How do viruses assemble in cells? Scientists reverse engineer a slow down version of rotavirus

Rotavirus is one of the main pathogens causing diarrhea in infants and young children. It mainly infects small intestinal epithelial cells, causing cell damage and diarrhea, causing the death of 130,000 infants and children under 5 years old every year.
It is the single leading cause of diarrhea in infants and young children, and nearly every child around the world has been infected with rotavirus at least once.

Viroplasms have been difficult to study because they usually form very quickly, but a chance observation led researchers at Baylor College of Medicine to discover new insights into viral plasmid formation.

The researchers created a mutated rotavirus that replicates unexpectedly much more slowly than the original virus, allowing them to observe the first step in virus assembly.

The findings, published in the Journal of Virology, open new possibilities for treating and preventing the viral disease and understanding how similar factories for other viruses work.

“Virulence formation is essential for successful rotavirus infection.
They quickly formed in infected cells, is composed of virus and cell protein, and interaction with lipid drops, but the details of how these ingredients are combined together are still unclear, “lead author Jeanette m. Criglar said, she is a former postdoctoral interns, now the department of molecular virology and microbiology baylor Mary laboratory scientist Dr Estes.

Lipid droplet is a complex and active multifunctional organelle, whose basic functions are mainly involved in lipid synthesis and storage. Lipid droplet related proteins are involved in a variety of lipid related biological processes, including cell signal transduction, lipid metabolism regulation, membrane transport, and the synthesis and secretion of inflammation-related proteins.

Many pathogens, including viruses, intracellular bacteria, and protists, target lipid droplets.
Pathogenic microorganisms can induce the increase of intracellular lipid droplets which in turn provide a platform for the proliferation of pathogenic microorganisms.

To shed new light on viral plasmid formation, Criglar and her colleagues studied NSP2, a viral protein essential for virus replication.
Without it, neither virulence nor new viruses can form.

Like all proteins, NSP2 is made up of amino acids that are strung together like beads on a necklace.
Bead’ 313 is serine.
And importantly, ser313 is phosphorylated — it has a phosphate group attached to it.

Protein phosphorylation is a mechanism by which cells regulate protein activity.
It works like a switch that activates or deactivates proteins.

Here, the role of NSP2 phosphorylation of serine 313 in virion formation was assessed.

Using a recently developed reverse genetics system, Criglar and her colleagues created a rotavirus with an NSP2 protein by converting serine to aspartic acid. The virus has a mutation of 313 amino acids, called a phosphate-like mutation.

The phosphoprotein name suggests that the mutant protein mimics the phosphorylated protein in the original rotavirus.

Reverse genetics starts with a protein, works backwards, makes a mutated gene, and then makes it part of a virus to study how the protein affects the behaviour of the virus.

“In laboratory experiments, our phospho-like mutant protein crystallizes faster than the original protein, not in days but in hours,” Criglar says.

“But surprisingly, compared to non-mutated rotaviruses, the phosphoviruses produce virions and replicate very slowly.”

“This is not what we expected.
We think the rotavirus with the mutant protein will also replicate faster.”

“We took advantage of the delay in viral plasmid formation to look at early events that are difficult to study.”

The first step in virality formation, the researchers found, was the binding of NSP2 to the lipid droplet, suggesting that NSP2 phosphorylated only at 313 can interact with the lipid droplet and not with other components of the virion.

Lipid droplets are an important component of viral virulence.
Rotavirus is known to trick infected cells into producing fat droplets, but how it does so is unclear.

New findings suggest that rotavirus may use phosphorylated NSP2 to trigger lipid droplet formation.

“It is very exciting to see that changing just one amino acid in the NSP2 protein affects the replication of the entire virus,” Criglar said.

“This phosphorous change changes the dynamics of virus replication without killing the virus.
We can use this mutated rotavirus to continue to study the sequence of events that lead to viral plasmid formation, including a long-standing question in cell biology about how lipid droplets form.”

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