Redesigning the Protein Sequence of the H5N1 Avian Influenza

[deleted-290678]

Hello Science Buddies,

For my school's annual science fair competition; all tenth graders are required to complete an independent research project and I have chosen to research a project in the category of Immunology/Virology. Upon looking through the science buddies site, I came across the project of BLASTING flu viruses, and I thought that would be a good place for me to start getting ideas. Thankfully, the page was very helpful, but the only flu virus they mentioned actually researching on were seasonal influenza's. I wanted to look onto something different, so I picked the Avian Influenza. Researching on the internet helped me understand the difference between this type of virus and a seasonal virus. Also, I read that there was a huge avian flu outbreak at many poultry farms throughout the US in 2014. Since this case was very recent, and the human to avian flu contact is not yet seen or treated; I thought it would be a great idea to create a science project that actually uses the BLAST system to redesign the protein sequence of the H5N1 Avian Influenza Virus, so it will be ineffective towards destroying the host cells in a human's body.
My project is due before August 24th, and I am asking the Science Buddies to advise me on what I could research on (other than information on just Avian Influenza and its outbreak), and how I can link my research to the BLAST technique. Overall, I wish to make my project very unique compared to other students who will be competing against me in December 2015, and also educate myself on something new and exciting because I do plan to major in Immunology/Molecular Biology in college. Please respond to my post, and Thank you for your time.

[deleted-141593]

Interesting idea.

I haven't used BLAST in a while due to the type of research I do, but here is a BLAST tutorial: http://digitalworldbiology.com/dwb/Tuto ... nners.html
There are several other good ones online as well.

As for H5N1, I suggest researching which of the two influenza A surface proteins ([H]emagglutinin and [N]euraminidase) is responsible for binding to human cells during an infection.

For example: http://www.nature.com/nature/journal/v4 ... 05264.html

Once you know WHY H5N1 binds to human cells, you can try to change that interaction.

Finally, I think you should think about broader implications of this idea. Lets say you redesign the flu proteins responsible for infection; how would this help treat or prevent H5N1 infection? Would it?
Is it feasible to re-engineer a virus that is out and about in the wild?

Perhaps think about flipping your idea a little bit: http://www.geneticliteracyproject.org/2 ... epidemics/
http://www.sciencedaily.com/releases/20 ... 141601.htm

Cheers,
Colin

[deleted-290678]

Thanks Colin,

Hemagglutinin are the molecules that bind to the host cell's surface by attaching themselves to the sialic acid that neuraminidase(the few carbohydrate enzyme) shoots out at the beginning to help the HA attach on. Following attachment, the virus envelope fuses with the host cell's membrane, and the viral genome spills inside the host cytoplasm. After researching on the viral entry, I also looked into how the interaction between the virus and host cell can be changed, and read that researchers have created neuraminidase inhibitors that inhibit the active site of the enzyme, which slow down the chemical reaction of the influenza. So, with that in mind; how am I able to alter the interaction between the 2 neutral cell surfaces, without making it seem like a copy of the research done already?
In addition, I also researched on the difference between 2 pandemic potential avian influenzas (H5N1 and H7N9), and found out that although they're quite similar in origin and disease spreading; H5N1 has a higher risk of mortality in blood related healthy people under the age of 50. H7N9 was recently discovered in 2013, so the information on it is very limited. Please respond and thank you for your time.

[deleted-141593]

So, a little clarification. Hemagglutinin is indeed the molecule that bind to the host cell's surface by attaching to sialic acid. However, neuraminidase actually cuts sialic acid off of glycoproteins . Neuraminidase is primarily important for viral replication and viral budding from host cells, although it also has a role early in infection via degredation of mucus that normally helps protect lung epithelial cells from infection: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC525087/

Neuraminidase inhibitors primarily work by preventing viral budding (sialic acid is not cleaved or cut off so viral particles remain attached to host cells where they were made and do not move to new un-infected cells).

As for differences between H5N1 and H7N9, it's not always clear why one virus is more deadly than another. Sometimes this is due to some virus-intrinsic property, for example, more efficient viral entry into host cells or more efficient viral replication etc. Differences can also be virus-extrinsic, for example, people over 50 could have been exposed to an earlier virus (or earlier vaccine) in a previous year that was structurally similar to H5N1 and thus have some immunity to it compared to younger people. I'm not saying this is true in this case, I am just saying that these are some possibilities in general.

