When the pandemic hit, Maddie Graham’s lab life shifted focus.
The junior biomedical engineering pre-med student suddenly started to find answers by extracting RNA out of wastewater to help detect SARS-CoV-2, the virus that causes Covid-19, which reiterated how important science is in our lives.
“I don’t think medicine would be anything without research,” Graham said. “I think it’s really important to see the other side of things, understand how things have come to be and how they’ve made these medical advances. It was cool to be able to do something related to Coronavirus when the pandemic started.”
That understanding is something Graham never thought she’d seek out when she first came to Mizzou.
“I wasn’t planning on doing research because I didn’t think I would like it,” Graham said. “Originally I thought, ‘No, that’s okay I’ll focus on other things like volunteering and stuff.’”
But as she was taking classes, her friend Braxton Salcedo suggested that she work in the lab he was in.
“She has a good personality and is very intelligent,” Salcedo said. “She was a good partner in class, she pulled her weight and was a good communicator. When the professor said that he wanted to bring on more undergrads, I knew she would be a good fit.”
Graham thought it sounded interesting. She decided to apply and got the job.
Now, Graham has spent just over a year as an undergraduate researcher in the Marc Johnson lab at the Bond Life Sciences Center.
“At first I imagined that undergraduates just wash the dishes and stuff, but it was cool when he told me that you actually get to be part of the science aspect,” Graham said.
Starting out pre-pandemic, the Johnson lab focused on HIV. Graham was making new plasmids to help manipulate genes for the graduate students and Johnson to use. Now she studies SARS-CoV-2 in wastewater and community trends associated with it.
Graham’s perspective on research has certainly changed since she started. Now that she is in upper-level courses, she is starting to see an overlap between her learning and her job.
“I’m in Cell Biology, and the things I’m learning are directly related to things I am doing in the lab,” Graham said. “It’s cool when there are moments where I see why we’re doing certain things and the reasoning behind it.”
While Graham is experiencing research, she is still unsure of what she specifically wants to do once she gets to medical school.
“I have a lot of time to figure that out,” Graham said. “So somewhere down the road, after I get experience in other areas, I’ll hopefully know.”
The research is going to help her wherever she ends up.
“She has good hands-on experience here,” Salcedo said. “Just in general, she’s a good worker and she’s nice to be around too. I think camaraderie is one thing that our lab really has that I’m not sure a lot of other labs have. For the most part, all of the students in the lab get along really well.”
Covid-19 hasn’t just impacted her job, she also ended up adopting a dog at the beginning of the pandemic.
“We fostered her for a bit and then I decided to keep her,” Graham laughed. “It’s nice taking her on walks or to the dog park — except when it’s cold.”
Kamal Singh was in the town of Allahabad in his native India, preparing for competitive exams to become a government official. As he craned his head to the left, he saw a highly respected official getting berated by an arrogant and disrespectful political leader. While Singh always knew that, in these positions, one was under the government’s thumb to an extent, that incident was what sealed the deal for him and made him realize he did not want to spend his life simply taking orders and being subject to verbal abuse by corrupt politicians.
It was at that moment — roughly 30 years ago — he decided he wanted to dedicate his life to something more personally fulfilling: research.
Singh was raised in a small village in rural India. While his primary and high school education came from a military school, he obtained a Master’s degree in physics from Agra University, in Agra, the city where Taj Mahal is located. Still, Singh thought he wanted to work as a public official until that incident served as the last straw. After that, he decided to complete his graduate degree in quantum physics, and committed fully to a life in academia.
That led him to cross the Atlantic in 1994 when he moved to the U.S. and joined Rutgers New Jersey Medical School. He realized that now was his chance to focus on a different area of science that he had always been fascinated with: biochemistry.
“I taught myself everything from scratch in New Jersey,” he said. “I had always wanted to work with DNA polymerase, and I felt like now was my chance to really get involved with that.”
Over the span of 15 years in New Jersey, he occupied a number of positions including postdoctoral fellow, instructor and assistant professor. His work transitioned from research related to DNA polymerase to his current focus on Human Immunodeficiency Virus (HIV) and other viruses including Foot-and-Mouth Disease Virus, SARS and MERS coronaviruses.
That’s where Singh’s story came to Mizzou where he arrived in Jan. 2009. While he has always been highly motivated and driven by collaborations, the bread and butter of his work for more than a decade has come at the Bond LSC focused on HIV.
The goal of Singh’s HIV-focused research, in a nutshell, is to find drugs that bind to the active site of the enzymes that replicate HIV, thereby blocking the spread of the disease. According to Kyle James Hill, a postdoctoral student working in Dr. Singh’s lab, the function of his research is twofold: to better inform human understanding of the viral life cycle and to help identify novel targets of drug identification to potential treatments.
