Continuing on antiviral drugs...
While viruses have fewer targets for drugs due to their smaller number of gene products, there are still many targets for potential drugs. Processes such as viral attachment, absorption, fusion, replication, transcription, translation, assmebly, anf virion release are a few such targets.
Entry inhibitors: One particularly effective drug target is that of virus entry into the cell. If the virus cannot enter a cell, then it canont cause an infection. One example is TAK799, a drug that blocks the HIV gp120 protein from attaching to the CCR5 co-receptor on T-cells. This prevents viral fusion, effectively blocking the entry of the virus. Other examples are Heparin and dextran sulfate, which are effective against many viruses, and Amantadine, which is used against influenza (specifically, it blocks the action of the M2 ion pump, thereby preventing the pH change in the endosome, resulting in the virion being trapped).
Replication inhibitors: Inhibiting viral replication is a favoured target of antiviral drugs. Most of the replication inhibitors are analogues of nucleosides, and are also chain terminators. They can be acyclic GTP analogues, acyclic nucleoside phosphonates, dideoxynucleotides, and pyrophosphate analogues. There are some non-nucleoside inhibitors as well. A well known example is that of Lamivudine which is given for Hepatitis B and HIV. Also known as 3TC, it has a sulphur at the 3' carbon instead of a hydroxyl group. This means that the growing DNA chain cannot be extended and elongation is terminated. It is only incorporated by reverse transcriptase, and not by cellular replication machinery, giving it specificity for infected cells.
Usully the mechanism by which replication inhibitors work is complex. First the drug is activated by the viral thymidine kinase enzyme, and then is selectively use by the viral replication machinery. This means that specificity is dependand upon two factors: that cellular kinases do not recognize it as a substrate, and that the cellular replication machinery does not use it as a valid nucleoside. Strains which are resistant, then, can have mutations in two different aspects.
Non-nucleoside analogue inhibitors work by some other method. They may affect the binding of a nucleoside to the polymerase or affect protein oligmerization.
Protease inhibitors: Many viruses require proteases to cleave their gene products from polyproteins (such as HCV, or the HIV protease). By inhibiting these, the assembly of the virus is also inhibited.
Neuraminidase inhibitors: In influenza, the virion is assembled at the cellular membrane and is attached to the cell surface by sialic acid. Neuaminidase cleaves the sialic acid, freeing the virion. Drugs which inhibit this chemical thus prevent virus release.
Viral Stability Inhibitors: Viruses with lipid envelopes are sensitive to detergents. Using detergents to break up the envelope will render the virus avirulent. Nonoxynol-9 is one such reagent. It is commonly used in spermacides to prevent HIV and HSV infections. Another is ST-246 which is used against poxviruses.
Antiviral Drug Resistance
Resistance to antiviral drugs is as big of a problem as that of bacteria to antibiotics. For example, before the introduction of HAART (higly active antiretroviral therapy), the drug Foscornet was used to treat HCMV which caused retinitis in 30% of HIV patients. Unfortunately, HCMV strains quickly became resistant to the drug and it can no longer be used.
How can viruses become resistant to drugs?
Polymerase fidelity: DNA Polymerase has a proofreading ability which allows it to remove incorrect nucleotides and nucleoside analogues. This means that viruses that use DNA Polymerase may be albe to avoid incorporating the drug into its growing DNA chain. RNA polymerase, however, lacks proofreading ability, meaning RNA viruses have a much higer error rate, and consequently, mutation rate, than DNA viruses. This could be the cause behind the aparent restriction in genome size of RNA viruses.
Also, no polymerase is 100% efficent at proofreading; if a mistake is made, it could potentially lead to a mutation that confers resistance to a drug.
Quasispecies: Viruses do not reproduce clonally, so each virus may be different. They are subject to evolutionary selection pressures so many different forms of a virus are produced; a drug is then not effective against all of them. An example is the resistance of Cidofovir in orthopoxvirus.
How can we determine if resistance will be a problem?
1) Pass the virus through a cell culture, increasing the concentration of a drug
2) Select the strains which gain resistance
3) Sequence the polymerase gene (or other gene target, depending on the drug) and determine where the mutations lie
4) make recombinate strains that contain the mutations
5) check for resistace in cell cultures and in mice
The last three steps are known as "marker rescue".
