Biophysics on World AIDS Day

December 1 is World AIDS Day. More than 35 million people worldwide are living with HIV, with 66% of new infections occurring in sub-Saharan African countries. Many biophysicists study HIV/AIDS, and we recently spoke with BPS member Bo Chen, University of Central Florida, and biophysicist Lesley Earl, a member of the Laboratory of Cell Biology, Biophysics Section, NIH/NCI/CCR, led by BPS member Sriram Subramaniam, about the research their labs have conducted relating to the HIV virus, and how biophysics research contributes to our understanding of HIV and potential therapies. 


 

Bo Chen, University of Central Florida

What is the connection between your research and HIV/AIDS?

My research is to understand the structure and formation of a protein shell called “capsid” that encloses the viral genome materials. Our results can potentially shed light to design antiviral drugs against HIV/AIDS. Even better, once we completely understand the formation of this protein shell, we can use it as a template to design customized nanoparticles with designated assembly morphology, stability, and affinity for biotechnologies in applications such as biosensors, drug delivery vehicles.

Why is your research important to those concerned about HIV/AIDS?

The integrity of the capsid is critical to the infectivity and function of the HIV particle.  Specifically, the capsid is formed by the self-assembly of hundreds of copies of the capsid proteins. When a virus infects a host cell, the capsid needs to disassociate at the right time and location to release the viral genome materials to initiate the replication process by hijacking the host cell machineries. Molecular biology and biochemistry experiments have shown that the capsid is more than just a protective armor. It is actively involved in multiple steps of the viral life cycles, including the reverse transcription of the viral RNA to DNA and subsequent importation of the DNA into the host nucleus. Afterwards, as new viral materials are produced by the host cells, hundreds of copies of these capsid proteins need to come together and assemble into the capsid in the same morphology, encapsulating the right amount of the viral genome materials. The infectivity of the HIV particle will be lost or attenuated if the capsid deviates from the normal morphology or stability. Therefore, the HIV capsid is considered as a promising antiviral drug targets, and elucidation of the capsid structure and assembly mechanism will help find new therapeutic strategies against the AIDS. Yet the structure and formation mechanism of the capsid is still not well-understood.

How did you get into this area of research and how long have you been working on it?

I started to characterize the HIV capsid assembly structure by solid state NMR when I was a postdoc in Dr. Robert Tycko’s group at NIDDK, NIH. We were looking for some large proteins for solid state NMR, to push the capability of the structural biology technique.  Two years into the project, I had a wild idea to simulate the self-assembly process in addition to my experimental efforts. It was inspired by the pioneering work of Dr. Gregory Voth, Dr. Michael Hagan, and Dr. David Chandler. With my passion for Mathematics and geometry, I came up with a novel model to simulate the self-assembly of the HIV capsid. Since then, it has been about eight years.

Do you receive public funding for this work? If so, from what agency?

I was awarded the 2013 Young Investigator Award by the Air Force Office of Scientific Research. I was so obliged to their support, without which our research could not be where it is today.

Have you had any surprise findings thus far?

We have two very interesting findings. Firstly, we enhanced our simulation model such that it not only captures the shape of individual capsid protein, but also provides sufficient simulation speed to simulate the self-assembly of the capsid proteins in 3D. In that sense, it is very unique. It enables us to accurately incorporate the experimental high resolution structure and dynamics information of the capsid protein into the simulation. We demonstrated that the capsid protein prefers to assemble into hexameric lattice with only mild curvatures in the majority of its time in the solution, where it samples an ensemble of different conformations, as assessed by solution NMR work from Dr. Marius Clore’s group. The conical tips with sharp curvatures are assembled by the capsid proteins in a highly selective conformation state, along a distinct assembly pathway.  Due to the ability to capture the protein structure at high resolution, we further probed the importance of the variations at the dimerization interface of the capsid protein. We showed that subtle variations of the crossing angle between helix 9 from the monomers in the dimer will not affect the curvature or morphology of the assembly,but dramatically change the assembly pathway. We are really excited with our results, published in BBA General Subjects, 1850,p2353-2367, and we are anxious to see if they can be confirmed by experiments and alternative theoretical work, as you know, a theory is only a theory until it is proven correct.

BoChenImage

Simulations with our novel coarse-grained model show that the tip of the conical HIV capsid can be formed by the capsid proteins in pentameric like conformation. The final assembled structure, exhibits co-existence of quasi-equivalent pentamers and hexamers as shown above. Figure from Xin, Q. et al. (2015) BBA-Gen Subjects 1850,2353-2367.

What is particularly interesting about the work from the perspective of other researchers?

To our knowledge, our work was the first 3D simulation of the HIV capsid assembly by a high resolution coarse-grained model that can capture accurately the protein structure. Our results therefore, can be directly compared with experimental observation. If it is correct, its knowledge will provide rational guidance to anti-HIV drug design targeting the capsid protein. As I mentioned above, our work brings us one step closer to constructing nano-architecture derived from the HIV capsid protein for a wide-range of applications in bio-nanotechnology. On an even broader scale, our model can be generalized to simulate other large biomolecular assembly, which is a very challenging topic. Such systems involve a large number of molecules, and the assembly process extends a time range that is too slow for simulation with all atom molecular dynamics but too fast for direct experimental analysis. So the protein molecular structure has to be coarse-grained in simulations to enable the simulation of such large systems. As you are probably aware, the function of the protein directly ties with its structure. So the unique strength of our model as we demonstrated on HIV capsid assembly is that we can retain the structural information quite well, without a prohibiting computational cost. We hope our work will help and inspire others with similar interests, just like my model was partly inspired by the pioneers in this field.