More precisely, Singh’s work is focused on enzymes/proteins that serve particular functions for the virus after the virus enters human body. Most enzymes have a suffix of -ase, and what comes before the suffix generally states the function of a given enzyme. For example, protease has to do with proteins, specifically the breakdown of proteins into smaller proteins or other subunits. Enzymes can have a positive function such as promoting growth and generation of muscle tissue, or a negative function, such as what happens when HIV highjacks their function for its own benefits. The HIV infection, in turn, weakens a person’s immune system and lowers his/her her ability to fight disease, in this case AIDS. Each enzyme has an active site, or a region where its specific function is carried out. All research that deals with enzymes, therefore, must center on what goes on at the active site.
Singh’s HIV-focused research is centered specifically on three HIV enzymes: reverse transcriptase, integrase and protease. Reverse transcriptase catalyzes reverse transcription: that is, the formation of DNA from an RNA genome of HIV. Reverse transcriptase converts RNA genome of HIV into DNA.
HIV then uses another enzyme called integrase to insert viral DNA into the genetic framework of the host cell. This allows the virus to further embed itself in what was once a healthy cell. A third enzyme called protease breaks down long proteins into smaller functional proteins, and these smaller proteins then combine with the genetic material of HIV to infect another cell.
The practical application of Singh’s work is that it is focused on moving research toward tangible results that physicians, in turn, can use to better diagnose and treatment plans for patients. Singh’s current lab work focuses most of all on finding drugs that are able to work to treat HIV patients from different parts of the world.
Singh became interested in investigating why certain drugs designed to treat HIV in the developed world, have less effect on the particular strains of HIV that exist in low- and middle-income countries. To help him with this process, Singh developed a collaboration with scientists at the renowned Karolinska Institutet in Stockholm, Sweden. Singh is also an Associate Faculty member at Karolinska.
While HIV infects people all over the world, there are actually many different strains of the virus, and some strains are more abundant in particular regions of the world. According to Singh, there are 10 subtypes of the virus, and more than 100 hybrid or combined types of it. Some drugs treat a particular subtype better than others. If a given drug does not work to treat a particular subtype, then it is useless in terms of treating patients that are infected with that strain of the virus. According to Singh, most drugs produced in the U.S. treat HIV subtype B, which is most prevalent in the developed world. However, patients in many other parts of the world, such as India, South Africa and Brazil are infected with subtype C of the virus. Singh says that 95% of patients in India are infected with subtype C.
“Because these patients are infected with a different strain of the virus, they, in many cases, do not respond to the type of drugs that are traditionally given to patients in the developed world,” he said. “Our job is figuring out why they don’t respond to that particular drug, and then coming up with something that can more effectively treat them.”
This practical application of his work is what’s most rewarding to Singh. His lab is able to provide concrete results that physicians then apply when making decisions about how to best treat their patients. Based on Singh’s work, physicians can better determine which type of drug would most likely effectively treat patients infected with a specific subtype of the virus, also considering the specific set of genetic variations and mutations that the patient may have. It is in this way that Singh’s work makes a very real difference in the world, and that is ultimately what keeps him so passionate about his work.
“To be able to apply what I work on in the lab and see it at work in real patients is very rewarding for me,” he said. “I am able to see the entire process unfold, starting with the very basic biochemistry all the way to direct application to the patients. Seeing that I’m making a real difference means everything to me.”
Researchers are one step closer to understanding HIV
By Danielle Pycior | Bond LSC
Usually, the human immune system is good at recognizing infected cells and then killing them, but in the case of the human immunodeficiency virus (HIV), the virus has ways to hide. One of the ways is by using a viral protein called Vpu.
Vpu helps HIV survive by hiding the fact that it is infected from its host cells. For the past few years, researchers at the University of Missouri have helped uncover how this works.
“If you delete Vpu, those virus-infected cells are killed more efficiently,” said Marc Johnson, a Bond LSC scientist and professor in the Department of Molecular Microbiology and Immunology at the MU School of Medicine.
Understanding the Basics
Johnson and his lab study the connection between viruses and their hosts, trying to understand how viruses convince the host to allow them to keep replicating. In the case of HIV, Johnson and his colleagues discovered that Vpu only functions with the help of B TrCP, another protein.
“The virus doesn’t do anything by itself,” Johnson said. “It only has nine genes, while we have 30,000. It works by tricking its host genes to doing its bidding for it.”
Yul Eum Song, a graduate researcher in Johnson’s lab, explained that CRISPR technology allowed them to alter the genome to see how the proteins operate. CRISPR is a gene editing technique adapted from the DNA of bacteria that allows scientists to add, remove or edit specific locations in a genome.
“So, we use this to target and knock out genes to see if they’re necessary for the mechanism of HIV,” Song said.
They used CRISPR to remove the two types of B TrCP strands from the genome to see if Vpu would still hide HIV without its help.
