very diverse peptides is generated. The objective is to choose those that will bind a desired target; the target can be a protein, a peptide, or a piece of DNA.

The most often used vector to build a random peptide display is the filamentous phage M13. In this display, the DNA which encodes the peptide or protein of interest is integrated into the pIII or pVI gene. To make sure that the fragments are completely inserted into the three possible reading frames, multiple cloning sites are sometimes employed, allowing the proper translation of the cDNA in its correct frame. The DNA hybrid and the phage gene are then put inside E. coli bacterial cells. Examples of these bacterial cells include XL1-Blue E. coli, SS320, TG1, and ER2738. The peptide or protein of interest is eventually expressed in either the minor or major coat protein

If another kind of vector is used, for example, a phagemid vector or simplified display construct vector, a helper phage must infect the E. coli cells; otherwise, the phage particles will not be separated from the E. coli cells. A helper phage activates the packaging of the phage DNA and assembles the mature virions with their corresponding protein fragments, which are included in the outer coating of the minor or major protein coat.

The generated phage library is then screened by addition into a microtiter plate containing immobilised target proteins or DNA sequences. Phages displaying a protein that bind to one target will remain, while the other phages can be discarded through washing. The remaining phage particles can be used to multiply the phage by infecting them into bacteria, thus increasing the diversity of the peptide display library.

Applications

The fast isolation of particular ligands through phage display has a wide variety of applications like epitope mapping, analyzing different protein interactions, vaccine development, drug design, and therapeutic target validation. Phage display is also used to pick inhibitors for the active and allosteric sites of G-protein binding modulatory peptides, enzymes, and receptor antagonists and agonists.

Determining the proper protein partners can be useful to identify the functions of various proteins. For drug discovery and design, Phage Display is employed in protein engineering or in vitro protein evolution. Therapeutic targeting with phage display is also primarily used to diagnose and determine tumour antigens, which is useful for cancer research.

Antibody Phage Display significantly improved the discovery and development of antibody drugs. Phage display for antibody libraries paved the way for rapid vaccine design and therapy. These libraries are used to learn more about the human immune system and to create human antibodies in vitro with the use of diverse synthetic substances.

Phage Display can be used in conjunction with other techniques, and with enough support and studies, more applications for it can be discovered.

Phage Therapy

Phage Therapy

What comes to mind when you hear the word “bacteria”? Most people, if not all, will answer “disease,” “sickness,” or “bad for the health.” What not all people know is there are actually both good and bad bacteria and some bacterial species are probiotic – bacteria that are helpful to its host. In fact, bacterial infections can be treated with bacteriophages: viruses that have the ability to infect and fight harmful bacteria, culminating in their destruction. Bacteriophage or phage therapy is therefore very useful in various fields like medicine, veterinary science, dentistry, and even agriculture.

History of Phage Therapy

Bacteriophages were discovered by two people: the English bacteriologist Frederick Twort in 1915 and the French-Canadian microbiologist Felix d’Herelle in 1917. Immediately after their discovery, the thought of using phages to fight bacterial infections was already apparent. D’Herelle began testing the therapeutic effects that phages may have on chickens and cows first and the tests were successful. Eventually, human tests were conducted and the development of phage therapy became more extensive especially with the foundation of the Eliavia Institute in 1923; the pharmaceutical company Eli Lilly began the commercialization of phage therapy in the US during the 1940s. During the Second World War, phages were used to treat bacterial diseases among soldiers of the Soviet Union, particularly gangrene and dysentery. The development of antibiotics in the 1950s led to a temporary setback on phage therapy as the use of antibiotics became more favourable. However, there has been a renewed interest in the development and employment of phage therapy in more applications.

Advantages over Antibiotics

Viruses and bacteria evolve over time and can develop a resistance to antibiotics. In theory, this resistance can also apply to phages, but it may be less difficult to overcome compared to antibiotics.

Because phages are target specific, meaning only a one or very few bacterial strains are targeted upon, it is easier to develop new phages than new antibiotics. A time period of only a few days or weeks is needed to acquire new phages for resistant strains of bacteria, whereas it can take years to obtain new antibiotics. When resisting bacteria evolve, the assigned phages also evolve, so when super bacterium appears, an equivalent super phage fights it as long as the phage is derived from the same environment.

