The secret Jedi life of the Virus - HIV as screwdriver for manipulating genes.

Wear your safety gown, two layer of gloves, a face mask and a head cap. Now pass through the air shower, clean everything with bleach and ethanol - and you are ready to explore HIV as a tool in biological research. So yes same virus that takes millions of lives every year, also hold the key to efficiently manipulate the genes of any living organism. When every conventional way of inserting genes in cells in a stable manner fails this guy comes to rescue, at least it does for me. So why not share its story? The story of how we turn a deadly virus in a tool that helps making landmark discoveries to for the good of the mankind.

Human embryonic kidney cells 293T updated their status - making virus tonight
Image of HEK293T cells that are producing virus. GFP gene was inserted with essential viral genes to make a replication incompetent virus that will be used to add GFP gene to genome of target cells.
Source: Taken by @scienceblocks on Olympus IX73 microscope

The Dark side of the force
What response does the name HIV or human immunodeficiency virus (virus which causes AIDS), evoke in you? Probably stress and fear. And it should. It's one scary fucker, who is really good at its job. The virus not only infects your cells, it integrates itself into your genomic DNA. Well you might have guessed it by now, HIV is a retrovirus, that means it has RNA as its genetic material, which it converts to DNA and insert it somewhere in between your genes (Schröder et al., 2002). And then? Then it just keeps making copies of itself. In fact if you sequenced your DNA you will find these sequences called reterotranaposons (Finnegan, 2012, Griffiths, 2001) , which.are remnants of ancient retroviruses(cousins of HIV) that might have infected us when we were not even human. They lost their ability to make you sick but they left their genes to jump around in your genome. These genes are now part of being who we are. (See this interesting article from Nat Geo. The genes of this virus have been lurking around in us since 6 million years.)

Just like any retrovirus, HIV becomes part of your own genes as well, and not in any good way. Within the retrovirus itself there is a family of freak viruses called Lentivirus. I call them freak because post infection and inserting their genes into your genome they can stay in there quietly, without causing any symptom, and spread for years. And then one day the hell breaks loose when it comes out of this latency and causes AIDS(Bacchetti and Moss, 1989). The fact that this rapidly mutating virus becomes part of your own genes and it stays there in your cells even when you try to treat the symptoms - makes it the worst bug to be fighting against. This is also why targeting it with drugs and developing a cure or vaccine against it have been so hard(Barouch, 2008).

Nonetheless, like you know every villian or antagonist of the movie has at least some positive side to it, so does HIV. I mean not really, it is more like we turn the tables in the lab and use its weapon against humanity to make tools to help humanity. It's like asking Darth Vader to help you carry your shopping bags.

You are really testing my force!
Image source: https://pixabay.com/en/star-wars-darth-vader-1724901/ | License - CC0 | Modified using open clipart via inkspace.

Alright then, How to politely ask Vader to carry your shopping bags?

Since the lentiviruses can stably insert their own genes into the cells, what if I just inserted a gene of my choice say insulin, or some mythical gene that makes you smart, or say Green fluorescent protein(GFP), before it could infect the cells? Well when the virus infects the cell it will also insert gene of your choice into that cell. If planned well enough, combined with CRISPR CAS this can be used to edit mutated genes, and help people with genetic disorders. But hang in there! wont that make you sick too. Well since I use that thing on my cells,I don't want them to fall sick either. So there is this inserting trick we use. We make the virus in the lab in such a way that it can infect the host cell or organism, it can integrate the desired gene at the right place, but it can't make anymore copies of itself.

But how?
In very simple terms we only use genes of the virus that are essential for what we want it to do. Then we break the virus DNA into parts. We keep the part that gets packed into the virus and also carries our gene, separate from part that carries enzymes and proteins needed by virus to replicate and to form its structure. Did you lose me there? OK, lets start with what all shit does the HIV carry with itself, anyway. And what structure does it have?

The secret Life of all retroviruses

Schematic of HIV structure
Created by @scienceblocks on inkspace. Any clipart used is taken from open clipart.