“CRISPR has lots of applications, but the simplest, and the one I use is basically just molecular scissors that you can put into cells. It will make cuts in the genome wherever you want,” Johnson said.
By removing a certain gene, researchers can see how cells will react with and without certain proteins. In this case, Johnson and his team discovered that without either type of B TrCP strand expressed in the genome, Vpu couldn’t function, so it couldn’t avoid being killed by the immune system.
The magnitude of the HIV problem
“Thirty-five million people are infected with HIV. There are more people living with HIV today than ever in history, and the number keeps going up every year because once they’re infected, they’re infected for life,” Johnson said.
Johnson said that in terms of public health, HIV is a huge concern. Though treatments have gotten substantially better, they can have nasty side effects and don’t always work for everyone, sometimes resulting in death.
“By understanding the mechanisms of HIV, researchers can help combat the virus and create treatments,” Song said.
Both researchers pointed out how discoveries in the area of HIV can translate into other fields. The knowledge that virologists researching HIV find can help other scientists figure out what questions to ask and what functions to look for.
“We’ve learned about the cell and how our own cells work by studying the virus, but the virus is constantly utilizing and counteracting in ways that we’re still figuring out,” Johnson said.
Looking forward, Johnson and his lab are trying to find compounds that will block Vpu, and consequently allow the immune system to kill the HIV infected cells. Though the virus can never be completely removed from someone’s system, virologists search for treatments that can target and kill those cells that are expressing HIV.
“Part of the process is nailing down how VPU works, and a big part of what my lab is doing now is actually screening and testing compounds that do block VPU activity,” Johnson said. “That means if you have an infected virus, you treat them with this compound, and then they behave just like VPU was not there.”
By understanding how various proteins function in cells, researchers can get closer to understanding ways to combat viruses, and closer to understanding the micro-complexities that exist inside the human body.
This research was published in the Journal“Viruses” in Oct. 2018 and was funded by the National Institute of Allergy and Infectious Disease of the National Institutes of Health.
“#IAmScience because I am fascinated by life on a molecular level and inspired that my research could positively impact medicine.”
As a graduate student in Donald Burke’s lab at Bond LSC, Paige Gruenke explores the role of ribonucleic acid, or RNA. That means her work involves a lot of test tubes. She looks at how specialized RNA molecules, called aptamers, bind tightly and specifically to proteins from HIV to prevent the virus from replicating. Her job is to locate the aptamers that bind to HIV proteins from a very large starting pool of RNA sequences by doing repeated cycles of removing the sequences that don’t bind and keeping the ones that do, until the strong binders dominate the population.
“A lot of the things I do don’t sound very exciting,” said Gruenke. “It’s throwing components into tubes and waiting for things to happen. It might sound mundane, but it’s all for the greater good.”
Gruenke hopes that her research will give scientists a better understanding of HIV, because understanding the virus will lead to better drug treatments and eventually, a cure. She is finishing up her second year as a Ph.D candidate in biochemistry, and plans to graduate by summer 2020. Gruenke has always been interested in the area of molecular medicine, but she has some advice for students who are just getting started.
“On a day to day basis, many experiments fail,” said Gruenke. “You’re always going to be learning something you didn’t know before. So, don’t be disheartened because something didn’t work out — just keep trying. Because whenever you have an ‘Aha!’ moment, it makes it all worthwhile.”
Work on HIV capsid proteins earns prestigious retrivology award
Science is all about structure in the work of Anna Gres.
For the past four years, she’s looked closely at one HIV protein to figure out its shape in order to stop the virus.
“Capsid protein is extremely important during the HIV life cycle. About 1,500 copies of it come together to form the protective core around the viral genome,” said Gres, a graduate student in the lab of Bond Life Sciences Center’s Stefan Sarafianos and a Mizzou Ph.D. candidate in chemistry. “So, if you are able to somehow disrupt the interactions between the proteins or make them different, the virus loses its infectivity.”
Gres takes her work on this protein to a national stage next month when she speaks at the Retroviruses meeting at Cold Spring Harbor Laboratory — one of the most prestigious international conference on retroviruses — as the recipient of the 2016 Uta Von Schwedler prize. The prize recognizes the accomplishments of one distinguished graduate student as they complete their thesis.
HIV capsid protein has been studied for almost 30 years, but it’s been tricky to get a precise depiction of what it looks like. Gres uses X-ray crystallography to essentially capture the protein in all its 3-D glory. This method gives scientists the higher resolution picture to study the molecular structure of capsid protein. Her work allows the Sarafianos lab and others to study how it interacts and connects with other capsid proteins and the host protein factors of the cell HIV is trying to take over.
