Proteins limit HIV-1 infection

Cells that expressed IFITM proteins (bottom row), showed much less spread of HIV-1 compared with cells lacking the protein. | courtesy Jordan Wilkins, Liu Lab

Cells that expressed IFITM proteins (bottom row), showed much less spread of HIV-1 compared with cells lacking the protein. | courtesy Jordan Wilkins, Liu Lab

By Jennifer Lu | MU Bond Life Sciences Center

For Shan-Lu Liu, thinking outside the box meant putting an antiviral protein inside HIV-infected cells, rather than into healthy ones.

Liu and his team of researchers studied how interferon-induced transmembrane (IFITM) proteins limit the infection of HIV-1, the primary strain of virus responsible for AIDS. The journal Cell Reports published their results on September 17.

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, the Liu laboratory demonstrated that these three IFITM proteins have the ability to thwart a variety of viral infections.

Shan-Lu Liu, Bond Life Sciences scientists and associate professor in the MU School of Medicine department of molecular microbiology and immunology. | Photo by Justin Kelley, University of MIssouri Health Care

Shan-Lu Liu, Bond Life Sciences scientist and associate professor in the MU School of Medicine department of molecular microbiology and immunology. | Photo by Justin Kelley, University of Missouri Health Care

“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.

Chemicals and couch potatoes

Endocrine disruptors impact physical activity and metabolism in mice

By Caleb O’Brien | MU Bond Life Sciences Center


Could your experiences in the womb make you lazy as an adult?

A recent study of California mice suggests that early exposure to environmental chemicals can later impact an animal’s metabolism and level of voluntary physical activity, according to new University of Missouri research.

“We found that if we developmentally exposed California mice to bisphenol A (BPA) or ethinyl estradiol (EE), the estrogen present in birth control pills, it caused later disruptions in voluntary physical activity,” said Cheryl Rosenfeld, a researcher in MU’s Bond Life Sciences Center and associate professor of biomedical sciences in the College of Veterinary Medicine. “What that means is they move around less in their home cage, they’re more likely to sleep, and they engage in less voluntary physical activity.”

Rosenfeld’s lab studies the ways that exposure to environmental chemicals such as BPA can affect other behaviors, including cognition and parenting. Endocrine-disrupting chemicals can accumulate in the environment and act like the hormones naturally produced by many organisms, including humans. To test the chemicals’ impact on metabolism and activity, the lab used California mice. This mouse model is a good model for metabolic diseases. And because these animals are initially derived from the wild, they may better replicate the genetic diversity of most human populations.

The researchers exposed the mice to BPA and EE in the womb and until weaning via the mom’s diet. A third group of mice whose mothers were placed on a phytoestrogen-free control diet was not exposed to either chemical. The scientists then placed all the mice on this same control diet and measured their energy expenditure, body composition and level of voluntary physical activity as adults.

To test those attributes, Rosenfeld’s lab relied on a variety of tools and techniques. They rigged bicycle computers to “hamster wheels” to track how far, fast and for how long the mice ran. Using a device called a “Promethion continuous measurement indirect calorimetry system” the researchers continuously monitored the mice’s energy expenditure by measuring oxygen consumption and carbon dioxide production and by using a three-beam system, tracked the rodents’ movements during the dark and light cycles.

Later, the researcher measured the animals’ body composition using an EchoMRI, a tiny MRI machine the size of a filing cabinet, and finally measured circulating concentrations of glucose and hormones that regulate metabolism.

Female mice exposed to BPA and EE were less active than control mice. They moved around their cages less at night (when the nocturnal California mouse is considered most active), moved more slowly, drank less water, and spent more time sleeping. In addition, BPA-exposed females burned more carbohydrates relative to fats, as compared to control mice. This is similar to the difference between obese and slender humans, and many researchers believe that burning more carbohydrates relative to fats can lead to fats gradually accumulating in the body.

“It’s worrisome that environmental chemicals we are exposed to in utero can override our genes and disrupt our neuro-circuitry,” said Sarah Johnson, a research specialist and graduate student in Rosenfeld’s lab and primary author on the study. “The net effect is that we can have behavioral disruptions into adulthood, including altered physical activity.”

The researchers are currently conducting follow-up studies to determine if the changes caused by exposure to BPA and EE predispose mice to obesity and other metabolic disorders. They also are interested in exploring if exposure could affect the children and grandchildren of these mice and examining the potential underlying neural mechanisms.

“Our findings are significant because decreased voluntary physical activity, or lack of exercise, can predispose animals or humans to cardiovascular diseases, metabolic disorders and even cancer,” Rosenfeld said.

Other authors on the study are Angela Javurek and Michele Painter (MU Biomedical Sciences), Mark Ellersieck (MU Agriculture Experimental Station- Statistics), Charles Wiedmeyer (MU Veterinary Medical Diagnostic Laboratory and Department of Veterinary Pathobiology) and John Thyfault (Kansas University Medical Center, Molecular and Integrative Physiology)

The study, “Sex-Dependent Effects of Developmental Exposure to Bisphenol A and Ethinyl Estradiol on Metabolic Parameters and Voluntary Physical Activity” was supported by NIH Grant 5R21ES023150 (to C.S.R.) and R01DK088940 (JPT) and was published in the Journal of Developmental Origins of Health and Disease.