Is it impossible for a retrovirus to be lysogenic?

Is it impossible for a retrovirus to be lysogenic?

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Is it impossible for retroviruses to be lysogenic? In the lysogenic cycle, the viral genetic material is incorporated into the host cell's DNA. Because retroviruses have RNA, it would be impossible for their genetic material to be incorporated into the host cell's DNA, unless the RNA is reverse-transcribed or something.

Is this the case? How does this work?



Both things are not exclusive of each other. Retroviruses are RNA-based viruses which need to reverse transcribe their RNA genome into DNA before they can multiply. This is done by the reverse transcriptase enzyme, which is part of the virus and is incorporated into the cell.

Once the reverse transcriptase has been active and made the DNA, this DNA can also be incorporated into the hosts genome. If you look into the Wikipedia article on Bacteriophages, you will find a list of different bacteriophages, with all kind of genomes, among them as well a number of RNA-based viruses.

RNA-based viruses which enter the lysogenic pathway are obviously either pretty rare or they haven't been discovered yet in bigger numbers. Two examples are the phages F2 and MS2 (which are closely related).

Virus latency

Virus latency (or viral latency) is the ability of a pathogenic virus to lie dormant (latent) within a cell, denoted as the lysogenic part of the viral life cycle. [1] A latent viral infection is a type of persistent viral infection which is distinguished from a chronic viral infection. Latency is the phase in certain viruses' life cycles in which, after initial infection, proliferation of virus particles ceases. However, the viral genome is not eradicated. The virus can reactivate and begin producing large amounts of viral progeny (the lytic part of the viral life cycle) without the host becoming reinfected by new outside virus, and stays within the host indefinitely. [2]

Virus latency is not to be confused with clinical latency during the incubation period when a virus is not dormant.

Historically they have been named for a variety of factors, including

  • the associated diseases (poliovirus, rabies) the type of disease caused (murine leukemia virus)
  • the sites in the body affected or from which the virus was first isolated (rhinovirus, adenovirus)
  • where they were first isolated (Ebola virus, Hantavirus)
  • the animal that carries the virus (bird flu, swine flu)
  • for the way people imagined they were contracted (dengue = &lsquoevil spirit&rsquo influenza = &lsquoinfluence&rsquo of bad air).

Newer Conventions

Example of an Influenza Virus Naming

Focus on Human Immunodeficiency Virus

- Causes the disease AIDS (Acquired Immune Deficiency Syndrome)

HIV Infection Cycle (animation) | HIV Life Cycle - drugs target specific viral processes

HIV Coloring Assignment *Make sure you understand the steps involved in infection and how drugs treat the disease.

Related to Viruses

Viroids - even smaller than viruses, consist of RNA strands that lack a protein coat
Prions - "rogue protein", believed to be the cause of Mad Cow Disease, also may cause Kuru in cannibal tribes

Treatment of Viruses


Antiviral Drugs



Viruses may enter a host cell either with or without the viral capsid. The nucleic acid of bacteriophages enters the host cell “naked,” leaving the capsid outside the cell. Plant and animal viruses can enter through endocytosis (as you may recall, the cell membrane surrounds and engulfs the entire virus). Some enveloped viruses enter the cell when the viral envelope fuses directly with the cell membrane. Once inside the cell, the viral capsid degrades, and then the viral nucleic acid is released and becomes available for replication and transcription.

Replication and Assembly

The replication mechanism depends on the viral genome. DNA viruses usually use host-cell proteins and enzymes to replicate the viral DNA and to transcribe viral mRNA, which is then used to direct viral protein synthesis. RNA viruses usually use the RNA core as a template for synthesis of viral genomic RNA and mRNA. The viral mRNA directs the host cell to synthesize viral enzymes and capsid proteins, and assemble new virions.

Of course, there are exceptions to this pattern. If a host cell does not provide the enzymes necessary for viral replication, viral genes supply the information to direct synthesis of the missing proteins. Retroviruses, such as HIV (group VI of the Baltimore classification scheme), have an RNA genome that must be reverse transcribed into DNA, which then is incorporated into the host cell genome. To convert RNA into DNA, retroviruses must contain genes that encode the virus-specific enzyme reverse transcriptase that transcribes an RNA template to DNA. Reverse transcription never occurs in uninfected host cells—the enzyme reverse transcriptase is only derived from the expression of viral genes within the infected host cells. The fact that HIV produces some of its own enzymes not found in the host has allowed researchers to develop drugs that inhibit these enzymes without affecting the host’s metabolism.

This approach has led to the development of a variety of drugs used to treat HIV and has been effective at reducing the number of infectious virions (copies of viral RNA) in the blood to non-detectable levels in many HIV-infected individuals.


The last stage of viral replication is the release of the new virions produced in the host organism, where they are able to infect adjacent cells and repeat the replication cycle. As you’ve learned, some viruses are released when the host cell dies, and other viruses can leave infected cells by budding through the membrane without directly killing the cell.

Biology 171

By the end of this section, you will be able to do the following:

  • List the steps of replication and explain what occurs at each step
  • Describe the lytic and lysogenic cycles of virus replication
  • Explain the transmission of plant and animal viruses
  • Discuss some of the diseases caused by plant and animal viruses
  • Discuss the economic impact of plant and animal viruses

Viruses are obligate, intracellular parasites. A virus must first recognize and attach to a specific living cell prior to entering it. After penetration, the invading virus must copy its genome and manufacture its own proteins. Finally, the progeny virions must escape the host cell so that they can infect other cells. Viruses can infect only certain species of hosts and only certain cells within that host. Specific host cells that a virus must occupy and use to replicate are called permissive . In most cases, the molecular basis for this specificity is due to a particular surface molecule known as the viral receptor on the host cell surface. A specific viral receptor is required for the virus to attach. In addition, differences in metabolism and host-cell immune responses (based on differential gene expression) are a likely factor in determining which cells a virus may target for replication.

Steps of Virus Infections

A virus must use its host-cell processes to replicate. The viral replication cycle can produce dramatic biochemical and structural changes in the host cell, which may cause cell damage. These changes, called cytopathic effects , can change cell functions or even destroy the cell. Some infected cells, such as those infected by the common cold virus known as rhinovirus, die through lysis (bursting) or apoptosis (programmed cell death or “cell suicide”), releasing all progeny virions at once. The symptoms of viral diseases result both from such cell damage caused by the virus and from the immune response to the virus, which attempts to control and eliminate the virus from the body.

Many animal viruses, such as HIV (human immunodeficiency virus) , leave the infected cells of the immune system by a process known as budding , where virions leave the cell individually. During the budding process, the cell does not undergo lysis and is not immediately killed. However, the damage to the cells that the virus infects may make it impossible for the cells to function normally, even though the cells remain alive for a period of time. Most productive viral infections follow similar steps in the virus replication cycle: attachment, penetration, uncoating, replication, assembly, and release ((Figure)).


A virus attaches to a specific receptor site on the host cell membrane through attachment proteins in the capsid or via glycoproteins embedded in the viral envelope. The specificity of this interaction determines the host—and the cells within the host—that can be infected by a particular virus. This can be illustrated by thinking of several keys and several locks, where each key will fit only one specific lock.

This video explains how influenza attacks the body.


Viruses may enter a host cell either with or without the viral capsid. The nucleic acid of bacteriophages enters the host cell “naked,” leaving the capsid outside the cell. Plant and animal viruses can enter through endocytosis (as you may recall, the cell membrane surrounds and engulfs the entire virus). Some enveloped viruses enter the cell when the viral envelope fuses directly with the cell membrane. Once inside the cell, the viral capsid degrades, and then the viral nucleic acid is released and becomes available for replication and transcription.

Replication and Assembly

The replication mechanism depends on the viral genome. DNA viruses usually use host-cell proteins and enzymes to replicate the viral DNA and to transcribe viral mRNA, which is then used to direct viral protein synthesis. RNA viruses usually use the RNA core as a template for synthesis of viral genomic RNA and mRNA. The viral mRNA directs the host cell to synthesize viral enzymes and capsid proteins, and assemble new virions.

Of course, there are exceptions to this pattern. If a host cell does not provide the enzymes necessary for viral replication, viral genes supply the information to direct synthesis of the missing proteins. Retroviruses, such as HIV (group VI of the Baltimore classification scheme), have an RNA genome that must be reverse transcribed into DNA, which then is incorporated into the host cell genome. To convert RNA into DNA, retroviruses must contain genes that encode the virus-specific enzyme reverse transcriptase that transcribes an RNA template to DNA. Reverse transcription never occurs in uninfected host cells—the enzyme reverse transcriptase is only derived from the expression of viral genes within the infected host cells. The fact that HIV produces some of its own enzymes not found in the host has allowed researchers to develop drugs that inhibit these enzymes without affecting the host’s metabolism.