“In the past scientists had been splitting the capsid protein in two halves and crystallizing them separately. Another approach was to introduce several mutations to make it more stable,” Gres said. “You would think that it shouldn’t really matter if we have a few mutations, but the protein behaves in such a way that even slight changes result in subtly different interactions that are enough for the virus to lose its infectivity. We were able to crystallize the native protein without any mutations and that should give us more accurate picture.”
Now that the Sarafianos lab and Gres have a good idea of what that native protein looks like, they’ve moved on to other mutated versions of the protein that impair virus infectivity. This could give them insight into how scientists can stop HIV.
“Many labs reported numerous mutations in the capsid protein over the past 25 years that either increase or decrease the stability of the core, which often results in a noninfectious virus,” she said. “Right now we are interested in seeing what structural changes accompany these mutations and how they can affect the overall stability of the core.”
Nineteen colorful foam flowers decorate the walls of Marc Johnson’s office, a memento from his lab members when they “redecorated” while he was out of town.
Each flower is labeled in bold Sharpie with the names of viruses and viral proteins that his lab studies—MLV, RSV, Gag, Pol, to name a few.
One flower stands out, marked in capital letters: H-I-V.
Johnson, an associate professor of molecular microbiology and immunology, is one of four researchers at Bond LSC who studies HIV, the virus that leads to AIDS. His research focuses on understanding how HIV assembles copies of itself with help from the cells it infects.
Like most viruses, HIV hijacks cellular functions for its own purposes.
“It has this tiny itty bitty little genome and yet it can infect 30 million people,” Johnson said. “It doesn’t do it by itself.”
To understand how viruses reprogram the proteins in our bodies to work against us, he said, you have to understand the cells they infect. If cells were a chamber, then viruses are the keyhole.
For example, cells use a protein called TSG101 to dispose of unwanted surface macromolecules by bending a patch of cellular membrane around the macromolecule until it is surrounded inside a membrane bubble. The process, like trapping a bug inside a sheet of tissue paper, is called budding.
The cell sweeps all the pinched-off bubbles into a larger receptacle, or multivesicular body. These bodies, Johnson said, act as the cell’s garbage collection system. To dispose of the trash, the compartments become acidic enough to disintegrate everything inside or fuse with the cell membrane so that the trash gets dumped outside the cell.
It’s like in the second Star Wars movie, “The Empire Strikes Back,” Johnson said. “They just drop all their garbage before they go into hyperspace, and that’s how the Millennium Falcon got out.”
HIV uses the same housekeeping mechanism to break out of infected cells and infect more cells, but it remains unclear which other host proteins HIV commandeers.
“It’s all part of the puzzle,” Johnson said.
THE GAME CHANGER
On his desk, Johnson keeps a white legal pad with a list of 16 projects written in blue ink.
“Things make it off the list or they’ll get added,” Johnson said. “Or they’ll spend years on the back burner. I have a lot of projects.”
One of the biggest projects involves using CRISPR/Cas9 — a precision gene-editing tool — to identify genes that make a cell resistant to viral infections.
“It’s a game changer. It really is,” Johnson said. “It’s so cool.”
The technology uses a missile-like strand of guide RNA to target specific sites in the genome for deletion. Before CRISPR, scientists had to suppress gene expression using methods that were neither permanent nor absolute.
But because CRISPR manipulates the genome itself, Johnson said, there’s less doubt about what is happening.
Using the CRISPR library, the Johnson lab can scan the effects of 20,000 unique gene deletions in a population of cells. When these cells, each of which contains a different deleted gene, are exposed to HIV, not all of them die. Those that survive can cue researchers in to which genes might be important for blocking HIV infection.
And if another researcher has doubts that a gene is truly knocked out, Johnson said, you can tell them, “I’ll just send you the cell line. You try it and see for yourself.”
A DAY IN THE LIFE
The Johnson lab is a tight-knit group that consists of a lab manager, two grad students, a postdoc and four undergrads.
Dan Cyburt — a third year grad student — studies molecules that interact with proteins that keep HIV from infecting the cell, such as TRIM5α. TRIM5α, a restriction factor, blocks replication of the viral genome.
Fourth year grad student Yuleum Song focuses on how the viral envelope protein, Env, is packaged into viruses before they break free from cells. While Env isn’t necessary for viral assembly and release, she said, it’s critical for the infection of new cells.
Undergrads work in a tag team, picking up where the other left off, to generate a collection of new viral clones.
And lab manager Terri Lyddon keeps day-to-day experiments on task.
Lyddon, who has been with the Johnson lab for ten years, spends much of her day working with cells inside the biosafety level 2 hood. The area is specifically designated for work with moderately hazardous biological agents such as the measles virus, Samonella bacteria, and a less potent version of HIV.
Normally, HIV contains instructions in its genome for making accessory proteins that help the virus replicate, but the HIV strains used in the Johnson lab lack the genes for some of these proteins. That means the handicapped viruses can infect exactly one round of cells and spread no further.