Compared to antibiotics, phages go deeper into the infected area. Antibiotics, on the other hand, have concentration properties that quickly decrease as they go below the surface of the infection. The replication of phages is concentrated on the infected area where they are needed the most, while antibiotics are metabolized and removed from the body. In addition, secondary resistance does not happen among phages, but happens quite often among antibiotics. Secondary resistance is acquired and occurs when there aren’t enough blood drug levels.

Certain infections in people and experimentally infected animals have been proven to be more effectively treated with phage therapy than using antibiotics. Since 1966, the average success rate of studies that used phages in various ways (systematically, topically, intravenously, or orally) is from 80 to 95%, with minimal or no allergic and/or gastrointestinal side effects. The infections studied are from E. coli, Acinetobacter, Psuedomonas, and Staphylococcus aureus. Multiple side effects like allergies, intestinal disorders, and yeast infections have been observed when using antibiotics.

Applications

Fighting and destroying bacterial infections (both in humans and animals) are the primary applications of phage therapy, but it can also be employed for other uses. It can be the key to fighting the NDM-1, a gene that can be included in the DNA of bacteria, enabling them to resist antibiotics. Waste water from sewage systems are not really considered waste because it is a rich source of phage strains for various kinds of bacteria that lead to the most up-to-date medicines. Skin grafting for extensive wounds, trauma, burns, and skin cancer can also be improved by using phage therapy to lessen the Psuedomonas aeruginosa infection. Some experiments for cells in tissue culture have also discovered antitumor agents in phages. Bacteria cause food to spoil faster, and phages have been studied for their potential to increase the freshness of food and decrease the incidents of food spoilage.

Phage therapy has many other potential benefits and giving it ample support can pave the way to a healthier future.

Papillomaviruses

  • Structure and Genome : Isocahedral virus with covalently closed circular double stranded DNA. Its virions are 50-55 mm large in diameter.
  • They are small DNA viruses of mammals and birds.
  • Most cervical carcinoma in women result from infection with papillomavirus.
  • Entry: They enter the body through small abrasions and infect keratin making cells keratinocytes in skin or mucous membrane.
  • Site of Infection: There are well over 100 human papillomavirus (HPV) types, differentiated by their DNA sequence. Each HPV type infects a preferred site, such as hands or the genitals, and infection may result in a benign wart or a cercinoma.
    The papilloma virus that infect the genitals are transmitted between individuals during sexual contact.
    Most papilloma virus infections do not become persistant, but in minority of the host the infection is not cleared by the host immune system and they have small risk of cancer developing. The risk is associated with about 15 of HPV types called ‘High-risk’ type such as HPV-16 and HPV-18. Other HPV types that infect genitals carries little or no risk of cancer, these ‘low-risk’ type include HPV-6 and HPV-2.
  • After infection: it has 3 possible results; immuned by body immune response, or remains longer period and cause genital warts or cause cervical cancer.
  • Detection: Changes can be detected by morphological observation of a cervical smear, If precancer cells are detected they can be killed or removed to prevent the development of tumor.
  • The presence of all or part of the genome of high risk HPV type can nearly always be demonstrated in precancer cells and in cancer cells; The virus DNA is integrated into a chromosome. The virus replication cycle is not complete in these cells and no progeny virions are formed.
  • HPVs are also involved in a very rare form of skin cancer; epidermodysplasia verruciforms. Patients are highly susceptible to infection with HPVs – mainly 5 and 8. Warts spread over entire body during childhood and 25-33% patients develop cancer (squamous cell carcinoma) in areas of the skin exposed to UV light. Contrast to cervical cancers, the DNA is rarely integrated into a cell chromosome.

How papilloma virus bring about the cancer?

Keratinocytes, the host cell of papilloma virus , stop dividing as they differenciate, but when infected presence of HPV induces an infected keratinocyte into the S phase of the cell cycle due to the fact that HPV need DNA replicating machinery of the host cell. The cell then undergoes cycles of cell division, which generally have a finite number but occasionally division of a cell infected with high risk HPV continues unchecked as a cancer.

Bacteriophage T4

T4 is a bacteriophage that infects Escherichia coli bacteria. The T4 phage is a member of the T-even phages, a group including enterobacteriophagesT2 and T6.

It is one of the largest phages, encoding roughly 200 genes and was the first prokaryotic organism providing evidence of gene splicing through presence of introns in the genome. T4 is capable of undergoing only a lytic lifecycle and not the lysogenic lifecycle.

History of T4.