This approach has led to the development of a variety of drugs used to treat HIV and has been effective at reducing the number of infectious virions (copies of viral RNA) in the blood to non-detectable levels in many HIV-infected individuals.


The last stage of viral replication is the release of the new virions produced in the host organism, where they are able to infect adjacent cells and repeat the replication cycle. As you’ve learned, some viruses are released when the host cell dies, and other viruses can leave infected cells by budding through the membrane without directly killing the cell.

Influenza virus is packaged in a viral envelope that fuses with the plasma membrane. This way, the virus can exit the host cell without killing it. What advantage does the virus gain by keeping the host cell alive?

Watch a video on viruses, identifying structures, modes of transmission, replication, and more.

Different Hosts and Their Viruses

As you’ve learned, viruses often infect very specific hosts, as well as specific cells within the host. This feature of a virus makes it specific to one or a few species of life on Earth. On the other hand, so many different types of viruses exist on Earth that nearly every living organism has its own set of viruses trying to infect its cells. Even prokaryotes, the smallest and simplest of cells, may be attacked by specific types of viruses. In the following section, we will look at some of the features of viral infection of prokaryotic cells. As we have learned, viruses that infect bacteria are called bacteriophages ((Figure)). Archaea have their own similar viruses.


Most bacteriophages are dsDNA viruses, which use host enzymes for DNA replication and RNA transcription. Phage particles must bind to specific surface receptors and actively insert the genome into the host cell. (The complex tail structures seen in many bacteriophages are actively involved in getting the viral genome across the prokaryotic cell wall.) When infection of a cell by a bacteriophage results in the production of new virions, the infection is said to be productive . If the virions are released by bursting the cell, the virus replicates by means of a lytic cycle ((Figure)). An example of a lytic bacteriophage is T4, which infects Escherichia coli found in the human intestinal tract. Sometimes, however, a virus can remain within the cell without being released. For example, when a temperate bacteriophage infects a bacterial cell, it replicates by means of a lysogenic cycle ((Figure)), and the viral genome is incorporated into the genome of the host cell. When the phage DNA is incorporated into the host-cell genome, it is called a prophage . An example of a lysogenic bacteriophage is the λ (lambda) virus, which also infects the E. coli bacterium. Viruses that infect plant or animal cells may sometimes undergo infections where they are not producing virions for long periods. An example is the animal herpesviruses, including herpes simplex viruses, the cause of oral and genital herpes in humans. In a process called latency , these viruses can exist in nervous tissue for long periods of time without producing new virions, only to leave latency periodically and cause lesions in the skin where the virus replicates. Even though there are similarities between lysogeny and latency, the term lysogenic cycle is usually reserved to describe bacteriophages. Latency will be described in more detail in the next section.

Which of the following statements is false?

  1. In the lytic cycle, new phages are produced and released into the environment.
  2. In the lysogenic cycle, phage DNA is incorporated into the host genome.
  3. An environmental stressor can cause the phage to initiate the lysogenic cycle.
  4. Cell lysis only occurs in the lytic cycle.

Plant Viruses

Most plant viruses, like the tobacco mosaic virus, have single-stranded (+) RNA genomes. However, there are also plant viruses in most other virus categories. Unlike bacteriophages, plant viruses do not have active mechanisms for delivering the viral genome across the protective cell wall. For a plant virus to enter a new host plant, some type of mechanical damage must occur. This damage is often caused by weather, insects, animals, fire, or human activities like farming or landscaping. Movement from cell to cell within a plant can be facilitated by viral modification of plasmodesmata (cytoplasmic threads that pass from one plant cell to the next). Additionally, plant offspring may inherit viral diseases from parent plants. Plant viruses can be transmitted by a variety of vectors, through contact with an infected plant’s sap, by living organisms such as insects and nematodes, and through pollen. The transfer of a virus from one plant to another is known as horizontal transmission , whereas the inheritance of a virus from a parent is called vertical transmission .

Symptoms of viral diseases vary according to the virus and its host ((Figure)). One common symptom is hyperplasia , the abnormal proliferation of cells that causes the appearance of plant tumors known as galls . Other viruses induce hypoplasia , or decreased cell growth, in the leaves of plants, causing thin, yellow areas to appear. Still other viruses affect the plant by directly killing plant cells, a process known as cell necrosis . Other symptoms of plant viruses include malformed leaves black streaks on the stems of the plants altered growth of stems, leaves, or fruits and ring spots, which are circular or linear areas of discoloration found in a leaf.

Some Common Symptoms of Plant Viral Diseases
Symptom Appears as
Hyperplasia Galls (tumors)
Hypoplasia Thinned, yellow splotches on leaves
Cell necrosis Dead, blackened stems, leaves, or fruit
Abnormal growth patterns Malformed stems, leaves, or fruit
Discoloration Yellow, red, or black lines, or rings in stems, leaves, or fruit

Plant viruses can seriously disrupt crop growth and development, significantly affecting our food supply. They are responsible for poor crop quality and quantity globally, and can bring about huge economic losses annually. Others viruses may damage plants used in landscaping. Some viruses that infect agricultural food plants include the name of the plant they infect, such as tomato spotted wilt virus, bean common mosaic virus, and cucumber mosaic virus. In plants used for landscaping, two of the most common viruses are peony ring spot and rose mosaic virus. There are far too many plant viruses to discuss each in detail, but symptoms of bean common mosaic virus result in lowered bean production and stunted, unproductive plants. In the ornamental rose, the rose mosaic disease causes wavy yellow lines and colored splotches on the leaves of the plant.

Animal Viruses

Animal viruses, unlike the viruses of plants and bacteria, do not have to penetrate a cell wall to gain access to the host cell. The virus may even induce the host cell to cooperate in the infection process. Non-enveloped or “naked” animal viruses may enter cells in two different ways. As a protein in the viral capsid binds to its receptor on the host cell, the virus may be taken inside the cell via a vesicle during the normal cell process of receptor-mediated endocytosis. An alternative method of cell penetration used by non-enveloped viruses is for capsid proteins to undergo shape changes after binding to the receptor, creating channels in the host cell membrane. The viral genome is then “injected” into the host cell through these channels in a manner analogous to that used by many bacteriophages.

Enveloped viruses also have two ways of entering cells after binding to their receptors: receptor-mediated endocytosis, or fusion . Many enveloped viruses enter the cell by receptor-mediated endocytosis in a fashion similar to that seen in some non-enveloped viruses. On the other hand, fusion only occurs with enveloped virions. These viruses, which include HIV among others, use special fusion proteins in their envelopes to cause the envelope to fuse with the plasma membrane of the cell, thus releasing the genome and capsid of the virus into the cell cytoplasm.

After making their proteins and copying their genomes, animal viruses complete the assembly of new virions and exit the cell. As we have already discussed using the example the influenza virus, enveloped animal viruses may bud from the cell membrane as they assemble themselves, taking a piece of the cell’s plasma membrane in the process. On the other hand, non-enveloped viral progeny, such as rhinoviruses, accumulate in infected cells until there is a signal for lysis or apoptosis, and all virions are released together.

As you will learn in the next module, animal viruses are associated with a variety of human diseases. Some of them follow the classic pattern of acute disease , where symptoms get increasingly worse for a short period followed by the elimination of the virus from the body by the immune system and eventual recovery from the infection. Examples of acute viral diseases are the common cold and influenza. Other viruses cause long-term chronic infections , such as the virus causing hepatitis C, whereas others, like herpes simplex virus, only cause intermittent symptoms. Still other viruses, such as human herpesviruses 6 and 7, which in some cases can cause the minor childhood disease roseola, often successfully cause productive infections without causing any symptoms at all in the host, and thus we say these patients have an asymptomatic infection .

In hepatitis C infections, the virus grows and reproduces in liver cells, causing low levels of liver damage. The damage is so low that infected individuals are often unaware that they are infected, and many infections are detected only by routine blood work on patients with risk factors such as intravenous drug use. On the other hand, since many of the symptoms of viral diseases are caused by immune responses, a lack of symptoms is an indication of a weak immune response to the virus. This allows the virus to escape elimination by the immune system and persist in individuals for years, all the while producing low levels of progeny virions in what is known as a chronic viral disease. Chronic infection of the liver by this virus leads to a much greater chance of developing liver cancer, sometimes as much as 30 years after the initial infection.

As already discussed, herpes simplex virus can remain in a state of latency in nervous tissue for months, even years. As the virus “hides” in the tissue and makes few if any viral proteins, there is nothing for the immune response to act against, and immunity to the virus slowly declines. Under certain conditions, including various types of physical and psychological stress, the latent herpes simplex virus may be reactivated and undergo a lytic replication cycle in the skin, causing the lesions associated with the disease. Once virions are produced in the skin and viral proteins are synthesized, the immune response is again stimulated and resolves the skin lesions in a few days or weeks by destroying viruses in the skin. As a result of this type of replicative cycle, appearances of cold sores and genital herpes outbreaks only occur intermittently, even though the viruses remain in the nervous tissue for life. Latent infections are common with other herpesviruses as well, including the varicella-zoster virus that causes chickenpox. After having a chickenpox infection in childhood, the varicella-zoster virus can remain latent for many years and reactivate in adults to cause the painful condition known as “shingles” ((Figure)).