How can resistance be prevented?
Use drugs more cleverly! Using two or more antiviral drugs which have independent mechanisms will reduce the chances of a strain becoming resistant. A "drug vacation" may also work by reducing the selective pressure on strains to become resistant (though this has the added risk of allowing virus titres to increase).
Showing posts with label 324. Show all posts
Showing posts with label 324. Show all posts
Sunday, 9 December 2007
Virus Prevention and Control Part I
As part of preperation for my immunology final, I'm going to write a bit about controlling and preventing virus outbreaks.
When people think of controling and preventing viruses, the first thought that comes to mind is, of cource, vaccines. But vaccines are only part of the picture. There are also "low tech" methods of virus control that can prove to be effective. First of all, by controling the quality of food and water, many viruses, like noravirus, can be prevented from spreading. Poor food and water quality is a major vector for virus spread. Likewise, insects can be a vector for viruses; controling insect populations thus controls these viruses. An example of this is the recent attempts at controling the mosquito populations in North America to stem the spread of West Nile Virus. Other animal populations also need to be controled - wild mammals, for instance, to keep rabies from being spread. A very important factor which also needs to be controlled is the use and sharing of needles to stop HIV and Hepatitis C infections. A few cities have taken the controversial option of providing "safe injection sites" to prevent drug users from using unsafe needles. Education about the dangers of sharing needles has also been used as a way of preventing these diseases. Quarintine is another method of keeping virus infections from spreading, as can be seen in the SARS outbreaks in Toronto a few years ago. These "low tech" solutions often prove to be as effective as vaccines.
When it comes to vaccines, however, two strageties are used. The first is to use a live, attenuated, vaccine and the other is to use a killed, subunit, or recombinant vaccine. Each has its benefits and hazards.
Attenuated vaccines: These vaccines are made up of living virus particles which have been "attenuated", or mutated, so that they are no longer a health risk. This provides a long lasting immunity that is both humoral (invokes antibodies) and cell-mediated. There is a risk, however, that the live virus can mutate back into the fully hazardous form. This risk is minimized by using multiple non-revertable mutations to make the attenuated form. Nonetheless, this is still a risky method to use in people who are immunocompromised.
A good example of an attenuated vaccine in action is the Sabin polio vaccine. Using three different strains of the polio virus, the strains were serially passed (ie, passed from one host to another) through multiple cell tissue types. The resulting virus had accumulated non-revertable mutations in the 5' UTR and in the viral VP3 capsid protein. This resulted in a avirulant strain which was used very effectively as a vaccine.
Killed/Subunit/Recombinant Vaccines: These viruses are not living, mutated forms. They are whole or partial subunits of viruses that have been killed by heat or chemicals. They are not as effective as attenuated viruses because the immunity they provide is not as long lived and often only provokes a humoral response. Nevertheless, they are safer to use.
An example of this is the HPV vaccine. The vaccine is a mix of VLPs (virus-like particles) which consist of the viral L1 capsid protein. It gives immunity to HPV types 6, 11, 16 and 16. Interestingly, this vaccine has created some ethical questions surrounding vaccinations. HPV poses a health hazard to women but not to men. However, men can act as carriers. Should men get vaccinated even though it gives no personal benefit to them?
Antiviral Drugs: If vaccines arent a feasible option, though, then there's the option of antiviral drugs. Antiviral drugs are not as easy to come by as antibiotics. Viruses dont have nearly as many genes as bacteria, so there are not as many targets for the drugs to work against. Also, many potential antiviral drugs have toxic effects on human cells. Only a handful of antiviral drugs are known, most of which have been discovered in the last 25 years, but the rate of discovery is increasing.
How are these drugs found? There are multiple ways:
Plaque reduction assays: The virus is plated onto a lawn on tissue, forming viral plaques. An increasing amount of the potential drug is added to the plates and the effect on the plaque number is observed. The higher the decrease in plaques, the better the drug works. To determine how effective the drug is to use practically, however, the Selectivity Index (SI) of the drug must be calculated: SI=CC50/EC50 where
CC50= the concentration of the drug that kills 50% of the host cells (Cytotoxic concentration)
EC50 = the concentration that kills 50% of the virus (Effective concentration)
The higher the SI, the more effective the drug is.