T4 has been extensively studied and has a rich history in the advancement of genetics. Some of the first essential ideas of genetics came from studies using T4 including: the basis of genetic code, even ribosomes, mRNA, and the codon. It is surprising that science has learned so much using a virus which is extremely complex. Nonetheless, we owe much of our current knowledge to T4.

Genome and Structure

The elongated isocahedral head of T4 phage contains linear double-stranded DNA genome about 169 kbp long and encodes 289 proteins including two accesory proteins, HOC (highly antigenic outer capsid proteins) and SOC (small outer capsid proteins). Binding of SOC stablizes the capsid and resist osmotic shocks.

The T4 genome is terminally redundant and is first replicated as a unit, then several genomic units are recombined end-to-end to form a concatemer. When packaged, the concatemer is cut at unspecific positions of the same length, leading to several genomes that represent circular permutations of the original. The genome is AT-rich and contains modified bases in the form of 5 hydroxy-methyl-cytosine, rather than cytosin, which protect the phage DNA from many host restriction systems and from phage-encoded nucleases that degrade cytosine-containing host DNA during infection. The T4 genome bears eukaryote-like intron sequences.

The T4 tail fibres, pins and base plates are involved in binding to the lipo-polysaccharide receptor of the E.Coli. The T4’s tail is hollow so that it can pass its nucleic acid into the cell it is infecting after attachment. The tail attaches to a host cell with the help of tail fibres.

The life cycle of T4

1. T4 adsorption and Injection

Phage binds to the lipo-polysaccharide and tryptophan receptor on the bacterium. T4 looks for a susceptible bacterium with its tail fibers. The tail fibers recognise the membrane first. The phage once bound to the cell, the base plate goes under conformational change which causes the sheath to contract piercing the cell membrane allowing the viral core to enter the cell, and release the dsDNA viral genome.  The outer sheath contracts driving the internal tail tube in to the cell. The Gene product, gp5 (known as tail lysozome) facilitates digestion of the peptidoglycon layer of the bacterium to reach the inner membrane.

The trans-membrane electrochemical potential is required for transfer of T4 DNA to cytoplasm. The empty capsid remains extracellular.

2. Replication and expression of genes.

Once T4 DNA is in the cytoplasm, it specifies a highly organised and coordinated program of gene expression with the help of 3 promoters; Pe (early genes), Pm (middle gens) and PL (late genes). Some of the first genes which are transcribed, called immediate-early, encode enzymes which break down host DNA. Host DNA is broken down in order to use host nucleotides to produce more viral DNA. It also encode the proteins needed for DNA synthesis and to build the capsid and tail structures.

The host enzyme, RNA polymerase, transcribe and translate the expression of early genes. Early genes rely on the transcription apparatus of the host, being transcribed from normal sigma70 promoters.

Delayed early and middle genes encode the 20 proteins which are involved in viral replication. In fact, T4 encodes nearly all of its own replicative machinery. There are two distinct replicative processes of T4 which are outlined below:

  1. Stage 1 – Replication occurs in a bidirectional manner with multiple origins of replication within the genome. The first several rounds of replication are initiated by RNA primers synthesized by the host RNA polymerase. These RNA primers can travel to their complimentary region in dsDNA and displace the other strand to produce a structure called an R-loop. The attached RNA can now act as a primer for the leading strand of DNA replication. The lagging strand is then synthesized using the replicative helicase, gp41. DNA replication is finished by gp30 which is the T4 encoded ligase as well as the host DNA ligase. Several minutes after infection, host RNA primers cannot be used because the promoting recognition specificity is altered on the host RNA polymerase and recombinant-dependent replication (RDR) is favored. The mechanism of RDR is outlined in stage 2.
  2. Stage 2 – At this point the virus uses its own replicative machinery to transcribe its late genes with a process known as the gp45 sliding clamp model. The primers for leading strand synthesis are recombination intermediates instead of RNAs made by RNA polymerase. 

Early gene products alter the host RNA polymerase in two ways for expression of middle genes. Modifies RNA polymerase will not stop translation and over shoot to reach Pm (middle gene promoter). The enzyme is there by able to read through a transcription terminator by anti-termination mechanism to express genes downstream of early genes and is modofied to recognise middle promoters.

Transcription of the late gene is coupled to replication. Promoters for these genes again differ from sigma70 promoters and require an alternative sigma factor, encoded by T4 regulatory gene, gp55, to activate transcription. Binding of gene, gp55 to host RNA polumerase allows the enzyme to recognise specifically T4 late gene promoters. Other T4 gene products, gp44, gp45 and gp62 are additionally required for late gene transcription.