To begin with, the genetic material of HIV is RNA (see figure above). In fact two identical strands of RNA per virus particles. The genetic material is bound to proteins of nucleocaspid. The nucleocapsid is packed within a layer of proteins making viral caspid. This is then packed within a layer of matrix protein and then a lipid bilayer, which anchors the envelope proteins. Also within the caspid the virus particles also pack other important enzymes which will come handy when they infect the cell(Frankel and Young, 1998). In any case,the story starts from the envelope.

Mechanism of HIV infection and replication in cell.
Created by @scienceblocks on inkspace. Any clipart used is taken from open clipart.

The envelope is what interacts with the proteins on the host cells. HIV envelope binds to CD4 protein on surface of T cell, for instance (Silberman et al., 1991). In fact this is where you can start to imagine about designing a host specific virus. If you modify the envelope you modify the cell and even the host the virus will infect. You can even make your virus specific to one host or many based on how you modify its envelope. Once the envelope is attached to the host cell, and the virus gains entry in the cell, and the RNA is released from the caspid as it breaks down(see figure above). This is where the holy reverse transcriptase enzyme comes into picture(yes the same molecule which broke the central dogma of molecular biology). This enzyme makes DNA copies of viral RNA. The DNA is then imported to nucleus of the cell, where with the help of viral integrase it gets incorporated into to host DNA. Once inside, the the host molecules make RNA and proteins of virus like they would of any of their own genes. (In fact this is the reason the virus needs you. It can't make copies of itself without you). Once the new proteins and RNA of virus is present in host cell there is a sequence on viral RNA called psi sequence. This is what the nucleocapsid proteins interact with to pack the virus.Once this interaction occurs the rest of the packing of virus follows. Finally new virus particles bud of the infected cells(Engelman & Cherepanov 2012, Frankel and Young, 1998)

The newly formed particles would then infect the nearby cells and the cycle will continue. And this cycle is what we need to break. Remember I told you, you can make this virus replication incompetent. I think we are ready to discuss that now.

The holy grail! the source code of virus - HIV genome

Genes of HIV virus and their functions The figure list the genes of HIV virus and its function. SInce many genes of the virus are encoded as single mRNA but has coding region for more than one protein you may find many loci having multiple subparts.
Note: I have made this figure this way for simplicity, you can look here to see more acurate organisation of the HIV genome.
Created by @scienceblocks on inkspace. Any clipart used is taken from open clipart.

As you can see in the above figure all the genes and important sequences that this virus harbours in order to infect the cell. Now imagine if I were to take this virus extract its RNA genome and make DNA out of it in a test tube in the lab. Now let me take this DNA and insert it inside some cell line in tissue culture or in some host. Will I get a functional virus if I took all the genes as DNA and inserted it in? The answer is yes you will. Nonetheless there is one problem, it will be fully functional virus. That is, it will keep on infecting new cells and making more copies of itself. We don't want that. We want it to make the virus particles just once and then when I use these particles to infect some other cells they should not multiply.

DNA sequence containing all genes of HIV if inserted in a cell, is capable of producing fully competent virus particles
Created by @scienceblocks on inkspace. Any clipart used is taken from open clipart.

Vasectomy of HIV - making it replication incompetent
I want you look at the HIV genome figure again. But this time pay attention to the sequences on genome. Note that LTR sequence on DNA is required by integrase to insert the viral DNA into genome, and psi sequence is required for packing fresh viral RNA into virus particles.

So if I deleted everything in between LTRs on both ends, except for psi sequence I can add anything in between these and it will be integrated into the host DNA. If I add Green fluorescent protein(GFP) gene in between these LTRs it would permanently become green (provided I added some sort of promoter sequence in front of the gene. Promoter is where gene expression start and it is essential to add). But hey I need this whole thing I made to be packed into new virus particles, so I can go infect some other cells, on some other day and time. I even have psi sequence on DNA I inserted in the cells. So it is hot and ready to be inserted into new virus particles. But I need viral assembly proteins and proteins the virus would need to be packed with to infect other cells.

Fantastic Jeans and how to cut them
Well here is a simple solution. Put those genes on separate piece of DNA. Let me put gag-pol on one(refer to viral genome figure). And lets add a promoter in front of it so it gets expressed in my cells. Also let me add Envelope proteins loci on a different piece of DNA with its promoter. Now let me insert the 3 separate pieces of DNA in the same cell and see what happens.