Some animal-infecting viruses, including the hepatitis C virus discussed above, are known as oncogenic viruses : They have the ability to cause cancer. These viruses interfere with the normal regulation of the host cell cycle either by introducing genes that stimulate unregulated cell growth (oncogenes) or by interfering with the expression of genes that inhibit cell growth. Oncogenic viruses can be either DNA or RNA viruses. Cancers known to be associated with viral infections include cervical cancer, caused by human papillomavirus (HPV) ((Figure)), liver cancer caused by hepatitis B virus, T-cell leukemia, and several types of lymphoma.

Visit the interactive animations showing the various stages of the replicative cycles of animal viruses and click on the flash animation links.

Section Summary

Plant viruses may be transmitted either vertically from parent reproductive cells or horizontally through damaged plant tissues. Viruses of plants are responsible for significant economic damage in both crop plants and plants used for ornamentation. Animal viruses enter their hosts through several types of virus-host cell interactions and cause a variety of infections. Viral infections can be either acute, with a brief period of infection terminated by host immune responses, or chronic, in which the infection persists. Persistent infections may cause chronic symptoms (hepatitis C), intermittent symptoms (latent viruses such a herpes simplex virus 1), or even be effectively asymptomatic (human herpesviruses 6 and 7). Oncogenic viruses in animals have the ability to cause cancer by interfering with the regulation of the host cell cycle.

Art Connections

(Figure) Influenza virus is packaged in a viral envelope that fuses with the plasma membrane. This way, the virus can exit the host cell without killing it. What advantage does the virus gain by keeping the host cell alive?

(Figure) The host cell can continue to make new virus particles.

(Figure) Which of the following statements is false?

  1. In the lytic cycle, new phages are produced and released into the environment.
  2. In the lysogenic cycle, phage DNA is incorporated into the host genome.
  3. An environmental stressor can cause the phage to initiate the lysogenic cycle.
  4. Cell lysis only occurs in the lytic cycle.

Free Response

Why can’t dogs catch the measles?

The virus can’t attach to dog cells, because dog cells do not express the receptors for the virus and/or there is no cell within the dog that is permissive for viral replication.

One of the first and most important targets for drugs to fight infection with HIV (a retrovirus) is the reverse transcriptase enzyme. Why?

Reverse transcriptase is needed to make more HIV-1 viruses, so targeting the reverse transcriptase enzyme may be a way to inhibit the replication of the virus. Importantly, by targeting reverse transcriptase, we do little harm to the host cell, since host cells do not make reverse transcriptase. Thus, we can specifically attack the virus and not the host cell when we use reverse transcriptase inhibitors.

In this section, you were introduced to different types of viruses and viral diseases. Briefly discuss the most interesting or surprising thing you learned about viruses.

Answer is open and will vary.

Although plant viruses cannot infect humans, what are some of the ways in which they affect humans?

Plant viruses infect crops, causing crop damage and failure, and considerable economic losses.

A bacteriophage with a lytic life cycle develops a mutation that allows it to now also go through the lysogenic cycle. How would this provide an evolutionary advantage over the other bacteriophages that can only spread through lytic cycles?

In a lysogenic cycle, the bacteriophage integrates into the host bacterium’s genome as a prophage, and is passed on to daughter cells every time a bacterium carrying the prophage replicates. This allows the prophage to be dispersed through a wide population without killing any of the host cells. Since the mutated bacteriophage also retains the ability to switch into the lytic cycle, it now has two methods to disseminate through the bacteria population.


Persistent Infections

Persistent infection occurs when a virus is not completely cleared from the system of the host but stays in certain tissues or organs of the infected person. The virus may remain silent or undergo productive infection without seriously harming or killing the host. Mechanisms of persistent infection may involve the regulation of the viral or host gene expressions or the alteration of the host immune response. The two primary categories of persistent infections are latent infection and chronic infection. Examples of viruses that cause latent infections include herpes simplex virus (oral and genital herpes), varicella-zoster virus (chickenpox and shingles), and Epstein-Barr virus (mononucleosis). Hepatitis C virus and HIV are two examples of viruses that cause long-term chronic infections.

Latent Infection

Not all animal viruses undergo replication by the lytic cycle. There are viruses that are capable of remaining hidden or dormant inside the cell in a process called latency. These types of viruses are known as latent viruses and may cause latent infections. Viruses capable of latency may initially cause an acute infection before becoming dormant.

For example, the varicella-zoster virus infects many cells throughout the body and causes chickenpox, characterized by a rash of blisters covering the skin. About 10 to 12 days postinfection, the disease resolves and the virus goes dormant, living within nerve-cell ganglia for years. During this time, the virus does not kill the nerve cells or continue replicating. It is not clear why the virus stops replicating within the nerve cells and expresses few viral proteins but, in some cases, typically after many years of dormancy, the virus is reactivated and causes a new disease called shingles (Figure 7). Whereas chickenpox affects many areas throughout the body, shingles is a nerve cell-specific disease emerging from the ganglia in which the virus was dormant.

Figure 7. (a) Varicella-zoster, the virus that causes chickenpox, has an enveloped icosahedral capsid visible in this transmission electron micrograph. Its double-stranded DNA genome becomes incorporated in the host DNA. (b) After a period of latency, the virus can reactivate in the form of shingles, usually manifesting as a painful, localized rash on one side of the body. (credit a: modification of work by Erskine Palmer and B.G. Partin—scale-bar data from Matt Russell credit b: modification of work by Rosmarie Voegtli)

Latent viruses may remain dormant by existing as circular viral genome molecules outside of the host chromosome. Others become proviruses by integrating into the host genome. During dormancy, viruses do not cause any symptoms of disease and may be difficult to detect. A patient may be unaware that he or she is carrying the virus unless a viral diagnostic test has been performed.

Chronic Infection

A chronic infection is a disease with symptoms that are recurrent or persistent over a long time. Some viral infections can be chronic if the body is unable to eliminate the virus. HIV is an example of a virus that produces a chronic infection, often after a long period of latency. Once a person becomes infected with HIV, the virus can be detected in tissues continuously thereafter, but untreated patients often experience no symptoms for years. However, the virus maintains chronic persistence through several mechanisms that interfere with immune function, including preventing expression of viral antigens on the surface of infected cells, altering immune cells themselves, restricting expression of viral genes, and rapidly changing viral antigens through mutation. Eventually, the damage to the immune system results in progression of the disease leading to acquired immunodeficiency syndrome (AIDS). The various mechanisms that HIV uses to avoid being cleared by the immune system are also used by other chronically infecting viruses, including the hepatitis C virus.

Think about It


Generally, tumor viruses cause little or no disease after infection in their hosts, or cause non-neoplastic diseases such as acute hepatitis for hepatitis B virus or mononucleosis for Epstein–Barr virus. A minority of persons (or animals) will go on to develop cancers after infection. This has complicated efforts to determine whether or not a given virus causes cancer. The well-known Koch's postulates, 19th-century constructs developed by Robert Koch to establish the likelihood that Bacillus anthracis will cause anthrax disease, are not applicable to viral diseases. Firstly, this is because viruses cannot truly be isolated in pure culture—even stringent isolation techniques cannot exclude undetected contaminating viruses with similar density characteristics, and viruses must be grown on cells. Secondly, asymptomatic virus infection and carriage is the norm for most tumor viruses, which violates Koch's third principle. Relman and Fredericks have described the difficulties in applying Koch's postulates to virus-induced cancers. [9] Finally, the host restriction for human viruses makes it unethical to experimentally transmit a suspected cancer virus. Other measures, such as A. B. Hill's criteria, [10] are more relevant to cancer virology but also have some limitations in determining causality.

