A team from Baylor College of Medicine in Houston Texas, created mutant rotavirus to study the formation of viroplasms or the initial stages of viral assembly. This will help scientists understand what triggers the rapid multiplication of this contagious virus.
Rotavirus is highly contagious, causing diarrhea and resulting in over 215,000 deaths of infants and children all over the world. The virus affects mostly younger ages, resulting in dehydrating diarrhea for up to eight days. It has several causes including consumption of contaminated food and putting unwashed hands into one's mouth after touching dirty surfaces, objects, or even poop.
Children who receive the vaccine only have partial protection from the virus and may still get infected multiple times. Besides the vaccine, those who contract infection are usually given rehydration solutions, medication, and treatment with intravenous (IV) fluids for severe cases.
Viral Assembly
Initial observations by the research team are that their mutant creation replicated slower than the original virus, allowing them to observe viral assembly. Viral assembly happens after infection occurs, where protein subunits assemble into a shell (capsid) around the viral nucleic acid. The capsid will penetrate another cell and this process multiplies, widely spreading infection.
Dr. Jeanette Criglar from the Department of Molecular Virology and Microbiology at Baylor said that 'the formation of viroplasms is indispensable for a successful rotavirus infection. They form quickly inside infected cells and are made of both viral and cellular proteins that interact with lipid droplets, but the details of how the parts are put together are still not clear.'
The team focused on a viral protein, NSP2, which allows the viroplasms to form and the virus to replicate itself. Along the NSP2's string of amino acids is the phosphorylated serine 313. Serine amino acids are attached to a phosphate chemical group, which activates and deactivates a protein.
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Mutant Form
Using a reverse genetics system, the scientists tested a sample of the rotavirus containing NSP2 and combined it with a mutated 313 amino acid, creating a phosphomimetic mutation. 'In laboratory experiments, our phosphomimetic mutant protein crystalized faster than the original, within hours as opposed to days," Criglar said. 'But surprisingly, when compared to non-mutant rotavirus, the phosphomimetic virus was slow to make viroplasms and to replicate.'
Dr. Mary Estes of the university said that the results were not what they had expected. The delay of viroplasm formation in their mutant virus had been advantageous to make new observations about how rotavirus progresses, she shared.
One observation they made in the initial stages of viroplasm formation is the NSP2's interaction with lipid droplets. As rotavirus infects cells, it produces lipid droplets for unknown reasons.
When the team observed how the NSP2 attached to 313 can interact with the droplets without interacting with the rest of a viroplasm's components, they concluded that the protein may be a trigger. Criglar expressed her excitement to see how the replication of an entire virus is affected by the change of one single amino acid.
'The phosphomimetic change altered the dynamics of viral replication without killing the virus,' said Criglar. The mutant rotavirus can help answer questions about the formation of lipid droplets. Estes expressed how this their first study on using a Japanese reverse genetics system for rotavirus.
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