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.”
Marc Johnson collecting pellets in his lab. | photo by Becca Wolf, Bond LSC
By Becca Wolf | Bond LSC
There is not much thought that goes into using the bathroom. You do your business, flush, and wash your hands. It is just a part of the daily routine.
Recently though, human waste has become a golden nugget to researchers. In fact, waste from toilets throughout the community are contributing to figuring out where the next COVID-19 outbreak could happen.
And Marc Johnson, Bond Life Sciences Center principal investigator and MU professor of molecular microbiology and immunology, is the person figuring out how this human waste can be an asset as he processes samples from the sewer systems of more than 70 communities across the state.
A map of the sewersheds Marc Johnson’s lab receives samples from. | map contributed by Marc Johnson
These samples may be able to serve as the canary in the coal mine before large outbreaks strike in a community, allowing health officials to prepare for and potentially prevent the spread of COVID-19.
SARS-CoV-2, the virus that causes COVID-19, appears in wastewater several days before an infected person shows symptoms, so Johnson wants to find a faster way to test for the virus.
“We’re very good at predicting what’s happening right now based on samples we collected five days ago,” Johnson said, “We are predicting the future, we just don’t know what our prediction is until the future is actually here.”
This data may not seem helpful, however, it proves that the COVID-19 case numbers are accurate and, if acted upon quickly, can prevent an outbreak in a concentrated population such as prisons or schools.
“With the students, when you’re talking about patients that don’t necessarily know they’re infected, it can be really helpful to have a heads up that this dorm has some infections even if they don’t know it,” he said.
Testing wastewater for SARS-CoV-2—an RNA virus—consists of two phases. Phase one is obtaining the sample and phase two is RNA extraction.
Starting phase one, Johnson receives a 24 hour composite from a wastewater testing facility, “So that no matter when, it doesn’t matter whenever people go, as long as they go once a day we’ll catch them,” he said. This gives his lab a representative sample of the population.
Once the sample is taken from a wastewater treatment facility, it is put into a courier system that brings it to the state lab in Jefferson City, Mo. After it arrives there, the Missouri Department of Natural Resources brings them to Johnson’s lab at Bond LSC so he can begin phase two, RNA extraction.
In Johnson’s lab, the samples are put through a filter that nothing bigger than a virus can get through. This eliminates the solids and other bacteria in the wastewater. Next, a chemical is added that allows the viral particles to stick together so they can be pelleted.
This is then put in a centrifuge and is spun for a few hours to get a small, invisible pellet. RNA is then extracted from this pellet in a QIAcube, a robot Johnson’s lab purchased for this project. The QIAcube is the same robot used by the Missouri Department of Health Services for COVID-19 testing in their own facilities.
Marc Johnson placing the samples in the QIAcube. | photo by Becca Wolf, Bond LSC
After Johnson extracts the RNA, the samples are sent to Chung-ho Lin, MU research associate professor at the College of Agriculture, Food and Natural Resources, who continues testing.
This whole process takes about five days.
Johnson hopes that test results will soon go public so people have an idea of where outbreaks are, although reading the data can be confusing.
Said Johnson, “I think we all agree we’ll be better if we put the data out the way we want it to be seen, because it’s just when you look at the raw numbers, without understanding how to interpret it, you can make any conclusion you want.”
It will be a learning curve for the public to be able to understand the data. Johnson even struggled with it at first, “We learned this lesson the hard way,” he said, “I freaked out several times where I thought I had just discovered Armageddon going to our state, but you have to put it in perspective. When you figure out how much it actually takes to get a signal and how big the sewer shed sample is, it’s like, ‘No okay, that’s normal.’”
Along with other testing sites, Johnson’s lab has been testing several universities and colleges in the state, including MU. With time and improvements, it is realistic that his lab will be able to predict the number of infected patients in an area.
“It’s a crap ton of work,” Johnson said, “But it’s kind of interesting. It worked far better than I would have expected.”
While research on COVID-19 was not on Johnson’s agenda earlier this year, he has been trying to have fun with it.
“The puns are unending,” he said, “There are just so many great puns I’ve gotten out of it.”
“It’s serious research but it’s nice that the topic has gotten a little bit of levity.”
Marc Johnson’s research focus changed suddenly one day this February when he received a shipment. That package of synthesized SARS-COV2 spike genes — the virus that causes COVID-19 — has now taken him down a new path.
“It was unusual, nothing like this has ever happened to me before,” Johnson said, an MU professor of molecular microbiology and immunology and Bond LSC investigator. “I’ve never had to switch directions so abruptly before but, you know, we’re always taking on new projects and shifting, it just usually doesn’t happen as fast.”