By separating what gets inserted into new virus, from the genes of the virus, you can create a virus, which just acts as a machine to deliver the gene of your interest. But the virus you produce would not make more copies of itself
Created by @scienceblocks on inkspace. Any clipart used is taken from open clipart.

As a result you have this cell throwing out virus particles with your gene of interest. But when I will take these virus particles and throw them on any other cell or try to modify gene expression in mouse, they just infect the nearby cells. However since genetic material of virus does not have genes to replicate or make caspid proteins anymore, these cells won't produce new viruses. It will just have the gene of your choice being expressed in your target cells.

Congratulation you have created your first engineered HIV virus, that will deliver gene of your choice into cell of your choice.

The method for using HIV for expressing, or knocking down genes of your choice, in your target cells evolved in early 2000s (Follenzi et al., 2000, Naldini et al., 1996). Today almost every lab that works on knocking down genes or even use CRISPR for that method prefers lentiviral transduction(Vijayraghavan and Kantor, 2017).

I hope next time headlines like this pops up saying a BioTech firm used HIV to cure cancer , you know before hand that they modified some genes somewhere. In this particular case they extracted T cell of the patient and then used HIV to change this killer T cells to recognise cancer cells better(Schuster et al., 2017).

References
Schröder AR, Shinn P, Chen H, Berry C, Ecker JR, Bushman F. HIV-1 integration in the human genome favors active genes and local hotspots. Cell. 2002 Aug 23;110(4):521-9. PubMed PMID: 12202041.

Finnegan DJ. Retrotransposons. Curr Biol. 2012 Jun 5;22(11):R432-7. doi:10.1016/j.cub.2012.04.025. Review. PubMed PMID: 22677280.

Griffiths DJ. Endogenous retroviruses in the human genome sequence. Genome Biology. 2001;2(6):reviews1017.1-reviews1017.5.

Bacchetti P, Moss AR. Incubation period of AIDS in San Francisco. Nature. 1989 Mar 16;338(6212):251-3. PubMed PMID: 2922052.

Barouch DH. Challenges in the Development of an HIV-1 Vaccine. Nature. 2008;455(7213):613-619. doi:10.1038/nature07352.

Frankel AD, Young JA. HIV-1: fifteen proteins and an RNA. Annu Rev Biochem. 1998;67:1-25. Review. PubMed PMID: 9759480.

Silberman SL, Goldman SJ, Mitchell DB, Tong AT, Rosenstein Y, Diamond DC, Finberg RW, Schreiber SL, Burakoff SJ. The interaction of CD4 with HIV-1 gp120. Semin Immunol. 1991 May;3(3):187-92. Review. PubMed PMID: 1888898.

Engelman A, Cherepanov P. The structural biology of HIV-1: mechanistic and therapeutic insights. Nature reviews Microbiology. 2012;10(4):279-290. doi:10.1038/nrmicro2747.

Follenzi A, Ailles LE, Bakovic S, Geuna M, Naldini L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet. 2000 Jun;25(2):217-22. PubMed PMID: 10835641.

Naldini L, Blömer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996 Apr 12;272(5259):263-7. PubMed PMID: 8602

Vijayraghavan S, Kantor B. A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells. J Vis Exp. 2017 Dec 12;(130). doi: 10.3791/56915. PubMed PMID: 29286484.

Schuster SJ, Svoboda J, Chong EA, Nasta SD, Mato AR, Anak Ö, Brogdon JL, Pruteanu-Malinici I, Bhoj V, Landsburg D, Wasik M, Levine BL, Lacey SF, Melenhorst JJ, Porter DL, June CH. Chimeric Antigen Receptor T Cells in Refractory B-Cell Lymphomas. N Engl J Med. 2017 Dec 28;377(26):2545-2554. doi:10.1056/NEJMoa1708566. Epub 2017 Dec 10. PubMed PMID: 29226764; PubMed Central PMCID: PMC5788566.

Declaration
Microscopy images used in this article were acquired by me.

The cartoons were created by and if any image was taken from other sources its link is provided. Only CC0 images were used for this purpose.

Wherever clipart was used to make the cartoons it was taken from openclipart.org, which provides open sourced Creative Commons Zero 1.0 Public Domain License work .
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