Untitled Flashcards Set

Lecture 8 

Understanding the life cycle of viruses led to the development of drugs 

A drug against a viral protein / a drug against targets in the virus - one of the advantages of this type of drug is a very low chance of harming the cell itself or side effects. One of the disadvantages is the development of a resistance mechanism to the drug used by the virus. 

A drug against targets in the host cell (protein or cellular mechanism) - the advantage is that the chance of developing resistance is low, however, sometimes when the protein of the virus cells is changed, it can learn to bind it in a different way and then a type of resistance is created. The downside is a high chance of side effects.

In order to develop a drug, you have to choose the exact cellular protein that will be suitable to be used as a target. 

It is possible to genetically manipulate RNA + viruses in the laboratory. 

As you remember, to study the virus, you can take its mRNA, turn it into cDNA and insert it into a plasmid. 

When the genetic material is in the plasmid, you can begin to mutate it and infect cells - and thus you can find which mutation prevents the virus from growing. 

The virus has all kinds of proteins, some of which are responsible for building the capsid and some are types of enzymes. When we look for a target for a drug, we will want to focus on damaging proteins with enzymatic activity. The enzyme has a catalytic site, so it is possible to synthesize a molecule that will inhibit it. In addition, in most viruses, the enzymes are found in a smaller amount compared to the capsid and envelope proteins (the structural proteins). 

Drug testing in vitro - in the polio virus, which codes for a long polyprotein, one of the proteins that can be used as a target is the 2A (protease). It was discovered that mutations in 2A prevent the virus from replicating. in order to know where the protease cuts and which amino acids are important for its activity, it is possible to make a second series of mutations around the cutting sites - so that the exact sequence in which it cuts is discovered. As soon as we have both data, we go through an assay. We will not work with the virus but with purified protein 2A. For example, after  we discovered that the virus cuts in the sequence of amino acids TYAG (^ the place of the cut), will be connected to a peptide bead with the sequence of amino acids, to which we will connect a fluorophore (a fluorescent molecule). The hands sink to the bottom of the test tube, which causes the test tube solution to not be illuminated (because the fluorophore has sunk to the bottom together with the hands. When we put the A2 protease into the solution, it will cut in the TYG sequence, which will cause the solution to be fluorescent. After that, the potential drug being tested is added - so that if you find a drug that delays this cutting (=no fluorescence\decreasing fluorescence), it will be considered to work. 

Drug test in bacteria - we will test the growth of bacteria. For Dodge, we will take bacteria and insert into them a receptor resistant to tetracycline - a membrane protein that confers resistance to the tetracycline molecule. Inside the receptor there is a loop, into which a bacterial sequence can be inserted. In this example, insert a sequence of protease of the HIV virus. Once the protease is expressed, it cleaves the receptor, which inactivates its activity. In this situation - there will be no resistance to the bacteria against the tetracycline, and the bacteria will die. Potential drugs are added to the engineered bacteria, until you see growth of bacteria = addition 

The drug →  inhibition of the protease → no cutting →there is tetracycline resistance→  there is growth. This method is cheaper than working with human cells. 

Computer drug testing in silico - with this method, crystallography is done for the viral protein to find its structure. After that, potential drugs are taken and the computer is allowed to analyze their structure and match it to the structure of the viral protein. It is important to remember that in Driech after finding the inhibitory molecule and finding the structure, it is atomized in order to find the best drug. 

Finding the optimal dose of the drug - we will introduce viruses and a potential drug into the culture, and we will check the amount of viruses in the solution over time. If we see a moderate decrease and then a slight increase - we will know that the dose is too low and most likely the viruses have had time to replicate and develop resistance to the drug. If we see a sharp decrease and then a sharp increase - we will know that the dose is not good enough, direction that when a mutant virus was created in the replication process that succeeded in multiplying. When we see a sharp drop  and then spit out the mirror on zero bacteria, meaning replication of the virus is not possible at this dose - we will know that we have found the optimal dose for the medicine. 

Inhibition of a cellular protein as a cure for AIDS - CCR5 - As you remember, CCR5 is a receptor line that is not necessarily essential for proper function. The drug binds to CCR5 and inhibits it, so that HIV infection is prevented. 