Viral growth inhibition assays: The growth of a virus versus the concentration of a drug can also be visualized and used to determine the drugs effectiveness. One method is to use 3H-Thymine (radioactive thymine) to measure the amount of viral DNA replication that occurs when different concentrations of drug are applied. Another method is to use a fluorescant protein like GFP or another visual marker like the lux operon or Lac Z operon to visualize the actual viral particles in stitu or in animal tissues. The effect of differeing concentrations can then be visualized.
Enzyme inhibition assays: These assays determine how effective a drug is on a certain virus enzyme. The test require a certain amount of knowledge concerning the pharmacology of the drugs to be tested because they have to use the form of the drug that is metabolically active within cells. One technique commonly used is FRET (fluorescene resonance energy transfer). This involves measuring the amout of light emitted by a fluorecant dye versus the drug concentration. For example, if the drug target being tested was an HIV protease, then a molecule would be constricted consisting of a protein (that the protease cleaves) with a dye on one end and a quench molecule on the other. Under normal circumstances, the fluorescant dye would not emit light because the quench molecule would quench it. With an active HIV protease however, the protein would be cleaved and the flourescant dye would be free to emit light. If the drug is applied, however, then the protease would not work and there would be no light emitted. By determining the amout of light given off (or lack thereof) one can determine how effective the drug is against that HIV protease.
This type of assay can be easily done by automated machines in multi-well titre plates, resulting in high throughput screening of potential drugs against particular drug targets.
When people think of controling and preventing viruses, the first thought that comes to mind is, of cource, vaccines. But vaccines are only part of the picture. There are also "low tech" methods of virus control that can prove to be effective. First of all, by controling the quality of food and water, many viruses, like noravirus, can be prevented from spreading. Poor food and water quality is a major vector for virus spread. Likewise, insects can be a vector for viruses; controling insect populations thus controls these viruses. An example of this is the recent attempts at controling the mosquito populations in North America to stem the spread of West Nile Virus. Other animal populations also need to be controled - wild mammals, for instance, to keep rabies from being spread. A very important factor which also needs to be controlled is the use and sharing of needles to stop HIV and Hepatitis C infections. A few cities have taken the controversial option of providing "safe injection sites" to prevent drug users from using unsafe needles. Education about the dangers of sharing needles has also been used as a way of preventing these diseases. Quarintine is another method of keeping virus infections from spreading, as can be seen in the SARS outbreaks in Toronto a few years ago. These "low tech" solutions often prove to be as effective as vaccines.
When it comes to vaccines, however, two strageties are used. The first is to use a live, attenuated, vaccine and the other is to use a killed, subunit, or recombinant vaccine. Each has its benefits and hazards.
Attenuated vaccines: These vaccines are made up of living virus particles which have been "attenuated", or mutated, so that they are no longer a health risk. This provides a long lasting immunity that is both humoral (invokes antibodies) and cell-mediated. There is a risk, however, that the live virus can mutate back into the fully hazardous form. This risk is minimized by using multiple non-revertable mutations to make the attenuated form. Nonetheless, this is still a risky method to use in people who are immunocompromised.
A good example of an attenuated vaccine in action is the Sabin polio vaccine. Using three different strains of the polio virus, the strains were serially passed (ie, passed from one host to another) through multiple cell tissue types. The resulting virus had accumulated non-revertable mutations in the 5' UTR and in the viral VP3 capsid protein. This resulted in a avirulant strain which was used very effectively as a vaccine.
Killed/Subunit/Recombinant Vaccines: These viruses are not living, mutated forms. They are whole or partial subunits of viruses that have been killed by heat or chemicals. They are not as effective as attenuated viruses because the immunity they provide is not as long lived and often only provokes a humoral response. Nevertheless, they are safer to use.
An example of this is the HPV vaccine. The vaccine is a mix of VLPs (virus-like particles) which consist of the viral L1 capsid protein. It gives immunity to HPV types 6, 11, 16 and 16. Interestingly, this vaccine has created some ethical questions surrounding vaccinations. HPV poses a health hazard to women but not to men. However, men can act as carriers. Should men get vaccinated even though it gives no personal benefit to them?