Tumor viruses come in a variety of forms: Viruses with a DNA genome, such as adenovirus, and viruses with an RNA genome, like the hepatitis C virus (HCV), can cause cancers, as can retroviruses having both DNA and RNA genomes (Human T-lymphotropic virus and hepatitis B virus, which normally replicates as a mixed double and single-stranded DNA virus but also has a retroviral replication component). In many cases, tumor viruses do not cause cancer in their native hosts but only in dead-end species. For example, adenoviruses do not cause cancer in humans but are instead responsible for colds, conjunctivitis and other acute illnesses. They only become tumorigenic when infected into certain rodent species, such as Syrian hamsters. Some viruses are tumorigenic when they infect a cell and persist as circular episomes or plasmids, replicating separately from host cell DNA (Epstein–Barr virus and Kaposi's sarcoma-associated herpesvirus). Other viruses are only carcinogenic when they integrate into the host cell genome as part of a biological accident, such as polyomaviruses and papillomaviruses. [ citation needed ]

A direct oncogenic viral mechanism [11] involves either insertion of additional viral oncogenic genes into the host cell or to enhance already existing oncogenic genes (proto-oncogenes) in the genome. For example, it has been shown that vFLIP and vCyclin interfere with the TGF-β signaling pathway indirectly by inducing oncogenic host mir17-92 cluster. [12]

Indirect viral oncogenicity involves chronic nonspecific inflammation occurring over decades of infection, as is the case for HCV-induced liver cancer. These two mechanisms differ in their biology and epidemiology: direct tumor viruses must have at least one virus copy in every tumor cell expressing at least one protein or RNA that is causing the cell to become cancerous. Because foreign virus antigens are expressed in these tumors, persons who are immunosuppressed such as AIDS or transplant patients are at higher risk for these types of cancers. [ citation needed ]

Chronic indirect tumor viruses, on the other hand, can be lost (at least theoretically) from a mature tumor that has accumulated sufficient mutations and growth conditions (hyperplasia) from the chronic inflammation of viral infection. In this latter case, it is controversial but at least theoretically possible that an indirect tumor virus could undergo "hit-and-run" and so the virus would be lost from the clinically diagnosed tumor. In practical terms, this is an uncommon occurrence if it does occur. [ citation needed ]

DNA oncoviruses typically impair two families of tumor suppressor proteins: tumor proteins p53 and the retinoblastoma proteins (Rb). It is evolutionarily advantageous for viruses to inactivate p53 because p53 can trigger cell cycle arrest or apoptosis in infected cells when the virus attempts to replicate its DNA. [13] Similarly, Rb proteins regulate many essential cell functions, including but not limited to a crucial cell cycle checkpoint, making them a target for viruses attempting to interrupt regular cell function. [14]

While several DNA oncoviruses have been discovered, three have been studied extensively. Adenoviruses can lead to tumors in rodent models but do not cause cancer in humans however, they have been exploited as delivery vehicles in gene therapy for diseases such as cystic fibrosis and cancer. [15] Simian virus 40 (SV40), a polyomavirus, can cause tumors in rodent models but is not oncogenic in humans. [16] This phenomenon has been one of the major controversies of oncogenesis in the 20th century because an estimated 100 million people were inadvertently exposed to SV40 through polio vaccines. [16] The human papillomavirus-16 (HPV-16) has been shown to lead to cervical cancer and other cancers, including head and neck cancer. [17] These three viruses have parallel mechanisms of action, forming an archetype for DNA oncoviruses. All three of these DNA oncoviruses are able to integrate their DNA into the host cell, and use this to transcribe it and transform cells by bypassing the G1/S checkpoint of the cell cycle. [ citation needed ]

Integration of viral DNA Edit

DNA oncoviruses transform infected cells by integrating their DNA into the host cell’s genome. [18] The DNA is believed to be inserted during transcription or replication, when the two annealed strands are separated. [18] This event is relatively rare and generally unpredictable there seems to be no deterministic predictor of the site of integration. [18] After integration, the host’s cell cycle loses regulation from Rb and p53, and the cell begins cloning to form a tumor. [ citation needed ]

G1/S Checkpoint Edit

Rb and p53 regulate the transition between G1 and S phase, arresting the cell cycle before DNA replication until the appropriate checkpoint inputs, such as DNA damage repair, are completed. [19] p53 regulates the p21 gene, which produces a protein which binds to the Cyclin D-Cdk4/6 complex. [20] This prevents Rb phosphorylation and prevents the cell from entering S phase. [20] In mammals, when Rb is active (unphosphorylated), it inhibits the E2F family of transcription factors, which regulate the Cyclin E-Cdk2 complex, which inhibits Rb, forming a positive feedback loop, keeping the cell in G1 until the input crosses a threshold. [19] To drive the cell into S phase prematurely, the viruses must inactivate p53, which plays a central role in the G1/S checkpoint, as well as Rb, which, though downstream of it, is typically kept active by a positive feedback loop. [ citation needed ]

Inactivation of p53 Edit

Viruses employ various methods of inactivating p53. The adenovirus E1B protein (55K) prevents p53 from regulating genes by binding to the site on p53 which binds to the genome. [13] In SV40, the large T antigen (LT) is an analogue LT also binds to several other cellular proteins, such as p107 and p130, on the same residues. [21] LT binds to p53’s binding domain on the DNA (rather than on the protein), again preventing p53 from appropriately regulating genes. [13] HPV instead degrades p53: the HPV protein E6 binds to a cellular protein called the E6-associated protein (E6-AP, also known as UBE3A), forming a complex which causes the rapid and specific ubiquitination of p53. [22]

Inactivation of Rb Edit

Rb is inactivated (thereby allowing the G1/S transition to progress unimpeded) by different but analogous viral oncoproteins. The adenovirus early region 1A (E1A) is an oncoprotein which binds to Rb and can stimulate transcription and transform cells. [13] SV40 uses the same protein for inactivating Rb, LT, to inactivate p53. [20] HPV contains a protein, E7, which can bind to Rb in much the same way. [23] Rb can be inactivated by phosphorylation, or by being bound to a viral oncoprotein, or by mutations—mutations which prevent oncoprotein binding are also associated with cancer. [21]

Variations Edit

DNA oncoviruses typically cause cancer by inactivating p53 and Rb, thereby allowing unregulated cell division and creating tumors. There may be many different mechanisms which have evolved separately in addition to those described above, for example, the Human Papillomavirus inactivates p53 by sequestering it in the cytoplasm. [13]

SV40 has been well studied and does not cause cancer in humans, but a recently discovered analogue called Merkel cell polyomavirus has been associated with Merkel cell carcinoma, a form of skin cancer. [24] The Rb binding feature is believed to be the same between the two viruses. [24]

In the 1960s, the replication process of RNA virus was believed to be similar to other single-stranded RNA. Single-stranded RNA replication involves RNA-dependent RNA synthesis which meant that virus-coding enzymes would make partial double-stranded RNA. This belief was proven to be incorrect because there were no double-stranded RNA found in the retrovirus cell. In 1964, Howard Temin proposed a provirus hypothesis, but shortly after reverse transcription in the retrovirus genome was discovered.

Description of virus Edit

All retroviruses have three major coding domains gag, pol and env. In the gag region of the virus, the synthesis of the internal virion proteins are maintained which make up the matrix, capsid and nucleocapsid proteins. In pol, the information for the reverse transcription and integration enzymes are stored. In env, it is derived from the surface and transmembrane for the viral envelope protein. There is a fourth coding domain which is smaller, but exists in all retroviruses. Pol is the domain that encodes the virion protease.

Retrovirus enters host cell Edit

The retrovirus begins the journey into a host cell by attaching a surface glycoprotein to the cell's plasma membrane receptor. Once inside the cell, the retrovirus goes through reverse transcription in the cytoplasm and generates a double-stranded DNA copy of the RNA genome. Reverse transcription also produces identical structures known as long terminal repeats (LTRs). Long terminal repeats are at the ends of the DNA strands and regulates viral gene expression. The viral DNA is then translocated into the nucleus where one strand of the retroviral genome is put into the chromosomal DNA by the help of the virion intergrase. At this point the retrovirus is referred to as provirus. Once in the chromosomal DNA, the provirus is transcribed by the cellular RNA polymerase II. The transcription leads to the splicing and full-length mRNAs and full-length progeny virion RNA. The virion protein and progeny RNA assemble in the cytoplasm and leave the cell, whereas the other copies send translated viral messages in the cytoplasm.

DNA viruses Edit

    (HPV), a DNA virus, causes transformation in cells through interfering with tumor suppressor proteins such as p53. Interfering with the action of p53 allows a cell infected with the virus to move into a different stage of the cell cycle, enabling the virus genome to be replicated. Forcing the cell into the S phase of the cell cycle could cause the cell to become transformed. [25] Human papillomavirus infection is a major cause of cervical cancer, vulvar cancer, vaginal cancer, penis cancer, anal cancer, and HPV-positiveoropharyngeal cancers. [7][26][27][28][29][30][31] There are nearly 200 distinct human papillomaviruses (HPVs), [29] and many HPV types are carcinogenic. [7][26] (HBV) is associated with Hepatocarcinoma[32] (EBV or HHV-4) is associated with four types of cancers (CMV or HHV-5) is associated with mucoepidermoid carcinoma and possibly other malignancies. [33] (KSHV or HHV-8) is associated with Kaposi’s sarcoma, a type of skin cancer. [34] – a polyoma virus – is associated with the development of Merkel cell carcinoma[24]

RNA viruses Edit

Not all oncoviruses are DNA viruses. Some RNA viruses have also been associated such as the hepatitis C virus as well as certain retroviruses, e.g., human T-lymphotropic virus (HTLV-1) and Rous sarcoma virus (RSV).