Influenza virus and M2 protein - Influenza virus enters the cell inside an endosome into which protons enter and lower the Hc. The decrease in acidity causes the fusion that releases the virus segments and another thing: 

In lesson 7 we mentioned that the RNA of the influenza virus is packaged in the NP protein which has a signal to reach the cell nucleus. In the last infection phase of the virus, the M1 protein binds to the NP and hides the signal to reach the nucleus, so during the maturation of the virus the segments leave the nucleus to the cytoplasm. At the beginning of the infection cycle, the virus wants to reach the nucleus. The decrease in pH causes M1 to dissociate from the NP so that the segments can reach the nucleus. 

The M2 protein is a membrane protein of the influenza virus. Its function is to introduce the protons into the cavity of the virus, which, as mentioned, causes the release of the NP from the 1M - something that both helps in uncoating and in transporting the segments to the nucleus. Dogi for a drug that inhibits M2 - the drug blocks the passage of protons into the virion. The drug inhibits influenza A. The drug is directed against M2 and prevents the uncoating and the release of the segments into the nucleus. The drug has been around for 60 years, so strains resistant to the drug have been created. 

Replication inhibitors chain terminators - the drugs have a partial sugar residue that lacks OH in position 3 which is used for elongation, which causes the elongation of the genetic material to stop in the replication process. 

A molecule that is not yet active will be called a pro drug 

• A medicine called ACV consists of a nucleoside (meaning no phosphates at all). There is no cellular enzyme that recognizes this molecule and can add the first phosphate to it. Only in the cells infected with the herpes viruses which are in the lytic pathway, which express thymidine kinase - will there be an initial phosphorylation of the ACV - which will cause the drug to be activated. A situation is created where in the cells that are infected with the virus, a nucleotide will be created that can undergo incorporation - so that it will inhibit the replication of the virus (and also the cellular replication which will cause the cell death of the infected cells). 

• A drug consisting of nucleotides/nucleosides (preferably) that will inhibit a cellular enzyme such as AZT (an anti-RT drug). The HIV virus replicates in the cytoplasm, which causes AZT to specifically inhibit the RT of HIV. 

• A-cyclic cytosine - 80 times more effective at inhibiting HCMV polymerase 

• A nucleotide that preferentially inhibits the RT of hepatitis B.

Hepatitis C- HCV  virus causes jaundice, chronic liver disease and hypocellular carcinoma and has an RNA+ genome that produces a polyprotein. As you remember, RNA + viruses do not bring enzymes with them and therefore create a polyprotein. succeeded in developing a drug that inhibits the protease, a drug that inhibits NS5A (an essential protein whose role is to bind RNA and help the NS5B protein) and a drug that inhibits rdRp (NS5B). When you take these drugs in a cocktail, you turn a chronic incurable disease into a curable disease. 

Medicines can attack at different stages of the virus' life

What do viruses help us with? 

• Genetic therapy - production of retroviruses / lentiviruses with the help of which it is possible to integrate a desired gene into the genome and thus cause its expression over time. 

• Treatment of phages 

  • why? 

    • Bacteriophages infect bacteria and kill them, thus killing harmful bacteria 

    • Compared to antibiotics, bacteriophages are specific to bacteria, there are no long-term side effects (for example, when babies are given antibiotics, their microbiome changes forever, etc.), and there is no danger of developing antibiotic resistance.

    • It is possible to use genetically modified phages, certain types of phages, a cocktail of several phages or a combination of phages and antibiotics (antibiotics increase sensitivity to phage). 

    • To insert the DNA into the bacteria, the phages secrete enzymes that help break down the peptidoglycan layer. In the laboratory, these enzymes can be isolated in order to damage the bacteria. 

  • Ways of treatment -

• Ingestion - through pills 

• Inhalation - if it is bacteria in the respiratory system 

• eye drops 

• Injection into a muscle or vein 

• Ointment with phages - if there is an open wound

• How do you do it? 

  • The desired bacteria are isolated from the patient and grown in the laboratory 

  • There are phage libraries in laboratories and hospitals from which you can take some phages, or alternatively collect them from sewage water. The idea is that every bacterium has a phage that can attack it. 

  • The bacteria culture is checked to see if the phage can multiply and kill the bacteria in the culture. 

  • It is possible to test the phage-bacteria ratio in animals 

  • The phages found are given to the patient.