Lyddon also ensures quality control for the lab by making sure students’ work is reproducible.
As a pet project, Johnson also independently confirms new findings reported in academic journals about HIV. Sometimes, Johnson says, the phenotypes that get published are not wrong, but they tend to represent the best outcomes, which might only exist in very specific scenarios.
“They’re only right by the last light of Durin’s day,” Johnson said, making a Lord of the Rings reference to a phenomenon in The Hobbit that reveals the secret entrance to a dwarven kingdom only once a year.
Because scientists base their work on the research of other scientists, he said, it’s always important to check.
A RECONSIDERED POSITION
According to the World Health Organization, 37 million people worldwide in 2014 have HIV or AIDS. The virus infects approximately two million new individuals every year. Breakthroughs in treatment have turned the autoimmune disease from a highly feared death sentence into a chronic and manageable condition.
For the longest time, HIV researchers scrambled to find better therapies against HIV why trying to develop a vaccine that could prevent AIDS.
But in the past five years, Johnson says he’s noticed a shift: researchers are gaining confidence in the possibility of finding a cure, something he once thought was impossible.
“Now it’s been demonstrated that it’s possible to cure a person,” Johnson said, referring to the Berlin patient. “So it’s only going to get easier.”
However, Johnson pointed out, most people would never undergo the kind of high-risk treatment that Timothy Ray Brown, the Berlin patient, received. Brown underwent a bone marrow transplant to treat his leukemia, and his new bone marrow, which came from an HIV-resistant donor, cured him of AIDS.
A “full blown cure” will be hard to attain, but Johnson believes there may be ways for HIV patients to live their lives without having to constantly take medication.
As an example, he points to certain “elite controllers” who are HIV positive but never progress further to show symptoms of AIDS. If scientists can figure out what’s different about their immune systems, Johnson said, then researchers could train the immune response in AIDS patients to resist HIV or keep it in check.
That’s a project for the immunologists. As a basic scientist, Johnson says he adds to the knowledge of how HIV works.
“I am not thinking about a therapy,” Johnson said, “but I’m also acutely aware that some of the best solutions come from basic science. “
Even though scientists haven’t discovered all the mechanisms behind cellular and viral function yet, Johnson said, the rules do exist.
“The sculpture is already there in the stone,” he said.
Johnson’s job is to chip away at the marble until the rules are found.
“They can inhibit influenza virus, Ebolavirus, HIV and SARS coronavirus,” said Liu, an associate professor in the Department of Molecular Microbiology and Immunology at the Bond Life Sciences Center.
Liu wanted to know why IFITM’s inhibition of HIV was uncharacteristically weaker than its inhibition of other viruses.
To study this conundrum, many researchers designed their experiments by expressing IFITM proteins in target or healthy cells. Then they infected these IFITM-bolstered cells with HIV, but saw minimal protection against viral infection.
In a twist, the Liu group put IFITM proteins in HIV-1 producer or infected cells instead of in healthy T-lymphocyte cells, a special kind of immune cell used specifically to study the viral infection by HIV.
They found that IFITM proteins, especially IFITM2 and IFITM3, interacted with the viral envelope protein (Env) that makes up the outer shells of virus particles.
For normal HIV infections to occur, Liu said, envelope proteins need to be cleaved into two parts.
Once processed, the resulting two portions, Env gp120 and gp41, can be incorporated into viral particles. The two processed envelope proteins protrude from the outer surface of the virus like mushroom-shaped pegs that help the virus latch on and fuse to target cells.
But when IFITM binds to envelope proteins they interfere with the viral envelope functions.
“It’s just unexpected,” Liu said, about this finding. In other viruses his group has studied, IFITM inhibited virus’ ability to fuse its outer shell with the membrane of a cell by making the cell membrane rigid during the infection process. He said he assumed IFITM would block HIV the same way.
Instead, they found evidence suggesting that IFITM blocks infection through direct contact with HIV’s envelope proteins.
“It is the first study that shows this kind of interaction,” Liu said. “That’s why this study is so surprising. We did not think about this.”
Liu does not yet know the mechanism behind IFITM and envelope protein interactions, but he said the outcome remains clear. “IFITM proteins inhibit this Env cleavage process and this makes HIV less infectious and less transmissible.”
To visualize IFITM’s inhibitory effects in action, Liu’s group tagged HIV-1 inside infected cells with a green fluorescent dye. Then they colored healthy target cells with a red fluorescent dye. When they mixed the two populations of cells together, they saw two days later a very tiny amount of cells exhibiting green signals within the red cells —a sign of spread of HIV cell-to-cell infection.
By comparison, cells in the control group—healthy red-tagged cells mixed with green HIV-infected cells that do not contain IFITM—showed a higher number of red cells lighting up green inside.