Antiviral Drugs: If vaccines arent a feasible option, though, then there's the option of antiviral drugs. Antiviral drugs are not as easy to come by as antibiotics. Viruses dont have nearly as many genes as bacteria, so there are not as many targets for the drugs to work against. Also, many potential antiviral drugs have toxic effects on human cells. Only a handful of antiviral drugs are known, most of which have been discovered in the last 25 years, but the rate of discovery is increasing.
How are these drugs found? There are multiple ways:
Plaque reduction assays: The virus is plated onto a lawn on tissue, forming viral plaques. An increasing amount of the potential drug is added to the plates and the effect on the plaque number is observed. The higher the decrease in plaques, the better the drug works. To determine how effective the drug is to use practically, however, the Selectivity Index (SI) of the drug must be calculated: SI=CC50/EC50 where
CC50= the concentration of the drug that kills 50% of the host cells (Cytotoxic concentration)
EC50 = the concentration that kills 50% of the virus (Effective concentration)
The higher the SI, the more effective the drug is.
Viral growth inhibition assays: The growth of a virus versus the concentration of a drug can also be visualized and used to determine the drugs effectiveness. One method is to use 3H-Thymine (radioactive thymine) to measure the amount of viral DNA replication that occurs when different concentrations of drug are applied. Another method is to use a fluorescant protein like GFP or another visual marker like the lux operon or Lac Z operon to visualize the actual viral particles in stitu or in animal tissues. The effect of differeing concentrations can then be visualized.
Enzyme inhibition assays: These assays determine how effective a drug is on a certain virus enzyme. The test require a certain amount of knowledge concerning the pharmacology of the drugs to be tested because they have to use the form of the drug that is metabolically active within cells. One technique commonly used is FRET (fluorescene resonance energy transfer). This involves measuring the amout of light emitted by a fluorecant dye versus the drug concentration. For example, if the drug target being tested was an HIV protease, then a molecule would be constricted consisting of a protein (that the protease cleaves) with a dye on one end and a quench molecule on the other. Under normal circumstances, the fluorescant dye would not emit light because the quench molecule would quench it. With an active HIV protease however, the protein would be cleaved and the flourescant dye would be free to emit light. If the drug is applied, however, then the protease would not work and there would be no light emitted. By determining the amout of light given off (or lack thereof) one can determine how effective the drug is against that HIV protease.
This type of assay can be easily done by automated machines in multi-well titre plates, resulting in high throughput screening of potential drugs against particular drug targets.
Tuesday, 30 October 2007
What I learned in school today Oct 30
Today's lecture was a guest lecture concerning viruses and their role in cancer, particularly how they can cause cancer and can be used to treat cancer.
What is cancer?
Cancer, in short, is an uncontrolled proliferation of cells. It is the 2nd leading cause of death in developed nations - 6 million people worldwide die from cancer every year. Three main therapies are used to treat it: radiation, chemotherapy and surgery. The first two are specific in that they target cells that are rapidly dividing. This may be problematic in that tumor cells are not the only cells that divide rapidly; certain bone cells, for example, quickly divide and may be targeted by these treatments.
The process whereby a normal cell becomes cancerous is called transformation. A transformation may be a change in the morphological, biochemical or growth patterns of a cell. Cells which are transformed become immortal, that is, they can grow and divide indefinately. Normal cells are subject to what is called the Hayflick Limit, at which a cell will stop dividing (this occurs when the telomeres are shortened to a length below which becomes deleterious to the cell). Cells which are transformed may have one or more altered phenotypes, such as a loss of anchorage dependance (normal cells will not grow unless attached to the extracellular matrix), loss of contact inhibition (normal cells will stop growing when they fill the available space and contact one another), a decreased requirement for growth factors (i.e. they will continue to grow even when not supplied with the signal to grow), or gross morphological changes.
Both RNA viruses and DNA viruses may transform cells into cancer cells. There are three ways in which viruses can do this: by expressing a modified version of a cellular proto-oncogene, by cis-activation of proto-oncogenes or trans-activation of proto-oncogenes.