Finally, I don't want to give you too much direction and influence your project too much. You have good ideas. The basic strategies for preventing or treating flu from a structural standpoint as I see it are, 1. to prevent HA biding to the host and prevent viral entry 2. Inhibit neuraminidase function to lessen spread of influenza particles within the infected host.

Under option 1, options include blocking the HA on the virus, or blocking the sialic acid on the host. There are good and bad things about both approaches. Current neuraminidase inhibitors also have some drawbacks including virus mutation resulting in resistance, and adverse side effects for the person taking the drugs: http://www.cdc.gov/flu/professionals/an ... icians.htm

Article on neuraminidase function: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3347517/
http://www.virology.ws/2013/11/05/the-n ... nza-virus/

Does any of this information give you any new ideas?

Cheers,
Colin

[deleted-290678]

Thanks for the clarification, it really helped me better understand the complicated information. As for my new idea: after watching a video on flu virus mechanism; I saw that host cell proteases actually cleaves the precursor hemagglutinin into two parts (HA1 and HA2)which later separate when the HA internalizes into the host cell. After that, HA1 opens up, and allows HA2 to form a triple alpha helix bundle which extends towards the endosomal membrane. Once the fusion peptides are anchored in the endosomal membrane, the whole molecule can fold back allowing the fusion of the viral and endosomal membranes. After fusion is done, the viral genome can make it way through the cytoplasm, and into the cell's nucleus. My solution: Since proteolytic cleavage by host cell enzymes is critical for the viral genome to enter the cytoplasm, why don't I just find a way block the enzyme from cleaving the HA molecules, so when it is time to separate after internalizing, they won't be able to perform that . This will all in turn prevent viral entry, which means no viral replication, finally meaning that the cell won't be infected, and since infection starts from one host cell; if that host cell isn't even infected/destroyed, how is the whole place going to get infected?
Thanks.

[deleted-141593]

Great idea. It looks like this has been done in some pre-clinical work: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC136409/
And here http://www.sciencedirect.com/science/ar ... 1X14011838

How were you thinking you might try to block this step?

[deleted-290678]

Just to be curious, since I am looking into blocking or altering the protease from cleaving the hemagglutinin, what outside forces cause the amino acid to mutate, and will one mutation cause a whole enzyme to stop functioning or possibly start functioning for something else? Also, can enzymes repel to any substance? Thanks.

[deleted-141593]

Just to be curious, since I am looking into blocking or altering the protease from cleaving the hemagglutinin, what outside forces cause the amino acid to mutate,

Chemical mutagens, radiation, mistakes during transcription or translation: http://www.uvm.edu/~cgep/Education/Mutations.html
https://en.wikipedia.org/wiki/Point_mut ... _mutations

and will one mutation cause a whole enzyme to stop functioning or possibly start functioning for something else?

It depends. Mutations can be silent, can cause loss of function, or gain of function: https://en.wikipedia.org/wiki/Point_mut ... gorization

Also, can enzymes repel to any substance? Thanks.

I'm not sure what you mean by this. Can you clarify?

Cheers,
Colin

[deleted-290678]

Thanks for the links. As for clarification for the my last question --For example, two opposite ends of a magnet repel each other, right? Basically, that was my question: is there anything that enzymes can't interact with?
More Questions:
1)Can specific mutations such as deletion, insertion, frameshift etc. be used to mutate the amino acid chain of the protease enzyme?
2)How is it possible to make all the proteases during the flu infection dysfunction, or is it done individually?
3)Can the viral envelope be covered with a protective film so the proteases can't cleave the hemagglutinin?
4)Can radiation or reduction in temperature be used to denature the enzyme?
5)What substances are enzymes unable to break down?

So far those are all the questions that I have, but I'm pretty I'll continue to have more as I further perform more research. Thank you and please respond.

[deleted-141593]

Thanks for the links. As for clarification for the my last question --For example, two opposite ends of a magnet repel each other, right? Basically, that was my question: is there anything that enzymes can't interact with?

Nothing categorically impossible. However, interactions of enzymes with substrates or antagonists depends upon the structure of both the enzyme and the other molecule.

More Questions:
1)Can specific mutations such as deletion, insertion, frameshift etc. be used to mutate the amino acid chain of the protease enzyme?