Typically, Johnson can be found in his lab studying viral glycoproteins, proteins with sugar attached to them involved in structural functions of the cell wall, and spikes, the knobby proteins on the surface of a cell. His main focus is studying HIV and its interaction with its host. However, his lab got to work right away to apply what they know to find a way to block SARS-COV2, and ultimately, COVID-19.
To do this, they focused on what they know best: glycoproteins and spike.
SARS-COV2 shares both of these features, and, in fact, Johnson has done previous research on other coronaviruses. Glycoproteins and spike allow the virus to attach to other cells. He infects cells with a safe, stripped virus containing the SARS-COV2 Spike and uses trial and error to see what works and what does not.
Early on making the glycoprotein functional was a challenge. Sometimes, when a protein of one virus is taken and stuck on another virus, it does not work. That was the case with SARS-COV2. He decided to cut of the tail of spike to see what happens.
“Most of its [spike] on the outside of the virus. There’s still a little piece on the inside, and if you make the virus smaller they don’t have that inside piece and often work better,” Johnson said, “I couldn’t get anything to work until I made that truncation.”
Without much of the tail, Johnson started looking for ways to block the virus. He has used many methods to find an effective blocking technique.
“We’ve thrown various peptides on and we’ve tested various small molecules,” Johnson said. “We’ve also tried plasma from patients who have recovered to see if they’re producing neutralizing antibodies and, no surprise, they are.”
Antibodies are crucial because they are a sign that someone has an effective defense against the virus. At the very least, antibodies allow the body to keep future infections in check. Taking antibodies from one patient and placing them in another patient passively transfers resistance to a virus and, often, immunity. Researchers are conducting studies to see if this is an effective way of blocking COVID-19.
Having only worked on COVID-19 for three months, there is still a lot Johnson—and the world—does not know.
“80% of people are just like, ‘Yeah, whatever, I’m fine,’ and then others just fall off the deep end. But, we don’t know what’s different. We don’t know why some patients do so poorly and others just shake it off,” he said. “It’s different than anything I’ve ever worked with before. I wake up every morning and it seems like there’s a new discovery every day.”
Working on COVID-19 is much different than working on other viruses, such as HIV. Many scientists are now putting manuscripts of their research online before they are published in hopes of aiding others.
While Johnson is aware that the sooner a vaccine is developed, the better, he knows not to rush things.
“It’s not about having a vaccine, it is about making sure that it’s safe and effective,” Johnson said. “We’re acutely aware that there’s this backlash against vaccines even when they are safe. If you put out one vaccine that wasn’t safe, you would ruin it for all vaccines for generations.”
For now, the search to stop COVID-19 continues and Johnson hopes his work helps come up with a treatment soon.
“We haven’t found a magic bullet yet, but we’ve seen some inhibition with various compounds,” Johnson said, “So it’s a starting point.”
On an average day, you can find post doctorate Norman Best surrounded by corn in the greenhouse or at his bench in the McSteen lab doing molecular work. However, since Columbia and state leaders issued a stay-at-home order on March 25 to prevent the spread of COVID-19, this means Bond LSC is mostly empty and researchers like Best are at home writing.
“It’s definitely made me appreciate what I had before,” Best said.
Coronaviruses are a family of viruses that can cause respiratory illness in humans. They’re found circulating among animals, and then passed to humans. While the world has seen dangerous coronavirus outbreaks including severe acute respiratory syndrome (SARS) in 2003 and Middle East respiratory syndrome coronavirus (MERS) in 2012, the 2019 emergence of COVID-19 has spread much more quickly.
According to the Centers for Disease Control and Prevention, COVID-19 is spread through respiratory droplets produced when an infected person coughs, sneezes or talks. Staying at home and avoiding contact with others will help prevent the spread.
Most researchers are working from a distance, like Best who is writing a paper on how the plant hormones auxin and brassinosteroid affect lateral meristem growth. However, some are deemed essential whether it’s to water plants, finish crucial experiments or study COVID-19 itself.
Marc Johnson, professor of molecular microbiology and immunology, is currently studying glycoproteins which are proteins on the surface of viruses that dictate what cell they’re going to infect. Even though Johnson usually works on HIV, he’s shifting his focus to COVID-19 and its glycoproteins called, spike.
Johnson is taking other viruses and replacing their glycoproteins with COVID-19 spike proteins to basically create a safe version of our current coronavirus. This will allow him to do multiple tests to try to inhibit viral entry.
In addition, these experiments can also test the effectiveness of antibodies. Recovered patients are donating their plasma — blood without red blood cells and just antibodies — to transfer their coronavirus-fighting antibodies to other patients.
“Of course, if you take plasma from a patient, you want to make sure that there are the antibodies you want in there, so that’s where my [experiment] would come into play to check whether there’s a high level of neutralizing antibodies in their serum,” Johnson said. “If there is, then you know it’s good for injecting. It might be helpful for the patient.”