• Advantages -

  • specific to species of bacteria, so that their use will not harm the bacteria that are beneficial to us, therefore the pathogenic bacteria should be characterized. 

  • Proliferate only on the storage - when the storage disappears, the phages will also disappear. 

  • Low chance of creating resistance 

•  Disadvantages -

  • It takes a long time to characterize the bacterium and find/engineer a phage against it. 

  • Phages have the ability to carry genes that give bacteria resistance to antibiotics 

  • There is a danger that the phages will kill a large amount of bacteria at once, while dispersing and dispersing the contents of the bacteria (endotoxins), which may cause a strong reaction of the immune system. 

  • A problem with using a foreigner because the immune system produces antibodies against them. 

• solutions -

  • Endolysins - use of proteins produced by phages to break down the bacterial wall.

Oncolytic viruses - against cancer 

A patient who had leukemia in the past also got corona. A few weeks after the infection, they saw that the cancer had shrunk significantly. It is important to note that this is temporary but this is an example of how viruses can help us. 

When it comes to a cancerous cell, the immune system knows how to recognize it - so most cancerous tumors are eliminated by the system. Tumors that manage to grow do so by releasing substances that overwhelm the immune system. Today, most anti-cancer drugs are based on damaging the DNA so that dividing cells die; The problem is that it hurts in a way that doesn't specific even in normal cells. Hence the idea of ​​using viruses, which are specific, as a cure for cancer. In addition, the body's antiviral response systems are known to inhibit cancer cell growth. Another thing - with the help of viruses, it is possible to restore immune activity to the cancer cells. 

how does it work 

• When a virus infects a cell, it does so with the help of a specific receptor = contributes to specificity. 

• In addition, it is possible to engineer a virus that will attack certain receptors on cancer cells. 

• It is possible to verify that the virus will undergo replication in certain cells (sensitive and permissive), which will cause the cell to be killed 

Adenovirus - a virus with two important proteins 

• E1A whose role is to inhibit retinoblastoma so that E2F can do transcription and push the cells into the S phase. When the cells are pushed into the S phase, they activate the P53 protein which causes the tumor to stop and the damage to be repaired. If the cells fail to repair the damage, the cell will undergo apoptosis. 

• Another protein is the E1B whose function is to inhibit P53. 

It is possible to clone the adenovirus and delete E1B. When the virus infects a normal wt cell, there will be activation of p53 which will cause the virus to stop replicating.  Conversely, when the cell infects cells with mutant P53, it will replicate, killing the cell and more viruses will be produced.  50% of cancer cells have a mutation in p53, and this is a way of treating them. 

Two types of oncolytic viruses -

• Viruses that naturally multiply and kill only cancer cells  

  • include viruses of the digestive system 

  • include viruses that do not infect humans that will preferentially multiply in cancer cells 

• Viruses that are engineered to multiply and kill only cancer cells. 

  • Like a mutation in adenovirus in E1B or the HSV virus. 

RIGVIR virus - a virus isolated from children and found to kill only cancer cells. Approved in Latvia. 

Endovirus- with deletion of E1B. Approved in China. It is important to note that the goal is to encourage an immune response to cancer. 

HSV virus - approved as a drug for colon cancer by the FDA. HSV is mutated in the proteins ICP61 ICP34.5 which will not cause damage to neurons. ICP34.5 inhibits PKR (which phosphorylates), therefore when the gene is deleted - in normal cells there will be an induction of PKR that will stop the virus, and in cancer cells as PKR is silenced the virus can grow. In addition, viruses have proteins that prevent the infected cell from recognizing it and activating the immune system. ICP47 prevents the presentation of the antigen to the immune system, so the virus was engineered so that the protein was deleted - creating a situation where the virus calls the immune system to fight the cancer cell. GM-CSF cell protein is inserted into these viruses whose function is to attract 

The echo cells of the immune system fight the cancer cell. This is how we created an effective and specific virus for cancer.

Endogenous viruses 

The human genome is divided into

• Less than 2% is protein coded. 

• About 45% are mobile elements (transposons) which are scattered over the genome, sometimes used as promoters or as other regulatory regions (change splicing of the gene, region responsible for polyadenylation, etc.) 

  • LINE 1- 17%

    • Too much of it in introns will cause a gene to be low bit. 

  • SINE 13%

  • LTR 8%

In plants, there are between 50-98% mobile elements. 