Sure. Good idea, though knowing what to mutate is difficult. If you want to go this route, the CRISPR/Cas9 system is great for this type of engineering: https://www.addgene.org/CRISPR/guide/
http://wormcas9hr.weebly.com/how-it-works.html

2)How is it possible to make all the proteases during the flu infection dysfunction, or is it done individually?

This is the hard challenge. What has been tried so far (in links I sent before) is to treat the animal getting the flu with small molecule inhibitors of the relevant protease/s. The problem with this is that these enzymes have normal functions that are disrupted by this. This could lead to side-effects of the treatment. The other problem is the specificity of the inhibitor; it it inhibits proteases other than those at work during flu infection, you could get even more side effects.

3)Can the viral envelope be covered with a protective film so the proteases can't cleave the hemagglutinin?

Interesting idea. How would you do this? In a way, this is what antibodies do; if you have antibodies specific to the flu, they bind to it, coat it, and mark it for destruction by immune cells.

4)Can radiation or reduction in temperature be used to denature the enzyme?

Which one? The proteases in the lungs? I don't think large temperature changes or irradiating a person are going to be viable strategies for preventing flu infection.

5)What substances are enzymes unable to break down?

Each enzyme is very specific for certain molecules or kinds of structures: https://blog.udemy.com/lock-and-key-hypothesis/
http://chemistry.elmhurst.edu/vchembook/571lockkey.html

Cheers,
Colin

[deleted-290678]

Can antibodies detect and coat proteases, just like it does to viruses? Or is that impossible because proteases by themselves are not foreign to the body?
Thanks.

[deleted-141593]

Good question. A person's own antibodies are unlikely to recognize his or her own proteases because, as you say, they are not foreign to their immune system. If they had antibodies that did do so, that might result in autoimmunity. What has been done in the past in some publications is use of short term treatment with chemical inhibitors of the proteases. Over time these inhibitors go away and allow them to function normally. Inducing antibodies against normal host proteins is probably not a good idea.

[deleted-290678]

This is kind of off topic, but how viable are these ideas that I am researching on? I am asking because my science fair coordinator is worried that they won't be possible to complete until I show something really unique to my research professor/mentor.
Thanks.

[deleted-141593]

Hmm, it depends on what the goal of the project is, how much time you have, and what resources you have access to. Many of these ideas would require lots of experimentation.

[deleted-290678]

Quick question: is it possible to genetically engineer enzymes (specifically proteases)?

[deleted-141593]

OK, what do you mean by that? Is it possible to modify the enzymes in a persons body? This kind of genetic therapy has not been very successful, and in this case it might not be a good idea to permanently change a normal protein in a healthy person (that is, it would not be ethically sound). The best strategy from my perspective would be to inhibit HA cleavage into HA1 and HA2 with a drug. However, that is not a trivial project to undertake.

[deleted-290678]

So you're saying that we can't make artificial enzymes?

[deleted-141593]

It is difficult to engineer people to make an "artificial enzyme" through gene therapy, and in this case, as we are talking about a normal, not dysfunctional, protein, it would be unethical in my opinion to do so. However, if you want to design a "decoy" protein (https://en.wikipedia.org/wiki/Decoy#In_biochemistry) that binds to HA but does not cleave it that is based on the structure of some protein you know can interact with HA, that might be possible, and it might be possible to treat patients by giving them this decoy as a drug. That's not a bad idea, though the advantage over a vaccine is not clear. Where I still think the interesting idea lies is in engineering chickens that are resistant to flu infection. Have you seen in the news that this is a big problem right now?

http://www.nytimes.com/2015/06/17/busin ... tages.html
http://www.theguardian.com/vital-signs/ ... en-farming

[deleted-290678]

Yes, I have and thank you for the links. But how do you engineer chickens that are resistant to bird flu; is it like mutating the bird's DNA when they're developing (before they hatch)? Thanks.

[deleted-141593]

There are a few variations. Usually it involves changing a gene in an embryonic stem cell, then injecting those cells into an embryo, and breeding the adult the comes from that embryo to normal animals. You then screen those progeny for the gene you changed. You breed those animals together to get animals that have two copies of the gene in question.
It is a fairly involved process, here are a few versions of the process for mice, it would be the same for a chicken:

http://jaxmice.jax.org/news/2014/CRISPR_Onestep.html
http://www.bio.davidson.edu/genomics/me ... ecomb.html
http://www.nature.com/srep/2015/150609/ ... 11221.html
http://www.genetics.org/content/199/1/1.full

This is not something you can actually do in the time you have, but you could design a gene to make chickens resistant to flu infection, with a mutant protease so there is no HA cleavage for example. You could then describe the process by which you would engineer such an animal. I think that would be a good project.