Overview table Edit

Virus Percent of cancers [7] Associated cancer types
Hepatitis B virus (HBV) Hepatocarcinoma [32]
Hepatitis C virus (HCV) HCV is a known carcinogen, causing hepatocarcinoma [35]
Human T-lymphotropic virus (HTLV) 0.03 Adult T-cell leukemia [36]
Human papillomaviruses (HPV) 5.2 HPV types 16 and 18 are associated with cancers of cervix, [7] [26] [27] [29] [30] anus, [7] [28] [29] penis, [7] [28] [29] vulva, [7] [28] [29] vagina, [7] [28] [29] and HPV-positive oropharyngeal cancers. [7] [28] [31] According to statistics in the United States, females are more impacted by HPV-associated cancers (83%) than males (74%). [37]
Kaposi's sarcoma-associated herpesvirus (HHV-8) 0.9 Kaposi’s sarcoma, multicentric Castleman's disease and primary effusion lymphoma
Merkel cell polyomavirus (MCV) NA Merkel cell carcinoma
Epstein–Barr virus (EBV) NA Burkitt's lymphoma, Hodgkin’s lymphoma, post-transplant lymphoproliferative disease, nasopharyngeal carcinoma [38] and a subtype of stomach cancer. [39]

Estimated percent of new cancers attributable to the virus worldwide in 2002. [7] NA indicates not available. The association of other viruses with human cancer is continually under research.

The main viruses associated with human cancers are the human papillomavirus, the hepatitis B and hepatitis C viruses, the Epstein–Barr virus, the human T-lymphotropic virus, the Kaposi's sarcoma-associated herpesvirus (KSHV) and the Merkel cell polyomavirus. Experimental and epidemiological data imply a causative role for viruses and they appear to be the second most important risk factor for cancer development in humans, exceeded only by tobacco usage. [40] The mode of virally induced tumors can be divided into two, acutely transforming or slowly transforming. In acutely transforming viruses, the viral particles carry a gene that encodes for an overactive oncogene called viral-oncogene (v-onc), and the infected cell is transformed as soon as v-onc is expressed. In contrast, in slowly transforming viruses, the virus genome is inserted, especially as viral genome insertion is an obligatory part of retroviruses, near a proto-oncogene in the host genome. The viral promoter or other transcription regulation elements in turn cause overexpression of that proto-oncogene, which in turn induces uncontrolled cellular proliferation. Because viral genome insertion is not specific to proto-oncogenes and the chance of insertion near that proto-oncogene is low, slowly transforming viruses have very long tumor latency compared to acutely transforming viruses, which already carry the viral oncogene. [ citation needed ]

Hepatitis viruses, including hepatitis B and hepatitis C, can induce a chronic viral infection that leads to liver cancer in 0.47% of hepatitis B patients per year (especially in Asia, less so in North America), and in 1.4% of hepatitis C carriers per year. Liver cirrhosis, whether from chronic viral hepatitis infection or alcoholism, is associated with the development of liver cancer, and the combination of cirrhosis and viral hepatitis presents the highest risk of liver cancer development. Worldwide, liver cancer is one of the most common, and most deadly, cancers due to a huge burden of viral hepatitis transmission and disease. [ citation needed ]

Through advances in cancer research, vaccines designed to prevent cancer have been created. The hepatitis B vaccine is the first vaccine that has been established to prevent cancer (hepatocellular carcinoma) by preventing infection with the causative virus. In 2006, the U.S. Food and Drug Administration approved a human papilloma virus vaccine, called Gardasil. The vaccine protects against four HPV types, which together cause 70% of cervical cancers and 90% of genital warts. In March 2007, the US Centers for Disease Control and Prevention (CDC) Advisory Committee on Immunization Practices (ACIP) officially recommended that females aged 11–12 receive the vaccine, and indicated that females as young as age 9 and as old as age 26 are also candidates for immunization. [ citation needed ]

The history of cancer virus discovery is intertwined with the history of cancer research and the history of virology. The oldest surviving record of on human cancer is the Babylonian Code of Hammurabi (dated ca. 1754 BC) but scientific oncology could only emerge in the 19th century, when tumors were studied at microscopic level with the help of the compound microscope and achromatic lenses. 19th century microbiology accumulated evidence that implicated bacteria, yeasts, fungi, and protozoa in the development of cancer. In 1926 the Nobel Prize was awarded for documenting that a nematode worm could provoke stomach cancer in rats. But it was not recognized that cancer could have infectious origins until much later as virus had first been discovered by Dmitri Ivanovsky and Martinus Beijerinck at the close of the 19th century. [41]

History of non-human oncoviruses Edit

The theory that cancer could be caused by a virus began with the experiments of Oluf Bang and Vilhelm Ellerman in 1908 at the University of Copenhagen. Bang and Ellerman demonstrated that avian sarcoma leukosis virus could be transmitted between chickens after cell-free filtration and subsequently cause leukemia. [42] [43] This was subsequently confirmed for solid tumors in chickens in 1910-1911 by Peyton Rous. [44] [45] Rous at the Rockefeller University extended Bang and Ellerman's experiments to show cell-free transmission of a solid tumor sarcoma to chickens (now known as Rous sarcoma). The reasons why chickens are so receptive to such transmission may involve unusual characteristics of stability or instability as they relate to endogenous retroviruses. [45] [46] Charlotte Friend confirmed Bang and Ellerman findings for liquid tumor in mice by . [47] In 1933 Richard Shope and Edward Weston Hurst showed that warts from wild cottontail rabbits contained the Shope papilloma virus. [41] In 1936 John Joseph Bittner identified the mouse mammary tumor virus, an "extrachromosomal factor" (i.e. virus) that could be transmitted between laboratory strains of mice by breast feeding. [48]

By the early 1950s, it was known that viruses could remove and incorporate genes and genetic material in cells. It was suggested that such types of viruses could cause cancer by introducing new genes into the genome. Genetic analysis of mice infected with Friend virus confirmed that retroviral integration could disrupt tumor suppressor genes, causing cancer. [49] Viral oncogenes were subsequently discovered and identified to cause cancer. [ citation needed ] Ludwik Gross identified the first mouse leukemia virus (murine leukemia virus) in 1951 [41] and in 1953 reported on a component of mouse leukemia extract capable of causing solid tumors in mice. [50] This compound was subsequently identified as a virus by Sarah Stewart and Bernice Eddy at the National Cancer Institute, after whom it was once called "SE polyoma". [51] [52] [53] In 1957 Charlotte Friend discovered the Friend virus, a strain of murine leukemia virus capable of causing cancers in immunocompetent mice. [47] Though her findings received significant backlash, they were eventually accepted by the field and cemented the validity of viral oncogenesis. [54]

In 1961 Eddy discovered the simian vacuolating virus 40 (SV40). Merck Laboratory also confirmed the existence of a rhesus macaque virus contaminating cells used to make Salk and Sabin polio vaccines. Several years later, it was shown to cause cancer in Syrian hamsters, raising concern about possible human health implications. Scientific consensus now strongly agrees that this is not likely to cause human cancer. [55] [56]

History of human oncoviruses Edit

In 1964 Anthony Epstein, Bert Achong and Yvonne Barr identified the first human oncovirus from Burkitt's lymphoma cells. A herpesvirus, this virus is formally known as human herpesvirus 4 but more commonly called Epstein–Barr virus or EBV. [57] In the mid 1960s Baruch Blumberg first physically isolated and characterized Hepatitis B while working at the National Institute of Health (NIH) and later the Fox Chase Cancer Center. [58] Although this agent was the clear cause of hepatitis and might contribute to liver cancer hepatocellular carcinoma, this link was not firmly established until epidemiologic studies were performed in the 1980s by R. Palmer Beasley and others. [59]

In 1980 the first human retrovirus, Human T-lymphotropic virus 1 (HTLV-I), was discovered by Bernard Poiesz and Robert Gallo at NIH, [60] [61] and independently by Mitsuaki Yoshida and coworkers in Japan. [62] But it was not certain whether HTLV-I promoted leukemia. In 1981 Yorio Hinuma and his colleagues at Kyoto University reported visualization of retroviral particles produced by a leukemia cell line derived from patients with Adult T-cell leukemia/lymphoma. This virus turned out to be HTLV-1 and the research established the causal role of the HTLV-1 virus to ATL. [41]

Between 1984 and 1986 Harald zur Hausen and Lutz Gissman discovered HPV16 and HPV18, together these Papillomaviridae viruses (HPV) are responsible for approximately 70% of human papillomavirus infections that cause cervical cancers. For the discovery that HPV cause human cancer the 2008 Nobel Prize was awarded. [63] In 1987 the Hepatitis C virus (HCV) was discovered by panning a cDNA library made from diseased tissues for foreign antigens recognized by patient sera. This work was performed by Michael Houghton at Chiron, a biotechnology company, and Daniel W. Bradley at the Centers for Disease Control and Prevention (CDC). [64] HCV was subsequently shown to be a major contributor to Hepatocellular carcinoma (liver cancer) worldwide. [41]

