HIV

Paige Gruenke #IAmScience

Paige Gruenke

Paige Gruenke, a Ph.D candidate in Dr. Donald Burke’s lab in Bond LSC. | Photo by Mary Jane Rogers, Bond LSC

By Mary Jane Rogers | Bond LSC

“#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.”

You shall not pass: the basic science of blocking HIV

 

Marc Johnson, associate professor of molecular microbiology and immunology at the Bond Life Sciences Center, studies viruses such as HIV. | photo by Jennifer Lu, Bond LSC

Marc Johnson, associate professor of molecular microbiology and immunology at the Bond Life Sciences Center, studies viruses such as HIV. | photo by Jennifer Lu, Bond LSC

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.

Marc Johnson observes cells modified with CRISPR under the microscope. | photo by Jennifer Lu, Bond LSC

Marc Johnson observes cells modified with CRISPR under the microscope. | photo by Jennifer Lu, Bond LSC

“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.

Graduate student Yuleum Song prepares cells for viral infection in the BL-2 hood. | Image by Jennifer Lu, Bond LSC

Graduate student Yuleum Song prepares cells for viral infection in the BL-2 hood. | Image by Jennifer Lu, Bond LSC

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.

Filling in the gaps of HIV

By Caleb O’Brien | MU Bond Life Sciences Center

The HIV capsid protein (shown above in an array of hexagons) plays a critical role in the virus life cycle. Bond LSC researchers recently developed the most complete model yet of this vital protein, a building block that forms the protective shell surrounding the virus’ genes. The journal Science published their findings online June 4. | Image by Karen Kirby

The HIV capsid protein (shown above in an array of hexagons) plays a critical role in the virus life cycle. Bond LSC researchers recently developed the most complete model yet of this vital protein.  Image by Karen Kirby and Anna Gres

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 protein the researchers imaged is a building block that forms the virus’ capsid, a protective shell surrounding the virus’ genes. The journal Science published their findings online June 4.

Stefan Sarafianos
Stefan Sarafianos

“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.

The protein building block of HIV capsid (top left) can assemble to form a hexamer (middle left). Crystals grown using this building block (top and middle left) contain an array or lattice of hexamers (bottom). | Image by Karen Kirby and Anna Gres

The protein building block of HIV capsid (top left) can assemble to form a hexamer (top middle). Crystals grown using this building block (bottom left and middle) contain an array or lattice of hexamers (right). | Image by Karen Kirby and Anna Gres

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.

17869819134_e42f708381_k
Karen Kirby

“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.

Sarafianos is an associate professor of molecular microbiology and immunology and Chancellor’s Chair of Excellence in molecular virology with MU’s School of Medicine and an associate professor of biochemistry in the MU College of Agriculture, Food and Natural Resources.

The study, “X-Ray Crystal Structures of Native HIV-1 Capsid Protein Reveal Conformational Variability,” was recently published in the journal Science. Research funding has been provided by the National Institutes of Health (Grants AI112417, AI120860, GM103368, AI076119, AI099284, and AI100890 (SGS), and GM066087 (OP)). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

Holding on: Bond LSC scientist discovers protein prevents release of HIV and other viruses from infected cells

Shan-Lu Liu and Minghua Li, HIV Research at the Bond Life Sciences Center

Shan-Lu Liu, Bond LSC scientist and associate professor in the MU School of Medicine’s Department of Molecular Microbiology and Immunology. Courtesy Justin Kelley, University of Missouri Health System.

Shan-Lu Liu initially thought it was a mistake when a simple experiment kept failing.

But that serendipitous accident led the Bond Life Sciences Center researcher to discover how a protein prevents mature HIV from leaving a cell.

Proceedings of the National Academy of Sciences published this research online Aug. 18.

“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.

Shan-Lu Liu and Minghua Li, HIV Research at the Bond Life Sciences Center

Minghua Li, coauthor of the study and an MU Area of Pathobiology graduate student. Courtesy Justin Kelley, University of Missouri Health System.

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.”

This model shows the  interaction between TIMs and PS among the round HIV virions, as well as that between viral producer cells. This collectively leads to accumulation of HIV virions on the plasma membrane on the outside of the cell. Courtesy Mingua Li.

This model shows the interaction between TIMs and PS among the round HIV virions, as well as that between viral producer cells. This collectively leads to accumulation of HIV virions on the plasma membrane on the outside of the cell. Courtesy Minghua Li.

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.

Read the full study on the PNAS website and browse the supplementary data for this work. See more news on this research from the MU School of Medicine.

Viruses as Vehicles: Finding what drives

Graduate students Yuleam Song and Dan Salamango inoculate a bacteria culture in Johnson's lab. The inoculation takes a small portion of a virus and multiplies the sample, allowing researchers to custom-make viruses.

Graduate students Yuleam Song and Dan Salamango inoculate a bacteria culture in Johnson’s lab. The inoculation takes a small portion of a virus and multiplies the sample, allowing researchers to custom-make viruses.

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.

 

Marc Johnson (left) with a post doctoral student that works in his lab. The lab does important research on the basic function and mechanisms of viral navigation and transport.

Marc Johnson (left) with Dan Salamango, a graduate student that works in his lab. The lab does important research on the basic function and mechanisms of viral navigation and transport.

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.

 

Viral navigation

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.”

Supervising editor is Paige Blankenbuehler

A drug that packs a punch: new compound works better against resistant HIV

Virologist Stefan Sarafianos stands in the atrium of the Bond LSC.

