Human lung cells were infected
with influenza A virus. In unmodified cells (left) and cells expressing
an irrelevant VHH control (middle), influenza A virus genome segments
decorated with nucleoprotein NP (green) accumulate in the nucleus
(blue), where virus replciation takes place. If expressed in cells, the
antiviral VHH 508 (right) prevents nuclear import of viral genomes.
Cells expressing VHH are stained in red.
Credit: Courtesy of Florian Schmidt/Whitehead Institute
Whitehead Institute scientists have
determined how to use alpaca-derived, single-domain antibody fragments
(also called VHHs or nanobodies) to perturb cellular processes in
mammalian cells, including the infection of human cells by influenza A
virus (IAV) and vesicular stomatitis virus (VSV). With improved
knowledge of protein activity, scientists can tease apart the roles
individual proteins play in cellular pathways, understand how disease
corrupts cellular function, and begin to design interventions to rectify
such aberrations.
Until now, researchers have relied largely on genetic approaches or
small molecules to inhibit protein function. However, these methods'
usefulness has limits -- genetic alterations may cause unintended
phenotypes. Only about 15% of proteins are "druggable" using small
molecules.
"Our method is an interesting and, in my opinion, an important
addition to the toolbox of the molecular biologist," says Whitehead
Member Hidde Ploegh, who is also a Professor of biology at Massachusetts
Institute of Technology and an affiliate member of the Koch Institute
for Integrative Cancer Research at MIT. "The approach allows you to work
in a wild-type protein environment -- you don't tinker with the host's
protein structure or the genetic makeup of the cell you wish to study,
but rather you add a highly specific perturbant."
Ploegh's lab has devised a screening strategy that employs VHHs or
nanobodies. These molecules are small, highly specific in what they
recognize, and sturdy enough to function in the environment of the
cytosol. In earlier work, the Ploegh lab used nanobodies to image the
immune system's function in real-time. Working with Whitehead Fellow
Sebastian Lourido's lab, VHHs made by the Ploegh lab helped decipher the
mode of action of a key enzyme used by the Toxoplasma gondii parasite
to invade cells.
In the current line of research, described online in the journal
Nature Microbiology, scientists led by postdoctoral researcher Florian
Schmidt have developed a rational screening approach that led to the
identification of nanobodies that interfere with the ability of IAV and
VSV to infect cells.
First, the scientists created nanobodies against IAV or VSV by
injecting alpacas with inactivated viruses. Millions of DNA sequences,
amplified from the immunized alpacas, were inserted into lentiviruses to
enable expression of VHHs in the cytosol of human cells. The transduced
human cells were then challenged with IAV or VSV. Any surviving cells
must have produced a VHH that interferes with virus replication. Indeed,
of the millions of cells transduced, about 260 contained nanobodies
that protected the cells against either virus and reduced viral
infection by more than 80%. When Schmidt analyzed these hits, he found
that the nanobodies jammed the viruses' infection machinery using
tactics specific to each virus -- anti-IAV VHHs targeted the viral
nucleoprotein NP, while the anti-VSVs recognized the viral nucleocaspid
N.
Using a similar, nanobody-based method, Schmidt determined the role
of the adaptor protein ASC in inflammasome assembly in myeloid cells,
but he envisions even broader applications for such screens.
"This technique is a very rapid way of identifying inhibitors of
essentially any biological process," he says. "And it allows us to look
at all the surfaces of a collection of proteins that we're interested in
and find the sites that are important for protein function."
By stabilizing their target molecules, nanobodies act as
crystallization chaperones, which allow scientists to more easily solve
the proteins' structure. The sites where VHHs bind to proteins are also
potential drug targets, as these locations impair the proteins'
activity.
This work was supported by the National Institutes of Health,
Fujifilm/MediVector, and the Swiss National Science Foundation (SNSF).