In 1994 Patrick S. Moore and Yuan Chang at Columbia University), working together with Ethel Cesarman, [65] [66] isolated Kaposi's sarcoma-associated herpesvirus (KSHV or HHV8) using representational difference analysis. This search was prompted by work from Valerie Beral and colleagues who inferred from the epidemic of Kaposi's sarcoma among patients with AIDS that this cancer must be caused by another infectious agent besides HIV, and that this was likely to be a second virus. [67] Subsequent studies revealed that KSHV is the "KS agent" and is responsible for the epidemiologic patterns of KS and related cancers. [68] In 2008 Yuan Chang and Patrick S. Moore developed a new method to identify cancer viruses based on computer subtraction of human sequences from a tumor transcriptome, called digital transcriptome subtraction (DTS). [69] DTS was used to isolate DNA fragments of Merkel cell polyomavirus from a Merkel cell carcinoma and it is now believed that this virus causes 70–80% of these cancers. [24]

All viruses bind to their hosts and introduce their genetic material into the host cell as part of their replication cycle. This genetic material contains basic 'instructions' of how to produce more copies of these viruses, hacking the body's normal production machinery to serve the needs of the virus. The host cell will carry out these instructions and produce additional copies of the virus, leading to more and more cells becoming infected. Some types of viruses insert their genome into the host's cytoplasm, but do not actually enter the cell. Others penetrate the cell membrane disguised as protein molecules and enter the cell.

There are two main types of virus infection: lytic and lysogenic. Shortly after inserting its DNA, viruses of the lytic cycle quickly produce more viruses, burst from the cell and infect more cells. Lysogenic viruses integrate their DNA into the DNA of the host cell and may live in the body for many years before responding to a trigger. The virus reproduces as the cell does and does not inflict bodily harm until it is triggered. The trigger releases the DNA from that of the host and employs it to create new viruses. [ citation needed ]

Retroviruses Edit

The genetic material in retroviruses is in the form of RNA molecules, while the genetic material of their hosts is in the form of DNA. When a retrovirus infects a host cell, it will introduce its RNA together with some enzymes, namely reverse transcriptase and integrase, into the cell. This RNA molecule from the retrovirus must produce a DNA copy from its RNA molecule before it can be integrated into the genetic material of the host cell. The process of producing a DNA copy from an RNA molecule is termed reverse transcription. It is carried out by one of the enzymes carried in the virus, called reverse transcriptase. After this DNA copy is produced and is free in the nucleus of the host cell, it must be incorporated into the genome of the host cell. That is, it must be inserted into the large DNA molecules in the cell (the chromosomes). This process is done by another enzyme carried in the virus called integrase. [ citation needed ]

Now that the genetic material of the virus has been inserted, it can be said that the host cell has been modified to contain new genes. If this host cell divides later, its descendants will all contain the new genes. Sometimes the genes of the retrovirus do not express their information immediately. [ citation needed ]

One of the problems of gene therapy using retroviruses is that the integrase enzyme can insert the genetic material of the virus into any arbitrary position in the genome of the host it randomly inserts the genetic material into a chromosome. If genetic material happens to be inserted in the middle of one of the original genes of the host cell, this gene will be disrupted (insertional mutagenesis). If the gene happens to be one regulating cell division, uncontrolled cell division (i.e., cancer) can occur. This problem has recently begun to be addressed by utilizing zinc finger nucleases [1] or by including certain sequences such as the beta-globin locus control region to direct the site of integration to specific chromosomal sites.

Gene therapy trials using retroviral vectors to treat X-linked severe combined immunodeficiency (X-SCID) represent the most successful application of gene therapy to date. More than twenty patients have been treated in France and Britain, with a high rate of immune system reconstitution observed. Similar trials were restricted or halted in the USA when leukemia was reported in patients treated in the French X-SCID gene therapy trial. [2] To date, four children in the French trial and one in the British trial have developed leukemia as a result of insertional mutagenesis by the retroviral vector. All but one of these children responded well to conventional anti-leukemia treatment. Gene therapy trials to treat SCID due to deficiency of the Adenosine Deaminase (ADA) enzyme (one form of SCID) [3] continue with relative success in the USA, Britain, Ireland, Italy and Japan. [ citation needed ]

Adenoviruses Edit

Adenoviruses are viruses that carry their genetic material in the form of double-stranded DNA. They cause respiratory, intestinal, and eye infections in humans (especially the common cold). When these viruses infect a host cell, they introduce their DNA molecule into the host. The genetic material of the adenoviruses is not incorporated (transient) into the host cell's genetic material. The DNA molecule is left free in the nucleus of the host cell, and the instructions in this extra DNA molecule are transcribed just like any other gene. The only difference is that these extra genes are not replicated when the cell is about to undergo cell division so the descendants of that cell will not have the extra gene. [ citation needed ]

As a result, treatment with the adenovirus will require readministration in a growing cell population although the absence of integration into the host cell's genome should prevent the type of cancer seen in the SCID trials. This vector system has been promoted for treating cancer and indeed the first gene therapy product to be licensed to treat cancer, Gendicine, is an adenovirus. Gendicine, an adenoviral p53-based gene therapy was approved by the Chinese food and drug regulators in 2003 for treatment of head and neck cancer. Advexin, a similar gene therapy approach from Introgen, was turned down by the US Food and Drug Administration (FDA) in 2008. [ citation needed ]

Concerns about the safety of adenovirus vectors were raised after the 1999 death of Jesse Gelsinger while participating in a gene therapy trial. Since then, work using adenovirus vectors has focused on genetically crippled versions of the virus. [ citation needed ]

Envelope protein pseudotyping of viral vectors Edit

The viral vectors described above have natural host cell populations that they infect most efficiently. Retroviruses have limited natural host cell ranges, and although adenovirus and adeno-associated virus are able to infect a relatively broader range of cells efficiently, some cell types are resistant to infection by these viruses as well. Attachment to and entry into a susceptible cell is mediated by the protein envelope on the surface of a virus. Retroviruses and adeno-associated viruses have a single protein coating their membrane, while adenoviruses are coated with both an envelope protein and fibers that extend away from the surface of the virus. The envelope proteins on each of these viruses bind to cell-surface molecules such as heparin sulfate, which localizes them upon the surface of the potential host, as well as with the specific protein receptor that either induces entry-promoting structural changes in the viral protein, or localizes the virus in endosomes wherein acidification of the lumen induces this refolding of the viral coat. In either case, entry into potential host cells requires a favorable interaction between a protein on the surface of the virus and a protein on the surface of the cell. [ citation needed ]

For the purposes of gene therapy, one might either want to limit or expand the range of cells susceptible to transduction by a gene therapy vector. To this end, many vectors have been developed in which the endogenous viral envelope proteins have been replaced by either envelope proteins from other viruses, or by chimeric proteins. Such chimera would consist of those parts of the viral protein necessary for incorporation into the virion as well as sequences meant to interact with specific host cell proteins. Viruses in which the envelope proteins have been replaced as described are referred to as pseudotyped viruses. For example, the most popular retroviral vector for use in gene therapy trials has been the lentivirus Simian immunodeficiency virus coated with the envelope proteins, G-protein, from Vesicular stomatitis virus. This vector is referred to as VSV G-pseudotyped lentivirus, and infects an almost universal set of cells. This tropism is characteristic of the VSV G-protein with which this vector is coated. Many attempts have been made to limit the tropism of viral vectors to one or a few host cell populations. This advance would allow for the systemic administration of a relatively small amount of vector. The potential for off-target cell modification would be limited, and many concerns from the medical community would be alleviated. Most attempts to limit tropism have used chimeric envelope proteins bearing antibody fragments. These vectors show great promise for the development of "magic bullet" gene therapies. [ citation needed ]

Replication-competent vectors Edit

A replication-competent vector called ONYX-015 is used in replicating tumor cells. It was found that in the absence of the E1B-55Kd viral protein, adenovirus caused very rapid apoptosis of infected, p53(+) cells, and this results in dramatically reduced virus progeny and no subsequent spread. Apoptosis was mainly the result of the ability of EIA to inactivate p300. In p53(-) cells, deletion of E1B 55kd has no consequence in terms of apoptosis, and viral replication is similar to that of wild-type virus, resulting in massive killing of cells. [ citation needed ]

A replication-defective vector deletes some essential genes. These deleted genes are still necessary in the body so they are replaced with either a helper virus or a DNA molecule. [4]

Cis and trans-acting elements Edit

Replication-defective vectors always contain a “transfer construct”. The transfer construct carries the gene to be transduced or “transgene”. The transfer construct also carries the sequences which are necessary for the general functioning of the viral genome: packaging sequence, repeats for replication and, when needed, priming of reverse transcription. These are denominated cis-acting elements, because they need to be on the same piece of DNA as the viral genome and the gene of interest. Trans-acting elements are viral elements, which can be encoded on a different DNA molecule. For example, the viral structural proteins can be expressed from a different genetic element than the viral genome. [4]