Bond LSC researcher Stefan Sarafianos stands in the LSC atrium. The virologist is an associate professor of molecular microbiology and immunology and Chancellor’s Chair of Excellence in Molecular Virology with appointments in MU’s School of Medicine and the Department of Biochemistry.

Resistance is the price of success when it comes to treating HIV.

Virologists at the Bond Life Sciences Center are helping to test the next generation of anti-AIDS medication to quell that resistance.

Stefan Sarafianos’ lab recently proved that EFdA, a compound that stops HIV from spreading, is 70 times more potent against some HIV that resists Tenofovir – one of the most used HIV drugs.

“HIV in patients treated with Tenofovir eventually develop a K65R RT mutation that causes a failure of this first line of defense,” said Sarafianos, virologist at Bond LSC. “Not only does EFdA work on resistant HIV, but it works 10 times better than on wild-type HIV that hasn’t become Tenofovir resistant.”

Sarafianos and a team of researchers found that EFdA (4′-ethynyl-2-fluoro-2′-deoxyadenosine) is activated by cells more readily and isn’t broken down by the liver and kidneys as quickly as similar existing drugs.

“These two reasons make it more potent than other drugs, and so our task is to look at the structural features that make it such a fantastic drug,” he said.

 

From soy sauce to virus killer

The path from EFdA’s discovery to current research is a bit unorthodox.

A Japanese soy sauce company named Yamasa patented this molecule, which falls into a family of compounds called nucleoside analogues that are very similar to existing drugs for HIV and other viruses. EFdA was designed and synthesized by Hiroshi Ohrui (Chem Rec. 2006; 6 (3), 133-143Org. Lett. 2011; 13, 5264) and shown by Hiroaki Mitsuya, Eiichi Kodama, and Yamasa to have potential usefulness against HIV. Samples sent for further testing confirmed EFdA’s potential usefulness against HIV. This started more than a decade of research to pinpoint what makes the compound special.

EFdA joins a class of compounds called nucleoside reverse transcriptase inhibitors (NRTIs) that includes eight existing HIV drugs. Like all NRTIs, EFdA hijacks the process HIV uses to spread by tricking an enzyme called reverse transcriptase (RT). RT helps build new DNA from the RNA in HIV, assembling nucleoside building blocks into a chain. Since EFdA looks like those building blocks, RT is tricked into using the imposter. When this happens the virus’ code cannot be added to the DNA of white blood cells it attacks.

“NRTIs are called chain terminators because they stop the copying of the DNA chain, and once incorporated it’s like a dead end,” Sarafianos said.

 

A little help from some friends

Sarafianos isn’t alone in studying EFdA.

The virologist’s lab works closely with University of Pittsburgh biochemist Michael Parniak and the National Institutes of Health’s Hiroaki Mitsuya to explore the molecule’s potential. Mitsuya had a hand in discovering the first three drugs to treat HIV and Parniak has spent years evaluating HIV treatments using cultured white blood cells.

Sarafianos’ focus requires him to take a very close look at EFdA to define how it works on a molecular level. He uses virology, crystallography and nuclear magnetic resonance to piece together the exact structure, bonding angles and configuration of the compound.

By looking at subtle differences in EFdA’s sugar-like ring, his lab identified the best structure that looks the most like actual nucleosides, doesn’t break down easily and is activated readily by CD4+ T lymphocyte white blood cells.

“The structure of this compound is very important because it’s a lock and key kind of mechanism that can be recognized by the target,” Sarafianos said. “We’re looking at small changes and the ideal scenario is a compound bound very efficiently by the target and activating enzyme but not efficiently by the degrading enzymes.”

 

Treatment for the future

The research of Sarafianos, Parniak and Mitsuya continue to uncover the magic of EFdA. In 2012, they showed that the drug worked incredibly well to treat the HIV equivalent in monkeys.

“These animals were so lethargic, so ill, that they were scheduled to be euthanized when EFdA was administered,” said Parniak. “Within a month they were bouncing around in their cages, looking very happy and their virus load dropped to undetectable levels. That shows you the activity of the molecule; it’s so active that resistance doesn’t come in as much of a factor with it.”

HIV prevention is the newest focus in their collaboration.

By recruiting formulation expert Lisa Rohan at the University of Pittsburgh, they are now putting EFdA in a vaginal film with a consistency similar to Listerine breath strips.

“The only way we are going to make a difference with HIV is prevention,” Parniak said. “If we can prevent transmission, this approach could make a huge difference in minimizing the continued spread of the disease when combined with existing therapies for people already infected.”

While AIDS in the U.S. occurs mostly in men, the opposite is true in sub-Saharan Africa where more than 70 percent of HIV cases occur. Since a film has a better shelf life than creams or gels, it could benefit those at risk in extreme climates and third-world countries.

“We have nearly 30 drugs approved for treating HIV infected individuals, but only one approved for prevention,” Sarafianos said. “Women in Africa would benefit from a formulation like this as a means to protect themselves.”

Despite this success, Sarafianos and Parniak aren’t slowing down in figuring out how EFdA works so well.

“We want to understand how long EFdA stays in the bloodstream and cells,” Parniak said. “If we understand structurally why this drug is so potent it allows us to maybe develop additional molecules equally potent, and a combination of those molecules could be a blockbuster.”

Grants from the National Institutes of Health fund this research.

In 2013 and 2014, the journals Retrovirology, Antimicrobial Agents and Chemotherapy and The International Journal of Pharmaceutics published this group’s work on EFdA. Sarafianos is an associate professor of molecular microbiology and immunology and Chancellor’s Chair of Excellence in molecular virology with MU’s School of Medicine and a joint associate professor of biochemistry in the MU College of Agriculture, Food and Natural Resources.