This suggested that having IFITM and HIV-1 inside the virus-producing cells somehow limited the virus’ infectivity and cell-to-cell spread at the same time.
His group also showed through a technique called co-immunoprecipitation that IFITM proteins bound specifically with envelope protein rather than with other proteins, such as Gag. Liu attributed this work to his two talented hardworking graduate students, Jingyou Yu and Minghua Li.
Unfortunately, the benefits of IFITM are short-lived. When the Liu laboratory let HIV-infected cells replicate again and again, they saw that HIV could evolve enough to circumvent the inhibitory effects of IFITM after 30 passages.
Liu said that his research on IFITM was still in its early stages, but that the next step would be to look at the IFITM’s function in HIV patients in order to move the basic research of IFITM from bench to bedside.
“Once we know better how this protein works, we can develop some inhibitors to block HIV, block Ebola, block other viruses,” Liu said. “So that’s our ultimate goal.”
Liu is a Bond Life Sciences Center investigator and an associate professor of molecular microbiology and immunology in the MU School of Medicine. He studies how viruses infect healthy host cells to cause illness and cell response to viral attacks.
The National Institutes of Health and the Canadian Institutes of Health Research partially supported this research. Additional collaborators include Eric Freed, PhD, senior investigator with the National Cancer Institute (NCI) HIV Dynamics and Replication Program; Chen Liang, PhD, at McGill University; and Benjamin K. Chen, PHD, at the Mount Sinai School of Medicine. Read the full study on the Cell Reports website and browse the supplementary data for this work. See more on this research from Mizzou News.
Seeing the whole picture can mean a lot when it comes to figuring out HIV.
Researchers at the University of Missouri Bond Life Sciences Center are gaining a clearer idea of what a key protein in HIV looks like, which will help explain the flexible protein’s vital role in the virus life cycle.
“The capsid acts as an invisibility cloak that hides the virus’ genetic information, the genome, while it is being copied in a hostile environment for the virus,” said Stefan Sarafianos, a virologist at Bond LSC and lead author of the study. “Fine-tuned capsid stability is critical for successful infection: too stable a capsid shell and the cargo is never delivered properly; not stable enough and the contents are detected by our immune defenses, triggering an antiviral response. Capsid stability is a key to the puzzle, and to solve it you have to understand its structure.”
This is the most complete model yet of an HIV-1 capsid protein. In a virus, the protein combines in groups of five or six — called pentamers and hexamers, respectively — that assemble into a mosaic that forms the capsid shell. Roughly 1,500 copies of the protein, grouped into about 250 hexamers and 12 pentamers, comprise the capsid.
HIV, or human immunodeficiency virus, is the retrovirus that leads to AIDS — acquired immunodeficiency syndrome. Roughly 1.2 million people live with HIV in the United States, according to the Centers for Disease Control and Prevention. Globally, about 35 million people were living with HIV in 2013.
A lucky break
Over the years, scientists have employed various techniques and tricks to figure out the structure of the capsid protein. But until now, the clearest image had been made of a mutated version of the protein. It was a compromise: the mutation made the protein stable enough that the scientists could get a good snapshot, but they couldn’t see the detailed interactions between hexamers.
Sarafianos’ lab figured out how to get the full picture: a detailed image of the unmodified proteins that filled all the gaps between hexamers.
The team used a technique called X-ray crystallography to unravel the protein’s secrets. Basically, they took many copies of the protein and coaxed them into forming a patterned, crystalline lattice. Next they shot high-powered X-ray beams at the crystal. By interpreting how the X-rays scattered when they ricocheted off the proteins, the researchers made a 3-D map of the protein.
“But it doesn’t make sense until we make an atomic model of the protein to fit in that map,” said Karen Kirby, a research scientist at Bond LSC. “The map is just a grid that you can’t really interpret unless you put a model into it to see ‘Ok, it looks like this part is here, and that part is there, and this is how the protein is put together.’”
The researchers altered, tested and honed their 3-D model until it exactly matched the map produced by the X-ray diffraction pattern. This can be difficult and painstaking, but the researchers’ greatest challenge was creating the protein crystals in the first place: Scientists had been trying to crystallize the unmodified version of the HIV protein for decades without success.
To make a crystal, proteins are suspended in a liquid then slowly precipitated out, just like a “grow your own crystals” kit. But there are a lot of variables that control the process, from salts and additives in the liquid to the amount of protein in the mixture.
“It’s a very delicate balance to grow crystals,” Kirby said. “Many people call it more of an art than a science. It’s frustrating because you can never predict which solution will grow crystals. There are a large number of variables.”
Initially, most arrangements the researchers tried resulted in useless brown junk, Kirby said, caused by the proteins forming solids too quickly. Anna Gres, an MU chemistry grad student who led the project, used a crystallization robot to screen roughly 2,500 conditions.