For those who aren’t working on COVID-19, the interruption has some feeling frustrated.
When Best was in the lab, he was doing molecular work on creating a CRISPR construct. CRISPR is a method of editing genes, essentially splicing DNA into a cell. Now, it’s sitting in the freezer half done.
“I’m working on finishing up a few publications that I’ve had data for that I’ve actually
not been able to analyze before,” Best said. “I had not taken the time to analyze as much as I have now because I am sitting all day on the computer…However, there are still a few things left to do in the lab that have been delayed because of quarantining.”
Jean Camden, senior research associate in the Weisman lab, goes into Bond LSC once a week to check on the mouse breeding colony in addition to working from home.
“For us, the timing was good,” Camden said. “We had just finished some large experiments, and we are now writing, all of us. We have plenty of work to do at home.”
For many researchers, as Camden describes, writing papers is one of the last tasks to do.
“Now there’s no excuse,” Camden said. There’s nothing else to do but write.”
However, even the mounds of data researchers have been sitting on will eventually run out.
“If [the end of social distancing] doesn’t happen within the next two or three weeks, then we will be getting behind on getting experiments done,” Camden said.
So far, MU has moved in-person summer classes online, but is hopeful to re-open campus in the fall under a “new normal.”
“[The end of social distancing] should depend on our preparedness and the resources because one of the reasons why I think we shut down is that we weren’t ready to cater for all the people that were going to be sick, and so the best option was to prevent it,” said Kwaku Tawiah, fifth year graduate student who studies Ebola in the Donald Burke lab. “I don’t know when normal life can return as we knew it before.”
Even though there’s a lot of uncertainty of what’s ahead, Bond LSC researchers are learning to adapt and are continuing their research.
“It is very fortunate that I have been able to work at home and keep my job,” Camden said. “A lot of people here in Columbia have been laid off, and I feel bad for the terrible things that have happened. So, I appreciate the position that I’m in.”
At the moment, Best is still sitting at home with his dog analyzing data and writing. He expects his paper on lateral suppressor1 to be published soon among many others in the works.
“I think we’re all ready to get back to normal life,” Camden said.
When Carolyn Robinson was a kid, she was fascinated by the world around her. She remembers putting scabs under magnifying glasses and squishing bugs to try and understand the oddities of the world.
“Science continuously blows my mind,” Robinson said. “There’s always something where you almost don’t believe it at first, and there is so much we still don’t know, even about something as simple as a virus.”
As a now 3rd-year graduate student working on her Ph.D. in molecular pathogenesis and therapeutics in Marc Johnson’s lab, she is still attempting to make sense of the puzzling things around her. She screens potential compounds that might block VPU, a protein that helps HIV escape the host.
Robinson grew up with parents who taught her the value of solving a puzzle on her own. She said this influenced her and gave her curiosity more direction throughout her life.
“My dad’s favorite quote was there’s more than one way to do anything, so if I was frustrated about something he’d encourage me to find a new way, or if I didn’t know how to spell a word he’d give me the first letter and make me find it in the dictionary,” she said.
Though she enjoys the small victories of solving puzzles, she said it can be easy to get lost in the day-to-day of research and lose sight of the big picture. When she faces a challenge or needs to clear her mind, she takes a 15-minute walk around campus to gain perspective, and the possibility of learning something new keeps her interested and excited throughout her research.
“You don’t expect to want to come in at 9 p.m. to check on some cells,” she said laughing.
During her undergraduate degree in cellular and molecular biology at Depaul University in Chicago, a mentor helped her realize her interest in doing research. After interviewing with different graduate programs, the experience she had with MU drew her in. She loves the atmosphere of not only her lab but of the community inside Bond LSC.
Though she isn’t certain where her studies will take her, she hopes to continue learning and participating in the community that science generates.
“I hope to be able to get science out into the community and explain science in a way that makes people who don’t have a degree in it to find it as interesting as I do,” she said.
As an undergraduate student, Yul Eum Song had experiences that put her on a path to help create change.
Now as an experienced and educated doctoral researcher, she studies the mechanisms of retroviruses in the lab of Bond LSC’s Marc Johnson, and she continues to love science and the way it answers questions and positively influences the world.
“I like viruses because they can be deadly, so it’s really fascinating for humans to learn about,” Song said.
Song received her undergraduate degree from Dongguk University in Korea. Though she started out as a philosophy major, she realized her love of science and decided to add an additional major of life sciences, or biology. While at Dongguk, she became interested in viruses, which ultimately influenced her decision to come to MU.
“Studying viruses can help get treatment, and that is really the big motivating thing for me to do science,” Song said. “Science makes me think that I can do small things every day to reach the goal of making the world a better place.”
