Slow-growing rotavirus mutant reveals early steps of viral assembly


Date:
June 23, 2020
Source:
Baylor College of Medicine
Summary:
Rotavirus is responsible for more than 130,000 deaths in infants and
young children younger than five years, every year. The virus causes
severe, dehydrating diarrhea as it replicates in viral factories
called viroplasms that form inside infected cells. Viroplasms have
been difficult to study because they normally form very quickly,
but a serendipitous observation led researchers to uncover new
insights into the formation of viroplasms.



FULL STORY ========================================================================== Rotavirus is responsible for more than 130,000 deaths in infants and young children younger than five years, every year. The virus causes severe, dehydrating diarrhea as it replicates in viral factories called viroplasms
that form inside infected cells. Viroplasms have been difficult to study because they normally form very quickly, but a serendipitous observation
led researchers at Baylor College of Medicine to uncover new insights
into the formation of viroplasms.


==========================================================================
The researchers created a mutant rotavirus that unexpectedly replicated
much slower than the original virus, allowing them to observe the first
steps of viral assembly. The findings, published in the Journal of
Virology, open new possibilities for treating and preventing this viral
disease and for understanding how similar factories of other viruses work.

"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," said
first author Dr. Jeanette M. Criglar, a former postdoctoral trainee
and now staff scientist in the Department of Molecular Virology and Microbiology at Baylor in Dr. Mary Estes's lab.

To get new insights into the formation of viroplasms, Criglar and her colleagues studied NSP2, one of the viral proteins that is required for
the virus to replicate. Without it, neither viroplasms nor new viruses
would form.

Like all proteins, NSP2 is made of amino acids strung together like beads
on a necklace. 'Bead' 313 is the amino acid serine. Importantly, serine
313 is phosphorylated -- it has a phosphate chemical group attached to
it. Protein phosphorylation is a mechanism cells use to regulate protein activity. It works like an on-and-off switch, activating or deactivating
a protein. Here, the researchers evaluated the role NSP2's phosphorylation
of serine 313 plays on viroplasm formation.

A serendipitous finding Using a recently developed reverse genetics
system, Criglar and her colleagues generated a rotavirus carrying an
NSP2 protein with a mutation in amino acid 313, called a phosphomimetic mutation, by changing serine to aspartic acid. The name phosphomimetic indicates that the mutant protein mimics the phosphorylated protein in
the original rotavirus. Reverse genetics starts with a protein and works backward to make the mutant gene, which then is made part of the virus
to study the function of the protein on viral behavior.



==========================================================================
"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."
"This is not what we expected. We thought that rotavirus with the mutant protein also would replicate faster," said Estes, Cullen Foundation
Endowed Chair and Distinguished Service Professor of molecular virology
and microbiology at Baylor. "We took advantage of the delay in viroplasm formation to observe very early events that have been difficult to study." Early steps: NSP2 and lipid droplets come together The researchers
discovered that one of the first steps in viroplasm formation is
the association of NSP2 with lipid droplets, indicating that NSP2 phosphorylated on position 313 alone can interact with the droplets,
without interacting with other components of the viroplasm.

Lipid droplets are an essential part of viroplasms. It is known that
rotavirus coaxes infected cells to produce the droplets, but how it does
it is unknown.

The new findings suggest that rotavirus may be using phosphorylated NSP2
to trigger lipid droplet formation.

"It was very exciting to see that just changing a single amino acid in
the NSP2 protein affected the replication of the whole virus," Criglar
said. "The phosphomimetic change altered the dynamics of viral replication without killing the virus. We can use this mutant rotavirus to continue investigating the sequence of events leading to viroplasm formation,
including a long-standing question in cell biology about how lipid
droplets form." "This is the first study in our lab that has used the
reverse genetics system developed for rotavirus by Kanai and colleagues
in Japan, and that's very exciting for me," Estes said. "There have
been very few papers that use the system to ask a biological question,
and ours is one of them."

========================================================================== Story Source: Materials provided by Baylor_College_of_Medicine. Original written by Ana Mari'a Rodri'guez, Ph.D.. Note: Content may be edited
for style and length.


========================================================================== Journal Reference:
1. Jeanette M. Criglar, Sue E. Crawford, Boyang Zhao, Hunter G. Smith,
Fabio
Stossi, Mary K. Estes. A genetically engineered rotavirus
NSP2 phosphorylation mutant impaired in viroplasm formation and
replication shows an early interaction between vNSP2 and cellular
lipid droplets.

Journal of Virology, 2020; DOI: 10.1128/JVI.00972-20 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/06/200623145358.htm

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