Transposons cause diversity, and even survival under stress conditions. 

The transposons are divided into 2

• copy and paste process 

  • Most of the transposons in the genome are in a broken or partial state which prevents them from jumping, so a new copy is made from them. 

  • RNA is created from the transposon, from which cDNA is created that undergoes integration at a new place in the genome. 

    • will be called retro transposon. 

• cut and paste mechanism 

  • Transposons that can jump from place to place. 

  • The transposon is cut, jumps to another place in the genome and undergoes recombination. 

  • Make up 3% of the human genome

There is a group of transposons that look like retroviruses (with poly gag) and will therefore be called ERVS, which make up 8% of the human genome, and have an LTR. 

Another group is called Non-LTR Retrotransposons 

The LINE1 group are the largest group, which encode two genes - ORF1 which binds RNA, and ORF2 which is the RT endonuclease which causes the integration of the transposon into the genome. 

The SINE group - short elements that do not code for any protein, so they need the ORF2 of LINE1 to jump. contain regulatory elements, and their entry into places in the genome can constitute enhancers and so on. 

Of the 5,000 copies in the genome of LINE1, only 80-100 of them are active and jump in the genome. Each embryo contains new jumps that were not present in its parents. 

When active LINE1 is expressed, it exits the nucleus into the cytoplasm, so that ORF1+2 is formed, the RNA returns back to the nucleus and there a piece of the nuclear DNA is cut. That piece will be a primer for preparing cDNA from the transposon RNA, and it will undergo integration at a new location. 

There can also be cases where the DNA jumps to a new place such as the SINE.

Where is LINE1 expressed?

• In the initial stages in the embryo (and then will be silenced). 

• In neurons - when they overlapped certain neuron cells they found that there were 80 jumps of transposons that were not found in neighboring cells. This is apparently important for the function of these neurons, and also for the expression of the specific genes of the neurons. In neuronal cells LINE1 is expressed. 

• In cancer cells - methylation on the DNA causes silencing of the transposons, and in cancer cells there is a decrease in methylation. This causes the expression of the transposons which will affect in 2 ways -

• LINE1 can be trapped on the cancer cells 

• LINE1 can be a driver for cancer 

The importance of endogenous viruses 

An endogenous retrovirus called HERVH is a long non-coding RNA that is important for determining the identity of stem cells in the embryo. 

When the HERVH is silenced - the factors of the stem cells are lowered, which causes a reduced amount of IPS cells 

IPS cells - cells that are not stem cells, such as fibroblasts, which turn them into stem cells. 

Transcription factors that are responsible for pluripotency (the ability to differentiate into all cells in the body) have microRNAs that target them and silence them. Expression of HERVS causes it to "capture" these RNA molecules, which makes these transcription factors work. 

HERVH is very essential for stem cells.

In the fertilization phase, after the zygote, cell-2 is formed. At this stage, a factor called DUX is expressed whose function is to activate genes. The DUX maintains the cells in the cell-2 stage. The cells will remain in this phase until LINE1 is expressed to silence DUX. After DUX is silenced, cell division progresses to cell-4 and from there to form an embryo. 

When LINE1 is silenced, both at the RNA level and by inhibiting the RT itself, the 2-cell 4 transition is stopped and the cells will remain in the 4-cell state 

Therefore - the expression of LINE1 is part of the control over the development of the embryo. 

Each of the mammals uses a retrovirus protein to produce a placental structure.

How was it created? Apparently, the virus interacted with germ cells, causing integration into the genome. After integration into the genome there was no need for an envelope and so it dropped from the gene. 

They saw that infection with hepatitis B+C causes an increase in LINE1, which is essential for the development of liver cancer.

In Meir's laboratory they showed that the KSHV virus (a type of HIV) causes an increase in LINE1 - you see a lot of RT activity. Many of the HIV drugs also inhibit LINERT in the experiment, they took cancer cells containing KSHV, and gave treatment with RT inhibitors so that there is a delay in the growth of the cancer cells. In order to show the specificity, they silenced LINE1 in the cancer cells, which caused an inhibition in the growth of the cancer cells. 

Thus they discovered that an increase in LINE1 is essential for the growth of cancer cells, and perhaps inhibition of LINE1 can stop the growth of cancer cells.