Song says her goals in life all center around one idea, making the world a better place. She believes that each small discovery can lead to big ones.
“Understanding the basic mechanisms is the first step to understanding the virus and how to apply the therapy,” Song said. “I am doing really basic science, and I can see how HIV research affects society, and it is really cool to see that. I can see how the little things form the big research.”
In Johnson’s lab, Song focuses her research on the mechanisms of retroviruses such as HIV, particularly Env glycoprotein. She is fascinated by how viruses utilize their hosts through very minimum genes and acknowledges that learning about how a virus operates at the basic level can give a scientist clues on what further questions to ask.
“It’s motivating to do something that’s useful for the world and also that’s interesting,” Song said. “HIV is one of the most interesting viruses to work with because of how it affects society.”
In May, Song plans to have finished her Ph.D. and to continue her education at St. Jude Research Center for her postdoc. While there, she will have the opportunity to observe doctors and put her research into context. She will be able to observe the ways in which doctors use research to create therapies. She is excited to work with translational research and capture the big picture.
Song thinks that understanding the entire context is vitally important. By seeing both the big picture of her own research and the larger picture of how that research gets used in the medical world, she and other virologists can avoid problems and create better solutions.
“I am still thinking about what I’ll be doing after my postdoc. I like science, so I want to keep doing science,” Song said. “My ultimate goal in life is to make the world a better place.”
Though she is still figuring out what specific steps she will take in the future, she holds tight to her goal of positively influencing the world through science.
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.
Viruses can be nasty things and scientists have to take precautions.
You might think of researchers in floor-length lab coats, safety goggles, and plastic gloves or even the more extreme look of bulky, yellow hazmat suits similar to what Jim Hopper wear in Stranger Things. But, depending on the type of viruses being handled, these stereotypes aren’t quite the truth.
For labs like that of Marc Johnson in Bond LSC, safety comes from the incomplete nature of the HIV viruses they study. The viruses in Johnson’s lab are defective, meaning they cannot reproduce themselves. It doesn’t mean the virus is completely safe to handle. If it were to come into contact with another living being it would only infect the cells exposed to the virus and could not expand further. With defective strains of HIV, the virus can be grown at biosafety level two.
This level two is one of four levels of biosafety that are used to define how a lab might be physically set up and how its researchers are equipped in order to contain a virus. The levels act as more of a scale than a concrete definition of the lab since the type of virus being handled by a lab can vary.
Johnson, a professor of Molecular Microbiology and Immunology, says that his lab teeters between BSL-2 and BSL-3 depending on what type of virus they are working with.
“Our lab is classified as a BSL-2 (Biosafety Level 2) because we work with pathogens that have a low chance of spreading and a low chance of doing significant damage if they do spread,” said Dr. Johnson. “This simply means that our lab is shut off from anyone outside the lab who might try to come in outside of business hours. We also equip our staff with safety goggles, and gloves while they work with a virus under a Laminar Flow Hood to keep the air sterile.”
The major difference between levels like BSL-2 and BSL-3 often comes down to the type of virus being handled, whether it be something like HIV or a more lethal infection like SARS. The Laboratory for Infectious Disease Research is one of the only facilities on the MU campus, that can be classified as a level three. Because the lab handles airborne viruses, they take extra precautions to regulate air and waste coming out of the facility.
The highest level of biosafety can be identified as BSL4, which typically handles deadly viruses such as Ebola that could cause significant harm if it spread. This is where the terrifying and bulky hazmat suits come into play. The virus being handled in a BSL-4 lab comes with a high risk of researchers being infected if the proper steps are not taken to properly handle or contain the virus.
There are no BSL-4 laboratories in Columbia. In fact, one of the nearest labs won’t be opening its doors until 2022 in Manhattan, Kansas. These levels of safety are simply put in place to protect those who wish to study a virus and further medical research for the rest of the world.
The different levels of Biosafety might seem frightening to some, but there is nothing really to fear. These precautions are put in places just as signs that remind us to wash our hands after using the restroom. They are ways to prevent the contamination and spread of viruses and disease. MU Researchers don’t just have these precautions in place to protect everyone around them. They also have these precautions in place so that viruses like HIV can be better understood and treated by medical professionals around the world.
“#IAmScience because the mysteries of the natural world aren’t going to solve themselves.”
Since the third grade, Marc Johnson never wanted to be anything else but a mad scientist. What began as experimenting with sprouting seeds and chemistry sets has blossomed into a career in virology. Specifically, he studies the “moves and countermoves” of viral components, a few hundred thousand at time! His advice for people wondering if science is for them: “If you’ve ever stayed up until 4 in the morning to finish a puzzle, you might be a scientist.”
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.