Researching HIV

If anyone has seen my previous post they will know that I’m spending my summer in Zurich, however, not everyone knows what I’m actually doing in here. Well, I’m in Zurich University for two months and I’m doing some research on Human Immunodeficiency virus (HIV).

 

N.B. after writing this post I noticed that it’s a bit lengthier than I intended it to be and I didn’t even scratched the surface of all the goodness that has to be said when talking about this virus, so this subject might need to be continued…

HIV particle
Artist depiction of HIV particle

One of the WHO Millennium Goals is to combat HIV and provide universal access to HIV-related AIDS treatment. Why is the fight against HIV so important? Well, 1.7M people died of HIV in and 34M were living with HIV in 2011, moreover, each year around 2.5M people get infected with it. With combined antiviral drug treatment (known as highly active antiretroviral therapy (HAART)) people infected with HIV can usually live into their 60s, however, no cure is yet present. The greatest difficulty in finding effective treatment against HIV lies in the fact that HIV is a retrovirus that has incredibly error prone reverse transcriptase (RT). RT is an enzyme that catalyzes ssRNAàdsDNA reaction but is very ‘careless’ and makes many mistakes while doing it. Over one day period every nucleotide in HIV genome (~9000kb) can potentially be changed. This RT ‘carelessness’ is the key to HIV immune evasion because errors in the genome lead to new protein patterns in various ‘parts’ of HIV after each replication. These new protein patterns either can no longer be inhibited by antivirals (e.g. nucleotide analogs that are often used in HAART can no longer bind and inhibit the RT) or can no longer be recognized by the immune system (antibodies are typically made by our immune system against foreign pathogens, however, HIV changes its appearance so quickly that by the time specific antibodies are made, new patterns in viral particle arise that can escape antibody recognition). I could actually rant on and on about HIV because of how *brilliant* of the pathogen it is, in fact error-prone reverse transcription is only a beginning of the story! There is lots to be told about HIV recombination, accessory proteins and specific life cycle, and then one can also look at infection from the host point- APOBEC family proteins, TRIM proteins, elite carriers etc. etc. etc. … oh gosh, it’s a treasure trove of incredible, beautiful and exciting stuff J But I should probably stop here because I’ll never get to point of  what research I am doing.

 

I am working on a project that tests a potential inhibitor of HIV entry. Before I tell what kind of inhibitor it is I need to provide a simplified explanation of HIV entry.

 

HIV uses its envelope glycoproteins (called gp120 and gp41) to bind cell-surface receptors. The first HIV receptor is CD4 molecule found on some of the immune cells. Firstly, gp120 binds CD4, which  in turn leads to conformational changes in the glycoproteins. The changes include a shift in gp120 variable regions, namely regions V1 and V2 shift and expose region V3. V3 can then bind a coreceptor (CxCR4 or CCR5) and this binding again causes conformational changes but this time in gp41. Gp41 forms a fusion peptide that is inserted into the target cell membrane an cause the viral  envelope to fuse with the host cell membrane. Please refer to the figure for illustration of HIV entry (it lacks V1 and V2).

HIV entry mechanism is considered to be a good target for various drugs and many have been designed to inhibit different entry steps, however, as mentioned previously resistance to these drugs arise faster then we are able to create any new ones. This summer I’m also working on such entry-inhibitor molecule. Biochemistry department in Zurich University have created a libraries of DARPin (=designed ankyrin repeat proteins) molecules that have different structures and can target and inhibit activity of different molecules. One of these libraries has been made for DARPins that can bind V3 region of gp120 and therefore, inhibit HIV binding to its coreceptor. Antibodies that target V3 are quite rare in HIV patients and are not very effective (I would guess mostly because V3 is kind of *hidden* inside gp120 and so the ‘bulky’ antibodies can hardly access it).  DARPins, on the other hand, are quite small (15-18kDa) and can access V3 and other hidden structures much more readily then the antibodies. Moreover, commercial DARPin production is much more viable as it can be made in prokaryotic systems (by simply expressing DARPin sequence containing plasmid in E.coli), whereas antibody production requires much more resources and eukaryotic systems. Obviously, because of the incredible HIV diversity (i.e. there is diversity among V3 regions as well) many different DARPins need to be tested for their activity against different HIV strains. So during my time here I’ll be introducing mutations in HIV gp120 and trying to inhibit the virus using different DARPins. The project involves making HIV with different V3 region sequences and making infectivity and neutralization assays to analyze how sequence variation affects DARPin effectiveness as well as general HIV infectivity.

Many people who know that I’m working with HIV have asked if I have to be very careful not to get infected with the virus. Well, not quite so (I mean certain carefulness is always good when handling pathogens) and that’s because such primary research is usually done on what’s called a pseudotyped virus. What that means is that scientist create a virus that is infectious only for one round. And what that means is that I transfect a eukaryotic cell with one plasmid that has envelope protein sequence and another that has the rest of the HIV genome (called backbone). Then the infected cell produces infectious viral particles that instead of having a full-length genome only have the backbone sequence (envelope sequence is not incorporated because it lacks the required *signal* to do that) and so during the next infection these pseudotyped particles will enter the next cell but will not be able to make viable viruses in it because they lacks envelope proteins needed to leave the cell. Because I work with a pseudotyped HIV the research is done in a biosafety level 2 lab (BSL-2) (same level as labs designed for any kind of microbe research). The Institute of Medical Virology, where I am based at, also has a BSL-3 lab and in it research is done on a wild type HIV that has been isolated from HIV patients. It’s not really a large lab more of a room really, but there’s great deal of security around it.  E.g. people working in it have to wear full overcoats, entrance into the lab has two metal doors with the space between them being sterilized by hydrogen peroxide and one of the walls has been made into huge autoclave so that everything that goes into the lab can get out only by passing through 121C and 100kPa test. The highest BSL is 4 and only a few labs in the world are of this level (BSL-4 labs are needed for the work with viruses like ebola and smallpox). Here’s a link to a documentary about BSL-4 lab that is being built in the middle of Boston- cool stuff.

I should stop here even though there’s great deal left to say. However, if anyone has any questions about my research or HIV in general please white a comment and I’ll try to answer them. I’m not an expert on HIV but it’s definitely one of my favorite viruses so I do know quite a bit about it  (more importantly, I am always happy to learn something new about the subject).

False-colour image of HIV infecting the cell

 

 

 

 

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