[deleted-290678]

Ok, I like your suggestions but how am I able to do that?

[deleted-141593]

Well, you need to research what chicken proteins are required for virus entry, or HA cleavage, or neuraminidase activity etc, and then design a new amino acid sequence that will interact differently with the flu based upon what is known about their interactions. Then you can describe the process for creating the modified chicken.

This is one example: http://www.nature.com/news/2011/110113/ ... 16.html#B1
https://bioethicsbytes.files.wordpress. ... 130111.pdf
http://www.sciencemag.org/content/331/6014/223

That one uses a variation of the decoy strategy I wrote about earlier.

[deleted-290678]

So am I supposed to find the amino acid sequence of the HA cleavage and neuraminidase etc.?

[deleted-141593]

If that is the project you want to do. You will need to find the sequences of the proteins of interest and design modifications to them to alter their functions.

[deleted-290678]

Well, you need to research what chicken proteins are required for virus entry, or HA cleavage, or neuraminidase activity etc, and then design a new amino acid sequence that will interact differently with the flu based upon what is known about their interactions. Then you can describe the process for creating the modified chicken.

This is one example: http://www.nature.com/news/2011/110113/ ... 16.html#B1
https://bioethicsbytes.files.wordpress. ... 130111.pdf
http://www.sciencemag.org/content/331/6014/223

That one uses a variation of the decoy strategy I wrote about earlier.

Okay... I did perform research on what chicken proteins are required for virus entry, but my Google search either came up with random science journals or nothing at all. And the same thing happened with the HA cleavage, and neuraminidase. Also, how am I supposed to design a new amino acid sequence? Can you please clarify on all of these things, because this project is really similar to what I originally intended as a project.

[deleted-141593]

The "random science journals" are probably your best sources of information! If you have problems accessing the journals we can try to figure out a way to fix that. Do you have access to an academic library? Some local libraries also have some scientific journal access. If you have found papers that you do not understand, you can give me the links so I can help you understand them. I cannot do the research for you, though.

Amino acid sequences for HA and N for H5N1 should be publicly available. Chicken protein sequences are accessible via BLAST and other resources. There are a number of directions you can take this. Good options in my opinion are A.) Design a protein that will bind to HA or N. Something like this would be based on the structure of a protein known to bind to HA or N. Then you would describe how to make a chicken that produces this decoy molecule. This is basically what the people who made the modified chicken I sent the article about did. B.) Decide whether to try to block HA or N. Then, find out what molecules HA or N binds to in a chicken. At this point you can research if there are closely related animals that are immune to the flu, and replace the chicken protein with the version of that protein from the other animal. This way it might still carry out its normal functions, but not bind to the flu.

Make sense?

Cheers,
Colin

[deleted-141593]

Do you want to talk about this idea with your adviser and see what feedback you get before you get any further along?

[deleted-290678]

Hi,

Sorry, I didn't respond right back. But I'm pretty sure if she will approve of that idea due to its feasibility and method within the time that I have.
So far, I have visited the links on the Science Buddies page that you have linked to me, so I did get an idea of what other people did. Also, since this project is based on genetic engineering; I researched on the basic concepts of genetic engineering so I can understand where my project is branching off from. Then, after I understood how they perform genetic engineering on vegetables, fruits, animals etc. ; I linked that method to genetically modifying my test subject: the chicken aka. Gallus Gallus. Afterwards, I researched online for the HA amino acid sequence and the neuraminidase amino acid sequence. I tried to find a picture of them on Google images just so I could see what I'm trying to alter, but I don't know what I'm supposed to be looking for because there was a thousands of pictures. Anyway, I went to a site called UniProt.org / blast ( I really don't know how to use the blast tool on NCIB because it looks so complicated), and I typed in the protein name of Hemagglutinin and Neuraminidase. For Hemaglutinin- Influenza A virus/Strain A/Scotland/Gallus Gallus H5N1 Type Q710U6. And for neuraminidase: Influenza A virus/Strain A/Scotland/ Gallus Gallus/ H5N1Type NRAM_159AO. With each of them, they had long sequences of letters which I'm inferring that is what you wanted me to change so that it would alter the original function of the protein?