3. Assembly and Release.

Once late genes are expressed, the viral base plate is first assembled, this then attaches to the tail and tail fiber proteins. These three different protein pathways combine to form a mature T4 phage capsid. DNA is packaged into the mature capsid protein by packaging and cutting the concatemers using a terminase complex found at the end of the concatemerized DNA strand. The terminase complex binds to the capsid head and moves DNA into the empty capsid head. The capsid also encases necessary enzymes for future infections such as virally encoded DNA polymerase.

The viral encoded enzyme holin (gpt) creates the holes in the inner membrane of the bacterial host-cell to allow lysozymes to exit and degrade the peptidoglycan cell wall. Cell lysis subsequently follows releasing a shower of bacteriophage progeny into the extracellular space.

Lambda Phage


Lambda phage 
is a temperate bacteriophage that infects Escherichia coli.. It have alternative replication pathways: Lysogenic or Lytic pathways.

History of Lambda Phage

In 1950, Esther Lederberg, an American microbiologist, was performing experiments on E. coli mixtures. She happened to observe streaks of mixtures of two types of E. coli strains that seemed to have been nibbled on and had viral plaque. One E. coli strain had been treated with ultraviolet light, so the damage was more apparent. It was later determined that this was caused by bacterial viruses, which replicated and spread resulting in cell destruction. The discovery led to the employment of Lambda phage as a model organism in microbial genetics as well as in molecular genetics.

Structure and Genome.

Lambda phage is a virus particle consisting of a Icosahedral head, measuring around 50-60 nanometers in diameter, containing double-stranded linear DNA as its genetic material, and a flexible tail that is around 150 nanometers long and may and may contain tail fibers.Lambda phage.jpg

Lambda contains a linear double stranded DNA genome of 48.5 KB.

Lambda phage exists in two form. Inside the phage it is linear double stranded DNA; inside host double stranded circular DNA molecule.

 

The life cycle of lambda

1. Lambda adsorption

Phage identify a host bacterium by binding or adsorption to a specific structure on the surface of the cell. Many different cell surface structures can be used as binding sites.

Lambda binds to an outer membrane protein called LamB via a protein that resides at the tip of the lambda tail called the J protein. LamB normally functions in the binding and uptake of the sugars maltose and Maltodextrin.

 

2. Lambda DNA Injection

Initially, lambda binds to LamB and the binding is reversible. This step requires only the lambda tail and the LamB protein.

Next, the bound phage undergoes a change and the binding to lag LamB becomes irreversible. This nature of the change is unknown but it requires that a phage head be attached to the phage tail.

Next, the lambda DNA is ejected from the phage and taken up by the bacterium. The DNA in the phage head is very tightly packed. If the condensed state of the phage DNA is stabilized, ejection if the DNA does not occur.

In addition to LamB, lambda also uses an inner membrane protein called Pstm to gain entry to the cytoplasm.

 

3. Protecting the lambda genome in the cytoplasm of the bacteria.

Lambda contains a linear double-stranded DNA molecule in its capsid. In the bacterium cytoplasm, dsDNA molecules are subjected to degradation by exonucleases that need a free end to digest the DNA.

The first event that happens to newly injected lambda DNA is that the DNA circularizes to prevent it from being degraded.

How circulation of DNA occurs?
Lambda has a specific site on its DNA, termed Cos sites (cohesive sites), which is used to circularize the DNA. The cos site is a 22bp sequence that is cut asymmetrically when the lambda DNA is packed. These cos sites are complementary to each other. A host enzyme, DNA ligase, seals the nicks at either end of the cos site generating a covalently closed, circular lambda genome. Basically two cos sites: cosL and cosR.

Restriction Alleviation.

This is a uniqueproperty of this virus.Lambda phage is very small.It mimics the methylation system and its own DNA is methylated. Because of this restriction enzyme will not cleave that DNA and are protected.

Restriction enzyme actually recognises a specific DNA sequence and cleaves the DNA on the both strands. The cut or digested DNA is sensitive to nucleases that degrade DNA. The modification part of the system is a protein that specifically modifies the DNA sequence recognise by the restriction enzyme and prevent the DNA from being digested.

This restriction or modification system allows a bacterium to tell DNA from its own species from foreign DNA.

