Rana's group used
the CRISPR Cas9 gene-editing tool in order to edit all the genes of human
(a type of
brain cancer) stem cells grown in 3D culture in tissue culture flasks. Each
variant of the gene-edited glioblastoma stem cells were exposed to Zika virus
to identify the specific gene and the respective encoded protein that are
required for entry of the virus into the cells. The virus entry process was
visualized by staining the viral particles with a green fluorescent protein
(GFP) coupled with fluorescence microscopy. Importantly, the researchers were
able to identify 92 specific human glioblastoma stem cell genes that are
essential for entry and replication of Zika virus in the cells. Of all these
genes, the most prominent was the gene that encoded the protein αvβ5 integrin. The findings have
been published in the journal
"Integrins are well
known as molecules that many different viruses use as doorknobs to gain entry
into human cells,"
says Rana. "I was
expecting to find Zika using multiple integrins, or other cell surface
molecules also used by other viruses. But instead, we found Zika uses αvβ5,
which is unique. When we further examined αvβ5 expression in brain, it made
perfect sense because αvβ5 is the only integrin member enriched in neural stem
cells, which Zika preferentially infects. Therefore, we believe that αvβ5 is
the key contributor to Zika's ability to infect brain
Rana's group also
used a mouse model to demonstrate that αvβ5 integrin could be blocked by daily
treatment with two experimental anticancer drugs - Cilengitide
SB273005 - both of which target αvβ5 integrin. The mice were treated
with the drugs, followed by Zika virus infection. Six days after virus
infection, the mice brains were harvested. The brains of the drug-treated mice
showed half the number of virus particles, compared to the brains of untreated
"The neat thing is that these findings not only help
advance the Zika virus research field, but also opens the possibility that we
could similarly block the entry of multiple viruses that use other integrins
with antibodies or small molecule inhibitors,"
Rana's group is
currently designing a mouse model in which αvβ5 integrin is absent in
the brain. This mouse model will help them to conclusively prove that αvβ5
integrin is absolutely vital for entry of Zika virus into the brain.
Study 2: Inhibition of Integrin -
Blocks the Entry of Zika Virus into the Brain
The second study
was led by Dr. Jeremy N. Rich, MD, MHS, who is a Clinical Professor of Medicine
and Director of Neuro-Oncology at UC San Diego School of Medicine. He also
holds joint appointments as Faculty Member in the Sanford Consortium for
Regenerative Medicine and Sanford Stem Cell Clinical Center at UC San Diego. He
is also Director of the Brain Tumor Institute, San Diego, California, USA.
Based on the fact
that viruses exploit integrins to gain entry into cells, Rich's group inhibited
each integrin molecule with a different antibody to establish, which produced
the most pronounced effect.
"When we blocked other integrins, there was no
difference. You might as well be putting water on a cell,"
says Rich. "But with αvβ5, blocking it with an antibody
almost completely blocked the ability of the virus to infect brain cancer stem
cells and normal brain stem
They also used a
mouse model of glioblastoma to demonstrate the inhibition of αvβ5 integrin
with an antibody or by deactivating the gene encoding it. Both these approaches
blocked Zika virus entry, thereby prolonging the life of the treated mice,
compared to untreated mice. Importantly, inhibition of αvβ5 integrin in human
glioblastoma tissue samples obtained from patients during surgery also blocked
Zika virus entry into brain cells.
Treatment of Brain Cancer by
Leveraging Zika Virus
The idea of
leveraging Zika virus for treating brain
came to Rich, from the observation that Zika infection causes
shrinking of the brain, which is seen in microcephalic babies born to Zika
virus-infected mothers. He subsequently established that Zika virus selectively
targets and kills glioblastoma stem cells, which couldn't be treated with
conventional anticancer drugs.
study added to his previous findings by establishing why Zika virus
preferentially kills glioblastoma stem cells while sparing healthy cells. His
group elucidated the underlying reason by analyzing the structure of αvβ5 integrin.
They found that the αv subunit was associated with the stem cells, while the β5
subunit was associated with cancer cells. High levels of these two subunits
collectively (in the form of αvβ5 integrin) play a crucial role in glioblastoma
stem cell survival, which resulted in a higher rate of killing of
glioblastoma stem cells compared to healthy stem cells or other types of brain
Rich indicates: "It turns out that the very thing that helps
cancer cells become aggressive cancer stem cells is the same thing Zika virus
uses to infect our cells."
Proof-of-Principle in a Human
of the cell culture studies of Zika virus infection of glioblastoma cells was
tested in a human organoid model. The human organoid model was developed in
collaboration with Professor Alysson Muotri, PhD, who is the Director of the UC San
Diego Stem Cell Program and a Member of the Sanford Consortium for Regenerative
organoid model was established by transplanting human glioblastoma tumors into
human brain organoids that essentially acted as "mini-brains", which could be
utilized for drug discovery studies. It was established that Zika virus
exposure of human organoids selectively destroyed the transplanted glioblastoma
cells, sparing other cells within the organoids. Importantly, when αvβ5
integrin was inhibited, the anticancer effect of Zika virus was reversed, which
proved beyond doubt the crucial role played by αvβ5 integrin in modulating Zika
The research team
is planning to identify novel drug molecules that are capable of blocking Zika
virus infection of brain cells more effectively. They are also interested in
genetically modifying the virus so that it can better target and specifically
destroy the cancerous brain cells, leaving behind the healthy ones.
Rich concludes: "While we would likely need to modify the normal Zika
virus to make it safer to treat brain
tumors, we may also be able to take advantage of the mechanisms the virus
uses to destroy cells to improve the way we treat glioblastoma."
He adds: "We should pay attention to viruses. They
have evolved over many years to be very good at targeting and entering specific
cells in the body."
The studies were
funded by the National Institutes of Health, California Institute for
Regenerative Medicine, and the International Rett Syndrome Foundation.
- Study 1: Integrin αvβ5 Internalizes Zika Virus during Neural Stem Cells Infection and Provides a Promising Target for
Antiviral Therapy - (https://doi.org/10.1016/j.celrep.2019.11.020)
- Study 2: Zika Virus Targets
Glioblastoma Stem Cells through a SOX2-Integrin αvβ5 Axis - (https://doi.org/10.1016/j.stem.2019.11.016)