What is particularly interesting about the work from the perspective of the public?

Well, they can see their tax money is well-spent :). Specific to the public concerns about health related issues, our results, if verified by other scientists, can be highly valuable for rational design of anti-HIV drugs and help fight AIDS. In fact, the capsid shell is a ubiquitous component for all viruses, whose formation mechanism is still debated. There is an abundance of viruses as pathogens that cause many other terrible diseases, including Hepatitis virus, Dengue virus, to name a few, and the list can go on and on. More broadly, many of the enzymes and proteins perform their duty not alone, but in cohort assemblies. Our model may be applied to investigate such systems of interest.


 

Lesley Earl, Laboratory of Cell Biology, Biophysics Section, NIH/NCI/CCR

What is the connection between your research and HIV/AIDS?

Our laboratory is generally focused on studying the 3D structure of cells, viruses, and proteins by electron microscopy. A primary focus for the lab over the last decade or so has been uncovering how HIV invades target cells. We have studied a number of aspects of this process, including structural analyses of the HIV envelope glycoprotein (the viral surface protein that binds to the target cell and allows the virus to enter), the formation of the HIV core (the protein structure that forms around and protects the virus’ genetic material), and the cellular structures of the HIV virological synapse (which allows much more efficient viral transfer between cells).

Why is your research important to those concerned about HIV/AIDS?

One of the most frustrating things about the many years of research into HIV has been our inability to design a vaccine that is effective against a broad range of HIV strains. In our structural studies we have been looking at what makes certain types of antibodies effective in neutralizing the virus and preventing infection. While we haven’t found a silver bullet yet for designing vaccine immunogens, we have uncovered some really interesting findings, such as the way in which certain neutralizing antibodies from patients can actually stop the envelope glycoprotein from opening up and binding to target cells. This type of information is critical for understanding how HIV functions, and for designing new therapies.

How did you get into this area of research and how long have you been working on it?

For the last 15 or so years, our group has been working on developing the ability to determine high resolution structures of proteins by cryo-electron microscopy. In 2006 or so, we were hunting around for something really interesting and important to work on, something where structure could really make a difference, and which we could also use as a platform to continue developing our technologies. We decided that working on the HIV envelope glycoprotein was just the thing – it’s a highly dynamic protein present in high concentration on the surface of the virus, and at the time, there were no structures at all of the native envelope glycoprotein. We published the first structure of the native unliganded envelope glycoprotein trimer in 2008, and have followed that with in-depth studies of the structural mechanism of HIV binding and entry into target cells.

Do you receive public funding for this work? If so, from what agency?

As a lab within the NIH Intramural Program, we primarily receive funding from the National Cancer Institute, as well as from the Intramural AIDS Targeted Antiviral Program.

Have you had any surprise findings thus far?

LesleyEarlImage

An uninfected T cell (blue) reaching around and attaching to an HIV-infected T cell (green), which has HIV virions (glowing yellow) on the surface. Image credit: Donald Bliss, National Library of Medicine.*

 

Truthfully, HIV continues to surprise us, no matter how long we work on it. Early on, we saw for the first time what seemed to be a twisting opening motion of the HIV envelope glycoprotein; in 2014, we found that even though the top portion of the trimer twists open, the base seems to stay still, suggesting that the interface between these two sections of the trimer must adjust as the trimer engages with receptors on the surface of the cell. Another surprising finding came from our study of the HIV core – we were looking at images of HIV virions from an infected culture, and realized that the generally accepted model for core formation, which suggests that the protein core subunits nucleate and build up from one end inside a virus particle, didn’t align with what we were seeing. Instead, we could see sheets of core material rolling off the membrane into the classic cone-like core shape. And finally, in our cellular studies, we were extremely surprised to see that uninfected cells seem to send out finger-like membrane extensions towards infected cells, apparently facilitating their own infection.

What is particularly interesting about the work from the perspective of other researchers?

I think the most interesting piece of our work in terms of what is of use to other researchers has been our cryo-electron tomographic studies of antibody-mediated neutralization of the envelope glycoprotein. By understanding what allows an antibody to prevent the virus from infecting cells, researchers working on designing an HIV vaccine can design novel protein variants what will elicit the right types of antibodies from patients, which is a key step in rational vaccine design.

What is particularly interesting about the work from the perspective of the public?

One of the great things about 3D electron microscopy techniques is that they give us the ability to actually see what’s going on at the cellular or even atomic level. The types of images being produced from our lab – whether they’re 3D models of proteins from single particle analysis, or models of viruses from cryo-electron tomography, or models of cells from focused ion beam scanning electron microscopy – really bring the biology of HIV to light.

 

*The data from which this image was derived is from the following publication: Do T, Murphy G, Earl LA, Del Prete GQ, Grandinetti G, Li G, Estes J, Rao P, Trubey C, Thomas J, Spector J, Bliss D, Nath A, Lifson JD, and Subramaniam S. 3D imaging of HIV-1 virological synapses reveals membrane architectures involved in virus transmission. J Virol. 2014 Sep;88(18):10327-39. doi: 10.1128/JVI00788-14.

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