That's all of what I performed so far, and please recommend anything that I might have forgotten.
Thanks.

[deleted-141593]

Sorry, I didn't respond right back. But I'm pretty sure if she will approve of that idea due to its feasibility and method within the time that I have.

OK.

So far, I have visited the links on the Science Buddies page that you have linked to me, so I did get an idea of what other people did. Also, since this project is based on genetic engineering; I researched on the basic concepts of genetic engineering so I can understand where my project is branching off from. Then, after I understood how they perform genetic engineering on vegetables, fruits, animals etc. ; I linked that method to genetically modifying my test subject: the chicken aka. Gallus Gallus.

Good start!

Afterwards, I researched online for the HA amino acid sequence and the neuraminidase amino acid sequence. I tried to find a picture of them on Google images just so I could see what I'm trying to alter, but I don't know what I'm supposed to be looking for because there was a thousands of pictures.

Not sure what you mean here, sorry.

Anyway, I went to a site called UniProt.org / blast ( I really don't know how to use the blast tool on NCIB because it looks so complicated), and I typed in the protein name of Hemagglutinin and Neuraminidase. For Hemaglutinin- Influenza A virus/Strain A/Scotland/Gallus Gallus H5N1 Type Q710U6. And for neuraminidase: Influenza A virus/Strain A/Scotland/ Gallus Gallus/ H5N1Type NRAM_159AO. With each of them, they had long sequences of letters

Were the letters all A, G, C, and T? If so this is the genetic sequence. If it was all kinds of different letters then it is the amino acid sequence. So, these are sequences for HA and N isolated from H5N1 from a checken in Scotland. Pretty neat, right? If you look in Uniprot you will see many other sequences from different isolates all over the world.

which I'm inferring that is what you wanted me to change so that it would alter the original function of the protein?

Well, I want it to be your project. But, yes, if you want to change the function you will have to change the amino acid sequence (by in turn changing the genetic sequence.) Now, if you want to alter the function of the chicken proteases that interact with influenza, then you will need to look up those sequences.

What do you want to alter, flu proteins or host (chicken or human) proteins? What is the rationale for the one you have chosen?

Do these papers help at all?
http://www.pnas.org/content/95/17/9713.full
This is an overview of HA cleavage. If this is what you want to interfere with, this is a good start. It turns out that, "how, and more specifically by what protease, the hemagglutinin protein of these various influenza strains is cleaved for viral activation," is very important for both how easily the virus is transmitted and how pathogenic it is (basically, how severe the symptoms are). Also important is that how aand by what HA is cleaved can vary from host to host (chicken vs human for example), and virus strain to virus strain (for example H2N3 vs H5N1).

So, if your interest is in HA cleavage, as you wrote several posts ago, what we need to do is see what is known about what protease cleaves H5N1 HA in the host of interest (chicken?), and learn about that protease.

Another good overview:
http://blog.h1n1.influenza.bvsalud.org/ ... the-virus/
"However, the Hemagglutinin of H5N1 and other highly pathogenic viruses have a cleavage region with several basic aminoacids, which allows the digestion of the hemagglutinin by several proteases, not only those found in our respiratory tract. Like the furin protease, which occurs in almost any type of cell. Then, the influenza HPAI is able to infect and replicate in various other tissues and cause a systemic infection. [3]"

Here are some other related papers:
http://jvi.asm.org/content/87/8/4786.full
"The pathogenicity of H5 viruses in poultry largely depends on the number of basic amino acids in their HA cleavage site: the higher the number of basic amino acids, the more readily proteins are cleaved and the higher the tissue tropism (11, 12). Because the HA cleavage amino acid residues (-RRRKK-) can be cleaved by furin-like protease (13), which is expressed in most organs of hosts, in HPAI H5N1 virus this site plays important roles in host pathogenicity and tissue tropism in hosts (14)."

Also: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2910732/

[deleted-290678]

I want to alter the amino acid sequence of the HA cleavage proteases that specifically cleave the hemagglutinin of the H5N1 Avian Influenza. Also, what kind of equipment will I need to accomplish my research project ?(my adviser wanted to know)