ineteen colorful foam flowers decorate the walls of Marc Johnson’s office, mementos from laboratory members who “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 a small but dedicated cohort of researchers at MU’s Bond Life Sciences Center who study HIV, the virus that causes AIDS. Johnson’s efforts shed light on a question that has perplexed HIV researchers for years: How is the virus able to assemble copies of itself 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 says. “It doesn’t do it by itself.”
Viruses, in fact, use proteins in our bodies to work against us, he says. To understand how, you have to understand the cells that viruses infect.
Cells use a protein called TSG101, for example, to dispose of unwanted surface macromolecules by bending a patch of cellular membrane around the macromolecule until it is isolated inside a “membrane bubble.” The process, like trapping a bug inside tissue, is called budding.
The cell then sweeps all of these pinched-off membrane bubbles into a larger receptacle, called the “multivesicular body.” These multivesicular bodies, Johnson says, act as the cell’s garbage collection system. To dispose of the “trash” they contain, the compartments become acidic enough to disintegrate everything inside or else fuse with the cell membrane so that the trash gets dumped outside the cell.
Johnson compares this latter process to the "garbage-dump ploy" that allows Millennium Falcon to escape its pursuers in The Empire Strikes Back: “They just drop all their garbage before they go into hyperspace.” HIV, he says, 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 says.
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 says. “Or they’ll spend years on the back burner. I have a lot of projects.”
One of the biggest projects involves using CRISPR/Cas9 — the precision "gene-editing" tool detailed in depth in Illumination’s previous issue — to identify genes that make a cell resistant to viral infections. “It’s a game changer. It really is,” Johnson says. “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 says, there’s less doubt about what is happening.
With CRISPR, researchers in Johnson's laboratory 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 become infected and die. Those that survive can cue researchers as to which genes might be important for blocking HIV infection.
And if another researcher has doubts that a gene is truly knocked out, Johnson says, you can tell them, “I’ll just send you the cell line. You try it and see for yourself.”
The Johnson laboratory is a tight-knit group that consists of a manager, two graduate students, a postdoctoral fellow four undergraduates.
Dan Cyburt, a third year graduate student, studies molecules that interact with proteins, such as TRIM5, that keep HIV from infecting cells. TRIM5, a so-called restriction factor, blocks replication of the viral genome.
Fourth-year graduate student Yuleum Song focuses on how the viral envelope protein, Env, is packaged into the viruses before they break free from cells. While Env isn’t necessary for viral assembly and release, she says, it’s critical for the infection of new cells.
Undergraduates work in a tag team, picking up where the other leave off, to generate a collection of new viral clones, while lab manager Terri Lyddon plays the crucial role of keeping 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, Salmonella 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 that these handicapped viruses can infect exactly one round of cells and spread no further.
According to the latest available data from the World Health Organization, some 37 million people worldwide are infected with HIV or have AIDS. Some two million new infections are recorded every year. Thankfully, breakthroughs in treatment have turned the autoimmune disease from a death sentence into a chronic and manageable condition, albeit one that is not easy on patients. As such, for the past several years researchers have spent much of their time working to improve therapies against HIV, while also 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 says, referring to Timothy Ray Brown, the now-famous "Berlin patient" who was cured of AIDS. “So it’s only going to get easier.”
However, Johnson pointed out, most people would never undergo the kind of high-risk treatment that Brown received. Brown underwent a bone marrow transplant at a Berlin hospital to treat his leukemia. The new marrow, which came from an HIV-resistant donor, left him AIDS free.
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 says, then researchers could train the immune response in AIDS patients to resist HIV or keep it in check.
As a basic scientist, Johnson says, he adds to the knowledge of how HIV works. “I am not thinking about a therapy,” Johnson says, “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 says, the rules do exist.“The sculpture is already there in the stone,” he says. Johnson’s job is to chip away at the marble until the rules are found.
Another bench researcher working to solve the puzzle of HIV/AIDS is MU virologist Shan-Lu Liu. For him thinking outside the box meant putting an antiviral protein inside HIV-infected cells, rather than into healthy ones.
Liu and his team of researchers are working to understand how interferon-induced transmembrane (IFITM) might limit the infectious potency of HIV-1, the primary strain of virus responsible for AIDS. IFITM proteins are biomolecules with broad antiviral properties. Although multiple versions of IFITM have been found in humans, three are known to have antiviral properties: IFITM1, IFITM2 and IFITM3.
In a 2013 paper published in PLoS Pathogens, Liu and his laboratory team demonstrated that these three IFITM proteins have the ability to thwart a variety of viral infections.
“They can inhibit influenza virus, Ebola virus, HIV and SARS coronavirus,” says Liu, also an associate professor of molecular microbiology and immunology at MU.
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 says, envelope proteins must 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 says, 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 says 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 says. “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 says 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 number 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’ infectiousness 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 proteins rather than with other proteins, such as Gag. Liu attributed the finding to his two 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 generations.
Liu says that his research on IFITM is still in its early stages. The next step will 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 says. “So that’s our ultimate goal.”
An earlier version of this story appeared in MU's DeCoding Science blog: http://decodingscience.missouri.edu/