Herpes simplex virus Edit

The herpes simplex virus is a human neurotropic virus. This is mostly examined for gene transfer in the nervous system. The wild type HSV-1 virus is able to infect neurons and evade the host immune response, but may still become reactivated and produce a lytic cycle of viral replication. Therefore, it is typical to use mutant strains of HSV-1 that are deficient in their ability to replicate. Though the latent virus is not transcriptionally apparent, it does possess neuron specific promoters that can continue to function normally. [ further explanation needed ] Antibodies to HSV-1 are common in humans, however complications due to herpes infection are somewhat rare. [5] Caution for rare cases of encephalitis must be taken and this provides some rationale to using HSV-2 as a viral vector as it generally has tropism for neuronal cells innervating the urogenital area of the body and could then spare the host of severe pathology in the brain. [ citation needed ]

Non-viral methods present certain advantages over viral methods, with simple large scale production and low host immunogenicity being just two. Previously, low levels of transfection and expression of the gene held non-viral methods at a disadvantage however, recent advances in vector technology have yielded molecules and techniques with transfection efficiencies similar to those of viruses. [6]

Injection of naked DNA Edit

This is the simplest method of non-viral transfection. Clinical trials carried out of intramuscular injection of a naked DNA plasmid have occurred with some success however, the expression has been very low in comparison to other methods of transfection. In addition to trials with plasmids, there have been trials with naked PCR product, which have had similar or greater success. Cellular uptake of naked DNA is generally inefficient. Research efforts focusing on improving the efficiency of naked DNA uptake have yielded several novel methods, such as electroporation, sonoporation, and the use of a "gene gun", which shoots DNA coated gold particles into the cell using high pressure gas. [7]

Physical methods to enhance delivery Edit

Electroporation Edit

Electroporation is a method that uses short pulses of high voltage to carry DNA across the cell membrane. This shock is thought to cause temporary formation of pores in the cell membrane, allowing DNA molecules to pass through. Electroporation is generally efficient and works across a broad range of cell types. However, a high rate of cell death following electroporation has limited its use, including clinical applications.

More recently a newer method of electroporation, termed electron-avalanche transfection, has been used in gene therapy experiments. By using a high-voltage plasma discharge, DNA was efficiently delivered following very short (microsecond) pulses. Compared to electroporation, the technique resulted in greatly increased efficiency and less cellular damage.

Gene gun Edit

The use of particle bombardment, or the gene gun, is another physical method of DNA transfection. In this technique, DNA is coated onto gold particles and loaded into a device which generates a force to achieve penetration of the DNA into the cells, leaving the gold behind on a "stopping" disk.

Sonoporation Edit

Sonoporation uses ultrasonic frequencies to deliver DNA into cells. The process of acoustic cavitation is thought to disrupt the cell membrane and allow DNA to move into cells.

Magnetofection Edit

In a method termed magnetofection, DNA is complexed to magnetic particles, and a magnet is placed underneath the tissue culture dish to bring DNA complexes into contact with a cell monolayer.

Hydrodynamic delivery Edit

Hydrodynamic delivery involves rapid injection of a high volume of a solution into vasculature (such as into the inferior vena cava, bile duct, or tail vein). The solution contains molecules that are to be inserted into cells, such as DNA plasmids or siRNA, and transfer of these molecules into cells is assisted by the elevated hydrostatic pressure caused by the high volume of injected solution. [8] [9] [10]

Chemical methods to enhance delivery Edit

Oligonucleotides Edit

The use of synthetic oligonucleotides in gene therapy is to deactivate the genes involved in the disease process. There are several methods by which this is achieved. One strategy uses antisense specific to the target gene to disrupt the transcription of the faulty gene. Another uses small molecules of RNA called siRNA to signal the cell to cleave specific unique sequences in the mRNA transcript of the faulty gene, disrupting translation of the faulty mRNA, and therefore expression of the gene. A further strategy uses double stranded oligodeoxynucleotides as a decoy for the transcription factors that are required to activate the transcription of the target gene. The transcription factors bind to the decoys instead of the promoter of the faulty gene, which reduces the transcription of the target gene, lowering expression. Additionally, single stranded DNA oligonucleotides have been used to direct a single base change within a mutant gene. The oligonucleotide is designed to anneal with complementarity to the target gene with the exception of a central base, the target base, which serves as the template base for repair. This technique is referred to as oligonucleotide mediated gene repair, targeted gene repair, or targeted nucleotide alteration.

Lipoplexes Edit

To improve the delivery of the new DNA into the cell, the DNA must be protected from damage and positively charged. Initially, anionic and neutral lipids were used for the construction of lipoplexes for synthetic vectors. However, in spite of the facts that there is little toxicity associated with them, that they are compatible with body fluids and that there was a possibility of adapting them to be tissue specific they are complicated and time consuming to produce so attention was turned to the cationic versions.

Cationic lipids, due to their positive charge, were first used to condense negatively charged DNA molecules so as to facilitate the encapsulation of DNA into liposomes. Later it was found that the use of cationic lipids significantly enhanced the stability of lipoplexes. Also as a result of their charge, cationic liposomes interact with the cell membrane, endocytosis was widely believed as the major route by which cells uptake lipoplexes. Endosomes are formed as the results of endocytosis, however, if genes can not be released into cytoplasm by breaking the membrane of endosome, they will be sent to lysosomes where all DNA will be destroyed before they could achieve their functions. It was also found that although cationic lipids themselves could condense and encapsulate DNA into liposomes, the transfection efficiency is very low due to the lack of ability in terms of “endosomal escaping”. However, when helper lipids (usually electroneutral lipids, such as DOPE) were added to form lipoplexes, much higher transfection efficiency was observed. Later on, it was figured out that certain lipids have the ability to destabilize endosomal membranes so as to facilitate the escape of DNA from endosome, therefore those lipids are called fusogenic lipids. Although cationic liposomes have been widely used as an alternative for gene delivery vectors, a dose dependent toxicity of cationic lipids were also observed which could limit their therapeutic usages. [11]

The most common use of lipoplexes has been in gene transfer into cancer cells, where the supplied genes have activated tumor suppressor control genes in the cell and decrease the activity of oncogenes. Recent studies have shown lipoplexes to be useful in transfecting respiratory epithelial cells.

Polymersomes Edit

Polymersomes are synthetic versions of liposomes (vesicles with a lipid bilayer), made of amphiphilic block copolymers. They can encapsulate either hydrophilic or hydrophobic contents and can be used to deliver cargo such as DNA, proteins, or drugs to cells. Advantages of polymersomes over liposomes include greater stability, mechanical strength, blood circulation time, and storage capacity. [12] [13] [14]

Polyplexes Edit

Complexes of polymers with DNA are called polyplexes. [15] Most polyplexes consist of cationic polymers and their fabrication is based on self-assembly by ionic interactions. One important difference between the methods of action of polyplexes and lipoplexes is that polyplexes cannot directly release their DNA load into the cytoplasm. As a result, co-transfection with endosome-lytic agents such as inactivated adenovirus was required to facilitate nanoparticle escape from the endocytic vesicle made during particle uptake. However, a better understanding of the mechanisms by which DNA can escape from endolysosomal pathway, i.e. proton sponge effect, [16] has triggered new polymer synthesis strategies such as incorporation of protonable residues in polymer backbone and has revitalized research on polycation-based systems. [17]

Due to their low toxicity, high loading capacity, and ease of fabrication, polycationic nanocarriers demonstrate great promise compared to their rivals such as viral vectors which show high immunogenicity and potential carcinogenicity, and lipid-based vectors which cause dose dependence toxicity. Polyethyleneimine [18] and chitosan are among the polymeric carriers that have been extensively studied for development of gene delivery therapeutics. Other polycationic carriers such as poly(beta-amino esters) [19] and polyphosphoramidate [20] are being added to the library of potential gene carriers. In addition to the variety of polymers and copolymers, the ease of controlling the size, shape, surface chemistry of these polymeric nano-carriers gives them an edge in targeting capability and taking advantage of enhanced permeability and retention effect. [21]

Dendrimers Edit

A dendrimer is a highly branched macromolecule with a spherical shape. The surface of the particle may be functionalized in many ways and many of the properties of the resulting construct are determined by its surface.

In particular it is possible to construct a cationic dendrimer, i.e. one with a positive surface charge. When in the presence of genetic material such as DNA or RNA, charge complementarity leads to a temporary association of the nucleic acid with the cationic dendrimer. On reaching its destination the dendrimer-nucleic acid complex is then taken into the cell via endocytosis.