[Proto-oncogenes are genes that, when mutated, result in the formation of tumors]
Expression of Modified Proto-oncogenes
An example of this method of cancer induction comes from the Rous Sarcoma Virus, the first oncogenic virus studied. This virus contains a gene called v-src. It is very homologous to a cellular gene called c-src (v=viral, c=cellular). The c-src gene product normally encodes for a tyrosine kinase. It contains two domains at one end of the protein (in the kinase domain), the SH3 and SH2 domains. The SH2 domain is of particular importance; it binds to phosphotyrosine residues. At the opposite end of the c-src protein lies a regulatory domain. The 527th amino acid in the protein lies in this region, and is a tyrosine. This tyrosine gets phosphorylated, and the SH2 region of the kinase domain binds to it. This results in an inactive protein. The usual fuction of c-src is to work in a signaling cascade to turn on transcription factors that will express certain genes involved in the cell cycle; when it is on, cells grow and divide. When inactive, these genes do not get expressed, and no cell growth.
The v-src gene, however, encodes for a truncated protein. It is cut off at the 526th amino acid residue. That is, it does not contain the Tyrosine residue required to keep the enzyme inactive. When expressed in a host cell, it is always turned on, resulting in contant expression of the downstream genes, and uncontrolled cellular growth and division - in other words, tumor formation.
Cis-activation of proto-oncogenes
This method involves the integration of a viral genome in to the host genome. Eukaryotic cells are often tightly regulated, with promotor sequences, and regulatory or enhancer elements controlling the expression of the gene. Insertion of the viral genome is often a random process, and in the event that the genome were to insert into the repressor element of a gene, then the repressor would become nonfunctional. This would lead to an inability to turn the downstream gene off when it is not needed. The gene would be constitutively expressed. If this gene is a proto-oncogene, then the result is likely to be the formation of a tumor.
[This is called CIS-activation because the viral agent that is causing the activation is in cis, that is, is on the same chromosome, as the gene it is infecting. "Cis" comes from "cistron", the old name for genes.]
Trans-activation of proto-oncogenes
This method involves a viral protein directly acting on a transcription factor, which causes proto-oncogenes to be upregulated. The resulting upregulation can lead to increased or unconrtolable cellular growth.
Examples
An example of how viruses can cause cancer can be seen in the Papillomavirus family of DNA viruses. There are over 106 types of Papillomavirus, and over 40 of these are able to infect the mucosal surfaces of the the genetail and aero-digestive tracts. HPV (Human Papillomavirus ) has been detected in some 99.7% of cervical cancers.
HPV has two primary viral oncoproteins: E6 and E7. They are involved in disregulating the cell cycle. During the cell cycle, cells go through a phase of DNA replication, known as S-phase. If there is damage to the DNA, or errors with improper cell division, replicating the DNA during S-phase can result in dire problems for the cell. Tumor supressor genes will stop the cell from completing S-phase if this is the case. The HPV E6 and E7 proteins block the action of these tumor supressor genes, allowing cells to go through S-phase, and thus DNA replication, growth and division, unregulated.
One tumor supressor that is affected by HPV is the Retinoblastoma protein (Rb). Rb binds a transcription factor called E2F, and keeps it from fuctioning. When E2F is needed, Rb is phosphorylated and E2F dissociates and is allowed to activate the transcription of genes involved in DNA synthesis, and consequently, progression through the cell cycle. The HPV E7 protein interferes with the E2F:Rb complex - E2F is constantly active, leading to unscheduled cellular DNA synthesis as well as viral DNA synthesis. Also, there is a "High Risk" form of E7 where Rb is not only bound but is targeted for proteosome-mediated degredation.
The cell has a safeguard against this, however. The p53 protein is translated under cellular stress or DNA damage, and it halts the proliferation of cells, and plays a role in apoptosis. This ensures that defective cells do not run the risk of becoming cancerous. This is where the viral E6 protein comes into play - it binds p53 and blocks it from fuctioning. Much like E7, E6 has a 'high risk' variant. This form of E6 recruits an E2 ubiquitin ligase which polyubiquinates the p53, marking it for degradation via the proteosome.
From the above, it can be seen that DNA and RNA viruses have deifferent roles in cancer. RNA viruses encode for viral concogenes, while DNA viruses have mechanisms evolved to counter cellular tumor-supressor genes.
