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Mutant protein sheds lights on viral propagation

March 30, 2017

Sarah McDonald, Ph.D.

Sarah McDonald, Ph.D.
Sarah McDonald, Ph.D., and her team found previously unknown functional sites on a rotavirus protein.

Some genetic mutations can cause a virus to flourish. Others make the virus wither away, unable to function normally and reproduce. Yet other genetic mutations only show their hand under certain conditions.

One such mutant protein, which malfunctions at high temperatures, has given scientists at the Virginia Tech Carilion Research Institute a better understanding of rotavirus —a common cause of diarrheal disease in infants and children.

Their study, which reveals previously unknown functional sites on a rotavirus protein, was recently published in the Journal of Virology.

Rotavirus infects every child under the age of five and can cause fatal dehydration if not properly treated.

The virus has three layers, with genetic material and enzymes at its center. The enzymes, called polymerases, help copy the virus’s genetic material. The details of this process have remained largely a mystery, limiting an understanding of rotavirus biology. 

To elucidate the details of this process, Sarah McDonald, an assistant professor at the Virginia Tech Carilion Research Institute who led the study, and her team utilized a strain of rotavirus with a temperature-sensitive mutation in the polymerase. When exposed to high temperatures, the virus is unable to propagate.

By studying how this mutation affects the polymerase’s behavior at high temperature, the researchers can begin to connect the dots of cause and effect between the mutation and the protein’s behavior.

“When the cells were incubated at low temperatures, the mutant polymerase trafficked just fine,” McDonald said. “Its enzymatic activity was the same as the normal protein. At high temperatures, we saw a big discrepancy between the two protein types. This provided biochemical validation that the temperature sensitivity characteristic of this virus is likely caused by this particular mutation.”

According to McDonald, the region around the mutation had no known function for the polymerase. Yet, when the mutated protein was exposed to high temperatures, it couldn’t properly interact with other proteins nor could it efficiently replicate the viral nucleic acid genome.

Previous scientific studies had revealed the existence of the temperature-sensitive mutation. The Virginia Tech Carilion Research Institute team was the first to show how the mutation might cause the virus’s temperature sensitivity.

“Whenever you have a mutation that causes a functional defect in a protein, there are often changes to the protein’s structural dynamics – how the protein moves,” said McDonald, who is also an assistant professor at the Virginia-Maryland College of Veterinary Medicine.  

To understand how the mutation may have changed the polymerase’s structure, McDonald teamed up with Leslie LaConte, a research assistant professor at the Virginia Tech Carilion Research Institute. LaConte compared the mutant proteins to normal proteins with computational simulations of how the proteins move at high temperatures.

The screenings were overlaid, and the most dramatic differences were at the site of the mutation, as well as one other spot.

“Surprisingly, we also found a lot of changes in the movement of a very distal loop on the polymerase, which also has no known function,” McDonald said. “It suggests that the original mutated region is important, but that perhaps this other location is also important for the function and interaction of the polymerase.”

McDonald said that they’ve gained some insight into rotavirus replication – that these regions are functionally important – and there’s still a lot to uncover.

“This study was part of our ongoing series of experiments to probe the structure and function of the viral polymerase and to understand how the virus replicates inside cells,” McDonald said.

McDonald also noted that both viral polymerases and temperature-sensitive viruses have been used for years as scientific tools to study how viruses operate. The information gleaned from them in her team’s current studies could help inform infection prevention and treatment for rotavirus.

Allison McKell, a graduate student in the Biomedical and Veterinary Sciences program at the Virginia-Maryland College of Veterinary Medicine, was the lead author on the paper, in addition to McDonald and LaConte.

The research was supported by the Virginia Tech Carilion Research Institute, the Biomedical and Veterinary Sciences Graduate Program of the Virginia-Maryland College of Veterinary Medicine, and the Virginia Tech Carilion Medical Scholars fund. 

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