A
team of scientists led by Ronald Harty, a professor of pathobiology and
microbiology at the University of Pennsylvania's School of Veterinary Medicine,
has identified a mechanism that appears to represent one way that host cells
have evolved to outsmart infection by Ebola and other viruses. In a new paper,
he and colleagues reveal that host cells sequester viral proteins away from the
plasma membrane within the cell, thus preventing viruses from spreading.
Harty
says that finding a way to amplify this molecular interaction could lead to a
novel antiviral strategy against Ebola that could temper the pathogen's damage.
The
scientists knew that, in infections with Ebola as well as
many other viruses, including Marburg, rabies and HIV, viral matrix proteins,
such as Ebola VP40, interacted with host proteins through short protein motifs:
the PY motif on the viral protein and the WW motif on the host cell protein.
Prior to this current study, all of the known interactions enabled the virus to
bud efficiently from the cell.
In
the current work, the researchers screened for new WW motifs from mammalian cell
proteins that bound tightly to the PY motif of Ebola virus' VP40 protein. Not
every PY motif binds to every WW domain; the interaction is specific, like a
lock and key.
The
screen turned up some proteins that the Penn team had explored before but also
a new one, a protein called BAG3, known as a chaperone protein, which under
normal physiological conditions acts to promote cell survival.
After
confirming that Ebola VP40 interacted with full-length BAG3 in mammalian cells
specifically via the WW domain, the researchers went on to test its
functionality in influencing budding. They used a test that avoids manipulating
the actual Ebola virus, which is too dangerous for the Penn Vet laboratory.
Instead, they examined virus-like particles, which are produced by the virus's
matrix protein, VP40, and are not infectious but accurately mimic the budding
step of infection.
When
the researchers examined cells expressing either Ebola or Marburg VP40 and then
added in BAG3, they found that VLP budding went down in a dose-dependent
manner. When they mutated the WW domain of BAG3 so it couldn't interact with
VP40's PY domain, budding levels remained unchanged.
Knocking
down levels of naturally occurring BAG3 with synthetic strands of RNA did just
the opposite, increasing budding.
"With
all of these assays, there was a consistent effect on budding levels, either up
or down," Harty said.
Enhancing
such an interaction, perhaps in combination with other therapies that attack
the virus at other stages of its life cycle, could give the immune system the
opening it needs to overcome an infection.
Using
confocal microscopy and fluorescently labeled proteins, the scientists
discovered that BAG3 appeared to be sequestering VP40 in the cell's cytoplasm
away from the plasma membrane where the viral particles would need to go in
order to bud off and spread to infect other cells. Their work identified BAG3
as the first WW containing host protein to negatively affect virus budding.
Although
the group has not yet tested the VP40-BAG3 interaction with live Ebola or
Marburg virus -- those experiments are planned -- they did find that BAG3
limits budding of a recombinant vesicular stomatitis virus that contains the
Ebola PY motif.
"We
used that to show that this works in a live virus infection," Harty said.
"Taken together, we're hoping and assuming that it works the same way with
the authentic Ebola virus."
In
addition to testing BAG3 interactions with the Ebola and Marburg virus, Harty's
lab also plans to further investigate what BAG3 is doing to VP40, whether it's
simply sequestering it or whether it's modifying or degrading it.
See:
Posted by Dr. Tim Sandle
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