Oncolytic viruses
Another area of cancer that viruses play a role is in cancer therapy. Oncolytic viruses are viruses that are used as a form of cancer therapy. In this method, live viruses are used to selectively lyse cancerous cells, while normal cells are left unaffected. The viruses infect tumor cells, replicate, lyse the cells and then spread to other uninfected tumor cells. For some viruses, this selectivity is not perfect and normal cells can be infected; however, the viruses will grow better in tumor cells than in normal cells and (hopefully) will pick tumor cells over normal ones. The ideal features of such a virus are that they cause only mild human diseases that are well characterized, have secondary inactivation mechanisms, do not damage normal cells, have low mutation and recombination frequencies, do not spread from host to host and cannot integrate into the host genome.
Once such oncolytic virus is Onyx 015, a modified version of Adenovirus. This variant lacks the adenovirus E1b55K and E1b19K proteins which block p53 and apoptosis. It is hypothesized that its E1a protein binds Rb and E2F leading to increased DNA replication of the viral genome, and lysis of the tumor cells. What keeps it from destroying normal cells is that this also activates p53, and p53 keeps the infected normal cells from spreading the virus. The infected tumor cells do not have p53 and thus are unable to stop lysis and spread of the virus to other tumor cells.
Another such virus is myxoma virus. This virus usually infects European rabbits and not humans. However, it has the ability to infect human tumor cells instead. The benefit of this virus over the Onyx 015 is that it is natural rather than engineered.
What is cancer?
Cancer, in short, is an uncontrolled proliferation of cells. It is the 2nd leading cause of death in developed nations - 6 million people worldwide die from cancer every year. Three main therapies are used to treat it: radiation, chemotherapy and surgery. The first two are specific in that they target cells that are rapidly dividing. This may be problematic in that tumor cells are not the only cells that divide rapidly; certain bone cells, for example, quickly divide and may be targeted by these treatments.
The process whereby a normal cell becomes cancerous is called transformation. A transformation may be a change in the morphological, biochemical or growth patterns of a cell. Cells which are transformed become immortal, that is, they can grow and divide indefinately. Normal cells are subject to what is called the Hayflick Limit, at which a cell will stop dividing (this occurs when the telomeres are shortened to a length below which becomes deleterious to the cell). Cells which are transformed may have one or more altered phenotypes, such as a loss of anchorage dependance (normal cells will not grow unless attached to the extracellular matrix), loss of contact inhibition (normal cells will stop growing when they fill the available space and contact one another), a decreased requirement for growth factors (i.e. they will continue to grow even when not supplied with the signal to grow), or gross morphological changes.
Both RNA viruses and DNA viruses may transform cells into cancer cells. There are three ways in which viruses can do this: by expressing a modified version of a cellular proto-oncogene, by cis-activation of proto-oncogenes or trans-activation of proto-oncogenes.
[Proto-oncogenes are genes that, when mutated, result in the formation of tumors]
Expression of Modified Proto-oncogenes
An example of this method of cancer induction comes from the Rous Sarcoma Virus, the first oncogenic virus studied. This virus contains a gene called v-src. It is very homologous to a cellular gene called c-src (v=viral, c=cellular). The c-src gene product normally encodes for a tyrosine kinase. It contains two domains at one end of the protein (in the kinase domain), the SH3 and SH2 domains. The SH2 domain is of particular importance; it binds to phosphotyrosine residues. At the opposite end of the c-src protein lies a regulatory domain. The 527th amino acid in the protein lies in this region, and is a tyrosine. This tyrosine gets phosphorylated, and the SH2 region of the kinase domain binds to it. This results in an inactive protein. The usual fuction of c-src is to work in a signaling cascade to turn on transcription factors that will express certain genes involved in the cell cycle; when it is on, cells grow and divide. When inactive, these genes do not get expressed, and no cell growth.
The v-src gene, however, encodes for a truncated protein. It is cut off at the 526th amino acid residue. That is, it does not contain the Tyrosine residue required to keep the enzyme inactive. When expressed in a host cell, it is always turned on, resulting in contant expression of the downstream genes, and uncontrolled cellular growth and division - in other words, tumor formation.