In recent years the benchmark for transfection agents has been cationic lipids. Limitations of these competing reagents have been reported to include: the lack of ability to transfect some cell types, the lack of robust active targeting capabilities, incompatibility with animal models, and toxicity. Dendrimers offer robust covalent construction and extreme control over molecule structure, and therefore size. Together these give compelling advantages compared to existing approaches.

Producing dendrimers has historically been a slow and expensive process consisting of numerous slow reactions, an obstacle that severely curtailed their commercial development. The Michigan-based company Dendritic Nanotechnologies discovered a method to produce dendrimers using kinetically driven chemistry, a process that not only reduced cost by a magnitude of three, but also cut reaction time from over a month to several days. These new "Priostar" dendrimers can be specifically constructed to carry a DNA or RNA payload that transfects cells at a high efficiency with little or no toxicity. [ citation needed ]

Inorganic nanoparticles Edit

Inorganic nanoparticles, such as gold, silica, iron oxide (ex. magnetofection) and calcium phosphates have been shown to be capable of gene delivery. [22] Some of the benefits of inorganic vectors is in their storage stability, low manufacturing cost and often time, low immunogenicity, and resistance to microbial attack. Nanosized materials less than 100 nm have been shown to efficiently trap the DNA or RNA and allows its escape from the endosome without degradation. Inorganics have also been shown to exhibit improved in vitro transfection for attached cell lines due to their increased density and preferential location on the base of the culture dish. Quantum dots have also been used successfully and permits the coupling of gene therapy with a stable fluorescence marker. Engineered organic nanoparticles are also under development, which could be used for co-delivery of genes and therapeutic agents. [23]

Cell-penetrating peptides Edit

Cell-penetrating peptides (CPPs), also known as peptide transduction domains (PTDs), are short peptides (< 40 amino acids) that efficiently pass through cell membranes while being covalently or non-covalently bound to various molecules, thus facilitating these molecules’ entry into cells. Cell entry occurs primarily by endocytosis but other entry mechanisms also exist. Examples of cargo molecules of CPPs include nucleic acids, liposomes, and drugs of low molecular weight. [24] [25]

CPP cargo can be directed into specific cell organelles by incorporating localization sequences into CPP sequences. For example, nuclear localization sequences are commonly used to guide CPP cargo into the nucleus. [26] For guidance into mitochondria, a mitochondrial targeting sequence can be used this method is used in protofection (a technique that allows for foreign mitochondrial DNA to be inserted into cells' mitochondria). [27] [28]

Due to every method of gene transfer having shortcomings, there have been some hybrid methods developed that combine two or more techniques. Virosomes are one example they combine liposomes with an inactivated HIV or influenza virus. This has been shown to have more efficient gene transfer in respiratory epithelial cells than either viral or liposomal methods alone. Other methods involve mixing other viral vectors with cationic lipids or hybridising viruses. [29]

A New Force

The idea that a symbiotic virus or any symbiotic relationship could have such a profound influence on the evolution of a new species is both new and controversial. For more than a century after Charles Darwin published On the Origin of Species , scientists focused on competition as evolution’s chief driving force. Biologist Lynn Margulis wasn’t convinced.

The late University of Massachusetts researcher believed that cooperation also played a role. Her evidence lurked in every cell of every plant and animal. Beginning in the late 1960s, Margulis argued that our cells contained symbiotic bacteria known as mitochondria and chloroplasts, which earned room and board by either supplying energy or producing food from sunlight. Margulis’s idea was ridiculed, and she struggled to find a journal that would publish her hypothesis.

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By the 1990s, however, enough genetic evidence had accumulated to show that Margulis was right. Symbiosis was responsible for some of the most significant evolutionary leaps in the history of the planet. Most scientists, however, viewed this event as an anomaly, a once-off freak occurrence that, although significant, didn’t play a role in the ongoing evolution of most species. Margulis, though, saw symbiosis everywhere and believed that this softer, gentler side of evolution was getting short shrift in research. Although most symbiosis research has focused on the role of the microbiome, the viruses tucked into our DNA can play a similar role in splitting apart two populations, turning one species into two. The first wedge scientists discovered was a protein called syncytin.

Viral Growth Curve

Unlike the growth curve for a bacterial population, the growth curve for a virus population over its life cycle does not follow a sigmoidal curve. During the initial stage, an inoculum of virus causes infection. In the eclipse phase, viruses bind and penetrate the cells with no virions detected in the medium. The chief difference that next appears in the viral growth curve compared to a bacterial growth curve occurs when virions are released from the lysed host cell at the same time. Such an occurrence is called a burst, and the number of virions per bacterium released is described as the burst size. In a one-step multiplication curve for bacteriophage, the host cells lyse, releasing many viral particles to the medium, which leads to a very steep rise in viral titer (the number of virions per unit volume). If no viable host cells remain, the viral particles begin to degrade during the decline of the culture (see Figure (PageIndex<8>)).

Figure (PageIndex<8>): The one-step multiplication curve for a bacteriophage population follows three steps: 1) inoculation, during which the virions attach to host cells 2) eclipse, during which entry of the viral genome occurs and 3) burst, when sufficient numbers of new virions are produced and emerge from the host cell. The burst size is the maximum number of virions produced per bacterium.

What aspect of the life cycle of a virus leads to the sudden increase in the growth curve?

Ebola is incurable and deadly. The outbreak in West Africa in 2014 was unprecedented, dwarfing other human Ebola epidemics in the level of mortality. Of 24,666 suspected or confirmed cases reported, 10,179 people died. 1

No approved treatments or vaccines for Ebola are available. While some drugs have shown potential in laboratory studies and animal models, they have not been tested in humans for safety and effectiveness. Not only are these drugs untested or unregistered but they are also in short supply.

Given the great suffering and high mortality rates, it is fair to ask whether unregistered and untested medications are better than none at all. Should such drugs be dispensed and, if so, who should receive them, in light of their extremely limited supplies? Is it ethical to treat untested drugs on patients with Ebola? On the other hand, is it ethical to withhold potentially life-saving drugs from dying patients? Or should the drugs perhaps be reserved for health-care providers working to contain the disease?

In August 2014, two infected US aid workers and a Spanish priest were treated with ZMapp, an unregistered drug that had been tested in monkeys but not in humans. The two American aid workers recovered, but the priest died. Later that month, the WHO released a report on the ethics of treating patients with the drug. Since Ebola is often fatal, the panel reasoned that it is ethical to give the unregistered drugs and unethical to withhold them for safety concerns. This situation is an example of &ldquocompassionate use&rdquo outside the well-established system of regulation and governance of therapies.

On September 24, 2014, Thomas Eric Duncan arrived at the Texas Health Presbyterian Hospital in Dallas complaining of a fever, headache, vomiting, and diarrhea&mdashsymptoms commonly observed in patients with the cold or the flu. After examination, an emergency department doctor diagnosed him with sinusitis, prescribed some antibiotics, and sent him home. Two days later, Duncan returned to the hospital by ambulance. His condition had deteriorated and additional blood tests confirmed that he has been infected with the Ebola virus.

Further investigations revealed that Duncan had just returned from Liberia, one of the countries in the midst of a severe Ebola epidemic. On September 15, nine days before he showed up at the hospital in Dallas, Duncan had helped transport an Ebola-stricken neighbor to a hospital in Liberia. The hospital continued to treat Duncan, but he died several days after being admitted.

The timeline of the Duncan case is indicative of the life cycle of the Ebola virus. The incubation time for Ebola ranges from 2 days to 21 days. Nine days passed between Duncan&rsquos exposure to the virus infection and the appearance of his symptoms. This corresponds, in part, to the eclipse period in the growth of the virus population. During the eclipse phase, Duncan would have been unable to transmit the disease to others. However, once an infected individual begins exhibiting symptoms, the disease becomes very contagious. Ebola virus is transmitted through direct contact with droplets of bodily fluids such as saliva, blood, and vomit. Duncan could conceivably have transmitted the disease to others at any time after he began having symptoms, presumably some time before his arrival at the hospital in Dallas. Once a hospital realizes a patient like Duncan is infected with Ebola virus, the patient is immediately quarantined, and public health officials initiate a back trace to identify everyone with whom a patient like Duncan might have interacted during the period in which he was showing symptoms.

Public health officials were able to track down 10 high-risk individuals (family members of Duncan) and 50 low-risk individuals to monitor them for signs of infection. None contracted the disease. However, one of the nurses charged with Duncan&rsquos care did become infected. This, along with Duncan&rsquos initial misdiagnosis, made it clear that US hospitals needed to provide additional training to medical personnel to prevent a possible Ebola outbreak in the US.

  1. What types of training can prepare health professionals to contain emerging epidemics like the Ebola outbreak of 2014?
  2. What is the difference between a contagious pathogen and an infectious pathogen?
Figure (PageIndex<9>): Researchers working with Ebola virus use layers of defenses against accidental infection, including protective clothing, breathing systems, and negative air-pressure cabinets for bench work. (credit: modification of work by Randal J. Schoepp)

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