 

4. What happens to the lambda genome after it is stabilized.

In case of lambda all the genes involved in specific functions are clamped together. SubattPxisint is the gene resp possible for recombination. The genome contain six major promoters. Function of Promoter is to promote transcription by binding to RNA polymerase.

PL – promoter leftward
PR – Promoter rightward
PRE – Promoter for repressor establishment.
PRM – Promoter for repressor maintenance.
PI – Promoter for integration
PR1 – Secondary rightward Promoter

CI – Repressor
Cro – Anti repressor
Q, N – Anti terminator proteins
Nut R – Nutrilisation site (rightward)
Nut L – Nutrilisation site (leftward)
tRI – Terminator right ward
tL – Terminator left ward
PL/OL – Promoter and operator (rightward)
PR/OR – Promoter and operator (leftward)
O, P – DNA synthesis
Paq – Promoter anti q
q nut – Q nutrilisation site

S helps in transcription of R to peptidoglycon, altimately weakening cell membrane for lysis.

After the genome is circularised and supercoiled (The host encoded enzyme, DNA gyrase, supwrcoils the lambda molecule), transcription begins from PL and PR. A series of genes known as early genes are transcribed and translated. These early genes are namely N and Cro. These genes products are the initial proteins needed for further phage development.

E coli RNA polymerase interacts with PL to give rise to short mRNA transcript that is translated into the N protein. Transcription from PR leads to Cro protein.

The N protein is able to extend transcription when RNA polymerase encounters a sequence in the DNA that tells it to stop. For that reason, N is called an anti termination protein. N allows RNA polymerase to transcript through tL and tR1 termination signals resulting in the synthesis of longer mRNA transcripts.

The longer transcript from PR encode the O, P and Cll proteins and a small amount of another anti terminator, the Q protein.

From PL, Clll the recombination proteins and a small amount of Xis and Int are made. Cll and Clll are reffered delayed early proteins. Cll helps in expression of promoters. Clll protects Cll from host proteases.

When N is bind to nut R1 / nut L1 the termination of R1 and L1 occurs. N protein anti terminates by binding to RNA polymerase after a specific base pair sequence, has been transcribed into mRNA. This sequence is called nut for Neutralization.

At this point, all of the players needed to make the lytic-lysogenic decision have been made. Cll and Clll are needed for lysogenic growth. The O and P proteins are used for replicating the lambda DNA.

Operater OR is further divided into OR3, OR2 and OR1. There are will be competition between Cro and Cl in competing in respect to binding. Cro likes to bind at OR3, if it bind it prevent binding of Cl. When N is bind to nutR1 and nutL1 the termination of R1 and L1 is not occurred and it continues dissociation. If no dissociation O,P,Q,S and R is produced. Function of O and P is to synthesise DNA, so phage DNA is synthesized.

DNA synthesis occurs on lytic cycle. Q protein is going to bind Qnut site, termination will not occur so expression of S and R occurs. S and R are responsible for lysis of the cell.

 

5. Lambda and the lytic-lysogenic decision:

At the most basic level, the decision depends on the amount of two phage encoded proteins called Cl and Cro, and their binding to their promoter control regions.

When Cl is bound, the expression of the lytic gene is repressed and the phage follows the lysogenic pathway. For this reason Cl is also known as Cl repressor or lambda repressor. The expression and binding of Cro will lead to lytic development. Cro is made from PR and Cl made from either PRE or PRM. Both Cro and Cl binds to the same DNA sequences called promoters.

Lambda contains two operators that bind Cro and Cl.
OR – overlaps PRM and PR promoters.
OL – overlaps with PL.

OR is the major player in the lytic-lysogenic decision, while OL is not a part of the decision. OR is composed of 17 base pair sequences called OR1, OR2 and OR3. Cl repressor binds to OR1, ten times better than it binds to OR2 and OR3. At increasing concentrations of Cl, it will bind to OR2 and eventually OR3.

When Cl repressor binds to OR, it stimulates the PRM Promoter and the production of Cl repressor and inhibits the PR promoter and the production of Cro, leading to lysogeny.

Cro binds to OR3 first, then OR2 and finally at high concentration to OR1. When Cro is bound to OR, it inhibits the PRM Promoter and the production of Cl, leading to lytic growth.

How does the phage switch between thses development pathways?

The major protein involved in the switch ia another phage -encoded protein called Cll. Cll activates the PRE and PI promoters. This leads to production of repressor, Cl and the integrase protein, which is also needed for lysogeny. Cll is unstable and rapidly degraded by the host-encoded HfIA protease. Inactivate Cll leads to lytic growth. Cll can be protected by the phage- encoded protein Clll. Active Cll leads to lysogrni. Cycle or growth.