Cis-activation of proto-oncogenes
This method involves the integration of a viral genome in to the host genome. Eukaryotic cells are often tightly regulated, with promotor sequences, and regulatory or enhancer elements controlling the expression of the gene. Insertion of the viral genome is often a random process, and in the event that the genome were to insert into the repressor element of a gene, then the repressor would become nonfunctional. This would lead to an inability to turn the downstream gene off when it is not needed. The gene would be constitutively expressed. If this gene is a proto-oncogene, then the result is likely to be the formation of a tumor.
[This is called CIS-activation because the viral agent that is causing the activation is in cis, that is, is on the same chromosome, as the gene it is infecting. "Cis" comes from "cistron", the old name for genes.]
Trans-activation of proto-oncogenes
This method involves a viral protein directly acting on a transcription factor, which causes proto-oncogenes to be upregulated. The resulting upregulation can lead to increased or unconrtolable cellular growth.
Examples
An example of how viruses can cause cancer can be seen in the Papillomavirus family of DNA viruses. There are over 106 types of Papillomavirus, and over 40 of these are able to infect the mucosal surfaces of the the genetail and aero-digestive tracts. HPV (Human Papillomavirus ) has been detected in some 99.7% of cervical cancers.
HPV has two primary viral oncoproteins: E6 and E7. They are involved in disregulating the cell cycle. During the cell cycle, cells go through a phase of DNA replication, known as S-phase. If there is damage to the DNA, or errors with improper cell division, replicating the DNA during S-phase can result in dire problems for the cell. Tumor supressor genes will stop the cell from completing S-phase if this is the case. The HPV E6 and E7 proteins block the action of these tumor supressor genes, allowing cells to go through S-phase, and thus DNA replication, growth and division, unregulated.
One tumor supressor that is affected by HPV is the Retinoblastoma protein (Rb). Rb binds a transcription factor called E2F, and keeps it from fuctioning. When E2F is needed, Rb is phosphorylated and E2F dissociates and is allowed to activate the transcription of genes involved in DNA synthesis, and consequently, progression through the cell cycle. The HPV E7 protein interferes with the E2F:Rb complex - E2F is constantly active, leading to unscheduled cellular DNA synthesis as well as viral DNA synthesis. Also, there is a "High Risk" form of E7 where Rb is not only bound but is targeted for proteosome-mediated degredation.
The cell has a safeguard against this, however. The p53 protein is translated under cellular stress or DNA damage, and it halts the proliferation of cells, and plays a role in apoptosis. This ensures that defective cells do not run the risk of becoming cancerous. This is where the viral E6 protein comes into play - it binds p53 and blocks it from fuctioning. Much like E7, E6 has a 'high risk' variant. This form of E6 recruits an E2 ubiquitin ligase which polyubiquinates the p53, marking it for degradation via the proteosome.
From the above, it can be seen that DNA and RNA viruses have deifferent roles in cancer. RNA viruses encode for viral concogenes, while DNA viruses have mechanisms evolved to counter cellular tumor-supressor genes.
Oncolytic viruses
Another area of cancer that viruses play a role is in cancer therapy. Oncolytic viruses are viruses that are used as a form of cancer therapy. In this method, live viruses are used to selectively lyse cancerous cells, while normal cells are left unaffected. The viruses infect tumor cells, replicate, lyse the cells and then spread to other uninfected tumor cells. For some viruses, this selectivity is not perfect and normal cells can be infected; however, the viruses will grow better in tumor cells than in normal cells and (hopefully) will pick tumor cells over normal ones. The ideal features of such a virus are that they cause only mild human diseases that are well characterized, have secondary inactivation mechanisms, do not damage normal cells, have low mutation and recombination frequencies, do not spread from host to host and cannot integrate into the host genome.
Once such oncolytic virus is Onyx 015, a modified version of Adenovirus. This variant lacks the adenovirus E1b55K and E1b19K proteins which block p53 and apoptosis. It is hypothesized that its E1a protein binds Rb and E2F leading to increased DNA replication of the viral genome, and lysis of the tumor cells. What keeps it from destroying normal cells is that this also activates p53, and p53 keeps the infected normal cells from spreading the virus. The infected tumor cells do not have p53 and thus are unable to stop lysis and spread of the virus to other tumor cells.
Another such virus is myxoma virus. This virus usually infects European rabbits and not humans. However, it has the ability to infect human tumor cells instead. The benefit of this virus over the Onyx 015 is that it is natural rather than engineered.
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