That was the easy part, Gres said: “The real challenge begins afterwards, as one needs to manually optimize the initial crystallization conditions to find the one that will produce protein crystals of desired quality. This process can take years. In our case, I think we were lucky: It took approximately 500 manual screenings and about 6 month.” But the hard work paid off when she was finally able to produce lovely, hexagonal crystals. Surprisingly, the crystals formed in groups of six proteins, which matched their formation in the viral capsid.
The transition from tiny, useless particulate to invaluable crystals was tremendously exciting, Kirby said. But even to Kirby and Sarafianos, why their attempts succeeded when many others failed remains a little mysterious.
“I still don’t know what are the fine details that made the difference,” Sarafianos said.
“That’s the million dollar question,” said Kirby. “We really don’t have a good answer for that.”
Although solving the enigmatic crystal structure of the native full-length capsid protein was really rewarding, Gres said, she will continue to tinker with her technique: “I am still trying to optimize crystallization conditions, hoping to improve the quality of the crystals and diffraction.”
Water, water everywhere
Once the researchers got a good look at the interactions between hexamers, they were surprised by what they found.
Based on the genetic sequence of the protein, scientists speculated that they would be hydrophobic, or repel water. Instead, they found that “ordered” water molecules at specific sites played a crucial structural role by bridging interactions at the interface between hexamers.
“We thought, ‘How could these lowly waters really be of consequence?’” Sarafianos said. “But if you think about it, there’s 256 of these hexamers in the whole capsid and all kinds of interfaces among them: There’s thousands of water molecules that stabilize the whole structure. We hypothesize that this is an essential part of the stability of the whole capsid molecule.”
To test that hypothesis, they took the crystals, dehydrated them and checked to see if their shape changed. Although the protein lattices may look like sturdy crystals, they’re more like jello, Sarafianos said.
“The protein molecules are precariously touching each other and forming a lattice that is very, very sensitive. It’s held together in this case by water molecules in addition to other interactions.”
The change in shape suggested that water molecules are important in that they allow the capsid to assume different shapes. Moreover, Sarafianos said, the capsid’s malleability and plasticity could be critical to the life cycle of the virus and allow it to act as a multi-functional molecular Swiss army knife.
Onward with research
A clearer image of the capsid protein, could help Sarafianos’ lab gain a better understanding of how the body combats the virus and to discover new ways to disrupt the viral capsid.
“Now we have a system to study effects of capsid-targeting compounds with novel mechanism of action,” Gres said.
Working with a medicinal chemist, Sarafianos’ lab will undertake an iterative process of making compounds, solving their structures, testing them against HIV and then refining the molecules, with the ultimate aim of producing new and effective antiviral drugs.
“It’s a striking phenomena caused by this particular protein,” Liu said. “The HIV is already assembled inside the cell, ready for release, but this protein surprisingly tethers this virus from being released.”
The TIM – T-cell/transmembrane immunoglobulin and mucin – family of proteins hasn’t received much attention from HIV researchers, but recent research shows the protein family plays a critical role in viral infections. From Ebola and Dengue to Hepatitis A and HIV, these proteins aid in the entry of viruses into host cells.
But its ability to stop the virus from leaving cells remained unknown until now. Liu’s lab stumbled onto this finding in November 2011 when trying to create stable cells for a different experiment. After two months of troubleshooting the HIV lentiviral vector – where genes responsible for creating TIM-1 proteins were inserted into a cell to create a stable cell line that expresses the protein – Liu was confident the vector’s failure was not only interesting but also important.
The lab spent the next two years trying to figure out what was happening. Minghua Li, an MU Area of Pathobiology graduate student, carried out experiments that confirmed the protein’s power to inhibit HIV-1 release from cells, reducing normal viral infection. His experiments showed TIM proteins prevent normal deployment of HIV, created by an infected cell, into the body to propagate.
TIM proteins stand erect like topiary on the outside and inside surfaces of T-cells, epithelial cells and other cells. When a virus initially approaches a cell, the top of each TIM protein binds with fats – called phosphatidylserine (PS) – covering the virus surface. This allows a virus, such as Ebola virus and Dengue virus, to enter the cell, infect and replicate, building up a population inside.
But as the virus creates new copies of itself, the host cell’s machinery also incorporates TIM proteins into new viruses. That causes problems for HIV as it tries to leave the cell. Now these proteins cause the viruses to bind to each other, clumping together and attaching to the cell surface.
“We see this striking phenotype where the virus just accumulates on the cell surface,” said Liu, who is also an associate professor in the MU School of Medicine’s Department of Molecular Microbiology and Immunology. “We consider this an intrinsic property of cellular response to viral infection that holds the virus from release.”
Further research is needed to determine overall benefit or detriment of this curious characteristic, but this discovery provides insight into the cell-virus interaction.