 

The lambda lysogenic pathway

If active Cll prevails, it activates PRE and PI promoters leading to production of repressor CI and the integrase protein. Eventually Cl activates PRM ensuring that a continuous supply of CI is made.

Lambda recombines into the chromosomes using a specific site on the phage called attP and a specific site on bacterial chromosome called attB. When the lambda DNA is in the chromosome, it is bound by attL and attR, which are hybrid attP/attB sites.

Once the lambda DNA is recombined into the chromosome, it is replicated and stably inherited by daughter cells as part of the bacterial chromosome. It stays in quiescent.

What prevent the expression of the late genes coding for lytic function?

The expression of late genes is prevented by the action of the lambda repressor, Cl. Lambda repressor binding to the operator sequences OR and OL blocks transcription from PL and PR. Since PR is blocked, the lambda Q protein is not made and transcription of the late genes does not occur.

 

The lambda lytic cycle.

If enough of the Q proteins accumulates in the cell, RNA Polymerase will continue its transcription from the third promoter, PR1 located infront of the Q gene. This extends transcription in to the late genes located downstream of Q. The late genes encode the proteins needed to complete the lytic infection including the head, tail, and lysis proteins.

The Q proteins which is made from PR, when N is present is a second anti-termination protein. It acts on the qut site and allows transcription through tR1. Q is necessary for synthesis of the head and tail genes.

After the infecting lambda DNA has been converted to a double-stranded circular molecule, it replicates from a specific origin using both the phage encoded O and P proteins and bacterial encoded proteins. This is called theta replication. Later in lytic development, lambda switches to a second mode of replication called rolling circle replication. Rolling circle replication produces long DNA molecules containing multiple phage genomes called concatomers. These concatomers are packed int to phage heads and mature phages are produced. 

The lambda R and S proteins are required for lambda to release progeny phages into the environment. The R protein is an endolysin that degrades the peptidoglycan cell wall and allow phage to escape from the cell. The S proteins forms a hole in the inner membrane to allow the endolysin to gain excess to cellwall. After the hole is made, approx 100 intact lambda phage particles are released. The entire lytic cycle lasts~ 35 minutes.

 

 

Induction of lambda by the SOS System.

When a lambda lysogen is treated with UV ~ 35 minutes later te cells lyse and release phages. UV damages the DNA and triggers a cellular response called SOS response to deal with this damage. The single stranded DNA from the damaged DNA activates RecA. Activated recA interacts with Cl, leading to cleavage of Cl and induction of the lambda lysogen. Activated RecA also interact with LexA and leads to LecA. Inactivation which leads to expression of number of genes, whose products repair the DNA damage in the cell. Cleavage of Cl, leads to expression of the phage lytic genes and phage production. The rational for this response is that lambda does not want to risk staying in the cell that has DNA damaged and may not survive. 

 

Superinfection:

If a cell is a lambda lysogen, another lambda phage that infects is not able undergo lytic development and produce phage. The incoming phage can inject its DNA, however the DNA is immediately shutdown and no transcription or translation of the lambda initiates. The Lysogens are immune to infection by another lambda phage particle, which is called superinfection. Superinfection is blocked because the lysogen is continuosly producing Cl repressor.

The lysogen actually produces more repressor than it needs to shut down one phage. This extra repressor binds to the superinfecting phage DNA at OL and OR and prevents transcription from PL and PR,

 

Applications of Lambda Phage 

The lambda phage has different applications, most of which are related to DNA cloning. This is because lambda phage can be used as a vector for generating recombinant DNA, which are combined DNA sequences that result from using laboratory techniques like molecular cloning to assemble genetic material from several sources. The site-specific recombinase of lambda phage can be used for shuffling cloned DNAs via the gateway cloning system, a molecular biology technique that ensures the effective transfer of DNA fragments between plasmids.

The lambda phage’s ability to mediate genetic recombincation is due to its red operon, which is a functioning unit of genomic DNA that has a cluster of genes controlled by a promoter or a single regulatory signal. This red operon can be expressed to yield the proteins red alpha (or exo), beta, and gamma, which can be used in recombination-mediated genetic engineering, a method commonly employed in bacterial genetics, generation of target vectors, and DNA modification.

Undoubtedly, the lambda phage is a powerful genetic tool that can be used in many important studies.