“This study shows that TIM proteins keep viral particles from being released by the infected cell and instead keep them tethered to the cell surface,” said Gordon Freeman, Ph.D., an associate professor of medicine with Harvard Medical School’s Dana-Farber Cancer Institute, who was not affiliated with the study. “This is true for several important enveloped viruses including HIV and Ebola. We may be able to use this insight to slow the production of these viruses.”
The National Institutes of Health and the University of Missouri partially supported this research. Additional collaborators include Eric Freed, PhD, senior investigator with the National Cancer Institute (NCI) HIV Drug Resistance Program; Sherimay Ablan, biologist with the NCI HIV Drug Resistance Program; Marc Johnson, PhD, Bond LSC researcher and associate professor in the MU Department of Molecular Microbiology and Immunology; Chunhui Miao and Matthew Fuller, graduate students in the MU Department of Molecular Microbiology and Immunology; Yi-Min Zheng, MD, MS, senior research specialist with the Christopher S. Bond Life Sciences Center at MU; Paul Rennert, PhD, founder and principal of SugarCone Biotech LLC in Holliston, Massachusetts; and Wendy Maury, PhD, professor of microbiology at the University of Iowa.
By Madison Knapp | Bond Life Sciences Center summer intern
Modern science has found a way to turn viruses —tiny, dangerous weapons responsible for runny noses, crippling stomach pains and worldwide epidemics such as AIDS— into a tool.
Gene therapy centers on the idea that scientists can hijack viruses and use them as vehicles to deliver DNA to organs in the body that are missing important genes, but the understanding of virus behavior is far from exhaustive.
Marc Johnson, researcher at the Christopher S. Bond Life Sciences Center and associate professor of molecular microbiology and immunology in the MU School of Medicine, has been building an understanding of viral navigation mechanisms which allow a virus to recognize the kind of cell it can infect.
Johnson’s research specifically explores the intricacies of the viral navigation system and could improve future direction of gene therapy, he said.
Turning a virus into a tool
Conceptualized in the 1970s, gene therapy was developed to treat patients for a variety of diseases, including Parkinson’s, leukemia and hemophilia (a genetic condition that stops blood from clotting).
To treat disease using gene therapy, a customized virus is prepared. A virus can be thought of as a missile with a navigation system and two other basic subunits: A capsule that holds the ammunition and the ammunition itself.
The viral genetic material can be thought of as the missile’s ammunition. When a cell is infected, this genetic material is deployed and incorporated into the cell’s DNA. The host cell then becomes a factory producing parts of the virus. Those parts assemble inside the cell to make a new virus, which then leaves the cell to infect another.
The capsule is made of structural protein that contains the genetic material, and the navigation system is a protein that allows the virus to recognize the kind of cell it can infect.
Gene therapy uses viruses to solve many problems by utilizing a virus’ ability to integrate itself into a host cell’s DNA; to do this successfully, researchers need to provide a compatible navigation component.
In the body, viruses speed around as if on a busy highway. Each virus has a navigation system telling it which cells to infect. But sometimes if a virus picks up the wrong type of navigation system, it doesn’t know where to go at all.
“What you can do is find a virus that infects the liver already, steal its navigation protein and use that to assemble the virus you want to deliver the gene the liver needs,” Johnson said. “You can basically take the guidance system off of one and stick it onto another to custom design your virus.”
But this doesn’t always work because of incompatibility among certain viruses, he said.
Johnson and his lab are working to understand what makes switching out navigation proteins possible and why some viruses’ navigation systems are incompatible with other viruses.
“I’m trying to understand what makes it compatible so that hopefully down the road we can intelligently make others compatible,” Johnson said.
The right map, the right destination
Johnson creates custom viruses by introducing the three viral components—structural protein, genetic material, and navigation protein—to a cell culture. The structural protein and genetic material match, but the navigation component is the wild card. It could either take to the other parts to produce an infectious virus, or it could be incompatible.
Johnson uses a special fluorescent microscope to identify which viruses assembled correctly and which didn’t.
A successful pairing is like making a match. If a navigation protein is programmed to target liver cells, it’s considered a successful pairing when the virus arrives at the liver cell target location.
The scope of gene therapy continues to widen. Improved mechanisms for gene therapy, and greater knowledge of how a navigation protein drives a virus could help more people benefit from the vehicles viruses can become.
Johnson uses several high-profile model retroviruses, including human immunodeficiency virus (HIV), which affects an estimated 35 million people worldwide each year, according to the World Health Organization.
Understanding nuances of HIV in comparison to other viruses allows Johnson to pick out which behaviors might be common to all retroviruses and others behaviors that might be specific to each virus.
Johnson said his more general approach makes it easier to understand more complex viral features.
“If there are multiple mechanisms at work, it gets a little trickier,” Johnson said. “My angle is more generic, which makes it easier to tease them apart.”